Proefschrift Lagerveld by Nicole Nijhuis

SMALL RENAL MASS CRYOSURGERY

SMALL RENAL MASS CRYOSURGERY Imaging and vascular changes B R U N O L F

L A G E R V E L D

Colophon: Cover and design by PIMMINK. Sponsors to the thesis: GELITA MEDICAL GmbH Galil Medical MML-Medical Montes Jura Medical Technologies BV Biomedic Nederland B.V. Janssen-Cilag B.V. GlaxoSmithKline BV Astellas Pharma BV Hoogland Medical B.V. Memidis Pharma BV Olympus Nederland B.V. Roche Nederland BV Ferring Geneesmiddelen BK Medical Benelux Ipsen Farmaceutica B.V. Pfizer bv Takeda Nederland BV Chipsoft B.V. PorgĂ¨s Coloplast Division Menarini Farma Nederland Printed by Gildeprint â&#x20AC;&#x201C; www.gildeprint.nl

SMALL RENAL MASS CRYOSURGERY: Imaging and vascular changes

CRYOCHIRURGIE VAN KLEINE NIERTUMOREN: Beeldvorming en vasculaire veranderingen

ACADEMISCH PROEFSCHRIFT Ter verkrijging van de graad van doctor aan de Universiteit van Amsterdam Op gezag van de Rector Magnificus Prof. dr. D.C. van den Boom ten overstaan van een door het college voor promoties ingestelde commissie, in het openbaar te verdedigen in de Aula der Universiteit op woensdag 9 april 2014, te 13.00 uur door Brunolf Walther Lagerveld geboren te Den Helder

PROMOTIECOMMISSIE Promotores:

Prof. dr. J.J.M.C.H. de la Rosette Prof. dr. ir. J.A.E. Spaan

Co-promotores:

Prof. dr. ir. H. Wijkstra Dr. M.P. Laguna Pes

Overige leden:

Prof. dr. J.S. LamĂŠris Prof. dr. P.F.A. Mulders Prof. dr. R.J.A. van Moorselaar Prof. dr. D.A. Legemate Prof. dr. A.G.J.M. van Leeuwen Prof. dr. M.J. van de Vijver

Faculteit der Geneeskunde

Paranimfen Mr. F.C. Lagerveld MSc. R.J.R. de Geus

Daniël 12:4

“ Maar gij, Daniël, houd de woorden verborgen, en verzegel het boek tot de eindtijd; velen zullen onderzoek doen, en de kennis zal vermeerderen.”

King James Bible

“ But thou, O Daniel, shut up the words, and seal the book, even to the time of the end: many shall run to and fro, and knowledge shall be increased”

The enemy inside

I have an enemy whom I am going to kill. Yes, to kill, it â&#x20AC;&#x2122;s a confession, no, donâ&#x20AC;&#x2122;t call the police my enemy is inside. Inside me, is it, what is the wonder, everybody has an enemy inside, a Trojan Horse. My horse rides on my kidney and is quite small this is my luck I can still kill him. The Bible says: whoever comes to kill you, stand up earlier and kill him. So it is him, or me to be or not to be, no choice, no alternative. I fought so many battles, so many wars, this fight will be the most important a duel for life only between us two. It is a tumor, yes, malignant the doctors said and it hides, the coward deep there, inside my body. You guessed: I have a cancer, a little one: early discovery, they call it and they will freeze him inside me so he will die, not me, and I will be. MoshĂŠ Liba The Hague, 28-09-2008

CONTENTS Chapter 1

General introduction and scope of the thesis Page 9 - 27

Chapter 2

Computerized 3D renal arterial reconstructions with a vascular casting and imaging cryomicrotome technique in a porcine model Page 29 - 43

Chapter 3

Cryosurgical injury: Acute vascular changes after renal cryosurgery in a porcine model Page 45 - 58

Chapter 4

Cryosurgical injury: Change of vascular anatomy and blood flow in a porcine kidney Page 59 - 74

Chapter 5

The utility of PADUA and RENAL nephrometry indices to predict for complications of laparoscopic cryoablation for small renal tumors Page 75 - 94

Chapter 6

Interpretations of alternative imaging methods of post-cryosurgical renal masses: Contrast-enhanced ultrasonography Page 95 - 110

Chapter 7

Interpretations of alternative imaging methods of post-cryosurgical renal masses: 18 F- fluordeoxyglucose positron emission tomography in combination with low-dose computed tomography Page 111 - 132

Chapter 8

Future prospects of renal mass cryosurgery Page 133 - 149 Summary & Samenvatting Abbreviations Publications & abstracts Dankwoord Curriculum Vitae

page 151 - 165 page 167 - 169 page 171 - 175 page 177 - 180 page 181 - 183

CHAPTER 1 General introduction and scope of the thesis

Introduction

The prefix “cryo-” meaning “very cold, freezing”, is the Latinized form originating from the Greek word “kryos“ (κρúος) meaning ‘icy cold’. It is used to describe the effects of low temperatures or activities carried on at very low temperature. “Ablation” originates from the Latin word “ablationem”, which is the noun of action from the past participle stem of auferre “ to carry away”, from ab- (“off”) and ferre (past participle of latum: “to bear”). The term “ablation” in the late Middle English in the general sense means, “taking * away, removal”. I n medicine, the word is used to describe surgical removal of body tissue. However, in nature science, the word is used to describe the process of removal of snow and ice from a glacier or iceberg by melting or evaporation. Therefore, the word “cryoablation” is not the most appropriate word to describe the process of lethally freezing tissues without instant removal of it. Thus, the term “cryosurgery” fits better with the devitalizing condition of freezing tissue. “Cryobiology” is the biology that studies the effects of low temperatures on living things. The biophysical response in the cell is directly related to the behavior of water molecules. Some organisms have adapted to survive extremely low temperatures. They can produce molecules preventing cellular fluids from freezing solid. Human cells do not bear these adaptive mechanisms. The dual nature of freezing to preserve and destroy has created the subfields of cryopreservation and cryosurgery. Cryosurgery is an old method that after technical developments currently plays a significant role in the minimal invasive management of urological malignancies such as renal cortical cancer and prostate cancer. James Arnott (1797–1883) from Brighton, United Kingdom, was the first to describe the medical use of freezing tissue. In his publication from 1850, he reported that several diseases could be treated with cryosurgery for which he even 1 developed a distinctive device . It is only in 1961 that the modern era of cryosurgery began. Then, Cooper and Lee developed an automated cooling 2 device using liquid nitrogen . With this, they could achieve temperatures as 0 low as -190 C used to freeze tissue. Approximately 30 years later, the thermodynamic principle for liquid or gas (origin 1895-1900), known as the Joule-Thomson effect * is applied in the development of a new generation cryoprobes described in figure 1. According to Joule-Thomson effect the temperature of a gas or liquid when it is forced through a small aperture to a region of lower pressure while kept insulated so that no heat is exchanged with the environment. As liquid or

* Named after James Prescott Joule and Sir William Thomson (1

Baron Kelvin).

gas expands, the average distance between its molecules grows, allowing an increase of its potential energy. The increase of potential energy implies a decrease in kinetic energy and, therefore, in temperature. For each gas, there is a temperature of inversion above which the change is positive and below which it is negative. The use of pressurized gas instead of liquids allowed the miniaturizing of the probes. Pressurized Nitrous Oxide or Argon can produce extremely low temperatures at the tip of the cryoprobe. Helium can be used to warm the probes.

Figure 1. The Joule-Thomson effect for high-pressurized Argon (freezing) and Helium (warming) running through an open-end capillary tube held within a closedtip needle. (With courtesy of Galil Medical速)

Some key points for monitoring and controlling the progress of tissue freezing were studied in the last 3 decades. In 1988 Onik et al., introduced the ultrasound-guided prostate ablation for the treatment of prostate cancer 3 . However, the application of extreme low temperature conditions to the prostate was not without the risk of injuring the rectal wall. Since 1990, thermosensors have been additionally applied to gather temperature feedback from tissue locations at risk. This combined use of monitoring techniques led to an improved monitoring of the freezing process and thus decreased the risk for rectal wall injury. Saliken et al. studied the clinical role 4 of using computed tomography for monitoring ice development . Afterwards in 1999, Tacke et al. reported an in vitro model to compare the use of ultrasound, computed tomography and magnetic resonance imaging for 5 monitoring the freezing process .

Third generation ultra-thin cryoprobes can easily be percutaneously introduced to the target tissue. The combination of this minimally invasive character and the improved monitoring of the freezing process have led to an improved application of cryosurgery of malignant tissues and is nowadays regularly carried out in urological clinical practice. Although a significant amount of basic research into the working mechanism of cryoablation has been done, there still remain fundamental questions. Several factors have to be taken into consideration. The lethal effects of administrating extreme-cold temperature to tissues consist of acute or direct and indirect cellular injury. Tissue cells, tissue structure, and tissue function will be damaged. This also applies to the vessel wall that runs through tissues. Freezing-induced arterial vascular injury provides a valuable 6 contribution to tissue destruction . These cryobiological mechanisms are summarized in this thesis. It is crucial to understand the involved pathophysiological mechanisms that lead to complete eradication of viable 7, 8 cancerous cells . Apart from direct effects of freezing of cells in general, vascular injury induces a cascade of processes that eventually lead to blood flow cessation, causing ischemia, which is considered a therapeutic effect 7-9 because of further tissue destruction . The vascular effect of freezing tissue has been especially studied in relation to frostbite but not to tumor 10, 11 ablation . Recent studies focussed on the cryogenic effect on the 12-15 structure and function of the microcirculation of superficial tissues .A more detailed insight into the vascular effects of cryoablation is useful. Improved insight into the processes involved may result in improved technique and aid in advancing the clinical assessment of cryoablation intervention for cancers. Presentation and staging of renal cancer

The classical presentation of renal carcinoma such as lumbar pain, a palpable mass and hematuria is uncommon in small renal masses. Currently, more than 50% of patients will be diagnosed with asymptomatic renal cancer as an incidental finding as a result of increased use of body16 imaging technologies for varying reasons . It has to be taken into account 17 that about 20% of renal lesions â&#x2030;¤ 4cm diameter are benign . The difficulty in predicting whether a radiographically diagnosed small renal lesion is benign or malignant requires the clinician to assess the natural history of these masses. The Dutch guideline committee for renal cancer advices that whenever one doubts the indication for surgical intervention image-guided core needle biopsies should be performed for histological examination. The International Union against Cancer has developed the 2010 TNMclassification for cortical renal cancer (table 1), which guides therapeutic 18 options and has prognostic value . However, some tumor-histological

features cannot be answered based on biopsies alone, and therefore it invalidates proper classification. Since 2002, the TNM-classification distinguishes pT1a for lesions ≤ 4cm and pT1b for lesion > 4cm diameter. Also, peripelvinal or renal sinus fat invasion is recognized as equally prognostic potential as the existence of renal capsular invasion. Risk stratification, or staging (table 2), of renal cancer is mandated for optimal therapeutically management of renal cancer. Several studies using the TNM-classification (1997) have reported the relation between staging 19, 20 and renal cancer specific survival . They show that there is an independent relation between renal tumor size and disease-free survival. Table 1. th

TNM-classification 2010 (7 ed) for renal cancer: Tumor, node and metastases and histopathological grading (Source: the International Union against Cancer). T – primary tumor Tx T0 T1 T1a T1b T2 T2a T2b T3 T3a T3b T3c T4

Primary tumor cannot be assessed No evidence of primary tumor Tumor ≤ 7 cm in greatest dimension, limited to the kidney Tumor 4 cm or less Tumor > 4 cm but ≤ 7 cm Tumor > 7 cm in greatest dimension, limited to the kidney Tumor > 7 cm but ≤ 10 cm Tumor > 10 cm, limited to the kidney Tumor extends into major veins or perinephric tissues but not into the ipsilateral adrenal gland and not beyond Gerota fascia Tumor grossly extends into the renal vein or its segmental (muscle containing) branches, or tumor invades perirenal and/or peripelvic fat Tumor grossly extends into vena cava below diaphragm Tumor grossly extends into vena cava above the diaphragm or invades the wall of the vena cava Tumor invades beyond Gerota fascia (including contiguous extension into the ipsilateral adrenal gland)

N – regional lymph nodes Nx N0 N1 N2

Regional lymph nodes cannot be assessed No regional lymph node metastasis Metastasis in a single regional lymph node Metastasis in more than 1 regional lymph node

M - distant M0 M1

No distant metastasis Distant metastasis

Histopathological grading Gx G1 G2 G3

Grade of differentiation cannot be assessed Well differentiated Moderately differentiated Poorly differentiated

Table 2. th

TNM-classification 2010 (7 ed.) for renal cancer: stage categories (Source: the International Union against Cancer). Stage I Stage II Stage III Stage IV

T1 T2 T3 T1, T2, T3 T4 any T any T

N0 N0 N0 N1 any N N2 any N

M0 M0 M0 M0 M0 M0 M1

Renal cancer epidemiology and etiology

The absolute incidence of renal cancer in The Netherlands has been increasing in the last decade (figure 2). Of all cancers diagnosed in The Netherlands in the year 2011, renal cancer is listed number 10 with 2.174 patients (2%). It is expected that in the year 2020 in The Netherlands, 1.600 men and 940 women will be diagnosed with renal cancer. The extensive use of radiographically body-imaging has led to an increased accidental identification of renal masses. The majority of those are without asymptomatic. Also, etiological factors such as obesity are considered to 21, 22 have positively influenced the incidence of RCC . Predisposing factors for the development of RCC are lifestyle (obesity, smoking and food pattern), 23-26 hypertension, genetic factors and medication (amphetamines, diuretics) . Several hereditary syndromes predispose one for the development of RCC. Algaba et al. describe 7 renal cancer syndromes that can be characterized (Von Hippel-Lindau, hereditary papillary RCC, familial papillary thyroid carcinoma, Birt-Hogg-DubĂŠ, tuberous sclerosis, constitutional translocation 26 chromosome 3, hyperparathyroidism: jaw tumor) .

Figure 2. The incidence of renal cell cancer (RCC) in The Netherlands from 1989 to 2011 (Source: Dutch Cancer Registry).

Within the cohort of new-diagnosed RCC, the percentage of stage 1 has increased from 40.5% in 2003 to 46.8% in 2009. During the same period, the percentage of stage 2 and 3 has slowly decreased, whereas that of stage 4 remained equal. Currently in The Netherlands, around 1000 patients are diagnosed with stage 1 disease. The prevalence of RCC in The Netherlands consists of all patients bearing the diagnosis of renal cancer that are still alive. The 20-years prevalence for RCC in 2012 was approximately 14.000 patients. In 2006, the 5-years prevalence of RCC was nearly 5000 patients, whereas this increased to more than 7000 patients 6 years later (figure 3). This increase in number may be influenced by the increased incidence of RCC and increased overall survival of the Dutch population in that period. The increase of absolute prevalence was particularly noted in the population of 65 years and older 27 (man 7% per year and women 3% per year) .

Figure 3. The 5-year prevalence of renal cell cancer in The Netherlands from January2006 to January-2013 (Source: Dutch Cancer Registry).

Table 3 shows the relative survival of RCC for different age groups. The survival is corrected for the expected mortality of the Dutch population and is stratified for gender, age and calendar year. Still, it shows that younger patients with RCC are expected to have a better survival. Figure 4 shows the relative survival rates per stage of renal cancer. The number of patients diagnosed with renal cancer in the period from 2003 to 2009 was for stage I, 4863; stage II, 1541; stage III, 1786; stage IV, 2581; unknown, 32. The follow-up of these patients is registered to February 2012. It shows that the 9-years relative survival for stage 1 disease is 87%.

Table 3. Relative survival rate of patients with renal cell cancer in The Netherlands in five age categories in the period from 2001-2010. (Source: Dutch Cancer Registry) Tumor number ď&#x192;Ş

Survival percentage in years after diagnosis 0

Patient age (1999-2008) 18896 100% 44 452307 100% 54 554584 100% 64 655556 100% 74 75+ 4629 100%

87%

83%

80%

79%

78%

76%

75%

74%

75%

82%

74%

70%

68%

65%

62%

61%

60%

57%

56%

79%

72%

67%

63%

61%

59%

57%

56%

54%

53%

75%

68%

64%

61%

58%

56%

53%

50%

48%

64%

57%

53%

50%

47%

45%

42%

41%

39%

38%

Figure 4. Relative survival rates per stage in years after diagnosis of renal cancer th (2003 to 2009). Staging is performed according to the 6 edition of the TNMclassification. (Source: Dutch Cancer Registry).

Renal cancer subtypes

The World Health Organisation classified the heterogeneous group of renal cell carcinoma based on histopathological and cytogenetic entities (table 4) 28 . In adults, different subtypes show a variance in natural history and, thus, variable clinical outcomes. Cheville et al. reported that the cancer-specific 29 survival is related to the histological subtypes . At 5 years, the cancerspecific survival rates for patients with clear cell, papillary and chromophobe RCC were 68.9%, 87.4% and 86.7%, respectively. This difference in survival highlights the need for accurate subtyping and staging now new-drug developments are taking place in the last decennium. Table 4. World Health Organisation Classification 2004 for the histological subtypes of renal cancer.

Clear cell Multilocular cystic clear cell Papillary Chromophobe Carcinoma of the collecting ducts of Bellini Renal medullary carcinoma Xp11 translocation carcinomas Carcinoma associated with neuroblastoma Mucinous tubular and spindle cell carcinoma Renal cell carcinoma, unclassified

Treatment of clinical T1 renal masses

The Dutch Guideline Committee for renal cancer 2010 describes decisive 30 conclusions regarding the surgical management of renal cancer . Level 3 evidence shows that the classical open radical nephrectomy, including lymphadenectomy and adrenalectomy, is no longer the gold standard for renal masses < 7cm and that laparoscopic radical nephrectomy of localized renal cancer is now preferred. Nephronsparing treatment is advised for cT1a tumors (â&#x2030;¤ 4cm). The gold standard is partial nephrectomy. Level of evidence

3 showed that a partial nephrectomy technically could be done laparoscopically. However, an increased risk for peroperative and postoperative complications using laparoscopic partial nephrectomy is reported. Therefore, the committee advises that the laparoscopic method can only be carried out in centres of expertise. The conclusions of the American Guideline Committee for T1 renal cancer (2009) are reasonably in 31 line with those of the Dutch guidelines . It mandates that nephron-sparing surgery should be considered first for the invasive treatment of cT1 renal masses. Under certain restrictions, both guideline committees now recognize cryoablative and radio frequency ablative therapies as alternative methods for the treatment of cT1a renal cancer. Patients with cT1b tumors are difficult to treat with ablation, and there is an increased risk for local recurrence and complications. A patient needs to be counselled that thermal ablation is a less-invasive treatment option, but local recurrence is more likely than with surgical excision, and measures of success are not well defined. Ablation may be advantageous in the group of high surgical risk patients. Klatte et al. 32 confirmed this in a systematic review . They reported that compared with partial nephrectomy, laparoscopic cryoablation results in a higher risk of local tumor progression (1.1% versus 8.5%, respectively). Cryoablation (CA) and radio frequency ablation (RFA) can be performed open, laparoscopically and in selective cases percutaneously. It is up for debate whether CA and RFA are expected to have the same outcome. A meta-analysis towards the difference between success of RFA and CA showed a 5.2% rate of local tumor progression for CA compared to 12.9% 33 with RFA . Also the difference in the outcome of percutaneous or laparoscopic thermal ablation needs to be distinguished. A meta-analysis performed by Hui et al. compared the surgical approach versus the 34 percutaneous approach of cryoablation and RFA . It showed that a percutaneous approach is safer but less effective than the surgical approach. Primary effectiveness rate for the percutaneous group was 87% and for the surgical group 94% (p <0.05). Laparoscopic assisted renal mass cryosurgery

Gill et al. reported the first series laparoscopic-assisted cryoablation of a 35 renal mass in 1998 . Since October 2003, approximately 250-300 laparoscopic renal tumor cryoablation are performed in the Netherlands. The surgical technique comprises genuine laparoscopy and is performed under general anaesthesia. Prophylactic antibiotic medication is only administered on indication. A transurethral catheter is introduced. The

patient will be positioned on the contralateral flank side in downward bend position and is fixated. We prefer a retroperitoneal approach to the kidney for all tumor locations except for those that are anteriorly located for which a transperitoneal approach is appropriate. The endoscopic ultrasound probe is essential in renal tumor cryosurgery for 3 reasons: 1. Identifying and measuring the target renal mass. 2. Guidance to biopsy and cryoprobe placement. 3. Monitoring ice progress. A bi-articular transducer-head needs space for an optimal freedom of movement, which is reduced when using a single-site approach. We use a 12mm trocar to give passage to the ultrasound probe. Although single-site approach of laparoscopic renal cryoablation is reported, 36 we advocate using a three-trocar approach . A single port access can be preferred for esthetic reasons, but it seriously compromises the intra-cavital mobility of the several instruments needed for the procedure. In a transperitoneal approach, the colon is mobilized first in order to reach the fascia of Gerota. From this point, the retroperitoneal and transperitoneal cryosurgery is similar. The contour of the kidney hidden in its perirenal fat often displays the tumor location. Nearby the suspected renal mass, the renal capsule will be approached. Dissection of the perirenal fat can sometimes be hazardous because of its volume or density. When the renal capsule is identified we follow this plane towards the tumor. The tumor capsule will be uncovered from its fat. Laparoscopic ultrasound is used to avoid biopsies from cystic or necrotic parts in the lesions. For tissue TM 速 sampling, a Magnum biopsy gun (Bard , Covington, Louisiana, USA), loaded with a disposable 16-gauge automatic side-cutting biopsy needle with a sample notch range of 19mm is used. It is fired through a trocar with the open end of the trocar close to the target. If the biopsy needle cannot reach the target, it is used percutaneously. A standard of three biopsies are taken at different locations in the mass. More biopsies, with a maximum of 5, are taken when the value of one or more of the first 3 biopsies is limited at macroscopic inspection. The laparoscopic-assisted cryoablation is performed using straight needle type probes (1.47mm diameter, 20cm length) selected for the volume of ice needed. The exact position and diameter of the tumor are confirmed by ultrasound. When the probe angle towards the tumor ax is perpendicular, the tip of the probe needs at least to reach the tumor margin. One or more needle type temperature sensors (1.47mm diameter, 20cm length) are placed just outside the tumor in normal renal parenchyma for monitoring the temperature to reach a sufficient range for adequate cell kill. Before freezing, we assure that no vital structures are near the target area. A standard of 10 minutes freezing is applied, after which a passive thaw will take place for 6

to 10 minutes. A second freeze period will be from 6 to 10 minutes. When, after the first freeze cycle the probe setting is not altered, we know that after approximately 6 minutes the ice progress will be at the level of that of the nd first freeze period after 10 minutes. From the 6-minute point in the 2 cycle, nd it is decided whether extending the cycle is needed. After the 2 freeze period, the ice will be thawed passively for a few minutes. Afterwards, the 0 0 probes will be actively warmed to 41 C to 43 C. The probes will loosen from the tissue after which they can be removed. When the ice has melted, a hyperemic phase of approximately 45 minutes will occur and bleeding can start. Most often this is at the places of probe insertion, however, sometimes a crack of the tumor tissue can appear. Compressing with gauze for several minutes attains hemostasis. In every case, we cover the lesion with a collagen sponge. No drain will be placed. Follow-up of clinical T1 renal tumor cryosurgery

All patients need to be informed of the histological findings. The majority of LCA-series in literature report their histological diagnosis based on intraoperative performed biopsies. This is expected to change now that latest guidelines of the European Association of Urology (EAU) and the American Association of Urology (AUA) have stated that ablation should only be performed on renal lesions that are proven to be malignant at 31, 37 preoperative image-guided biopsies . The downside of intraoperative biopsies is that the ablative procedure was unnecessary when a benign diagnosis is assessed afterwards. It can be debated whether cryosurgically-treated benign masses like oncocytoma or angiomyolipoma need intermediate and long term follow-up. One could be sustained with one time intravenously contrast radiographic imaging. No further follow-up is recommended when the treatment is classified as successful. All types of ablation for renal masses have in common that the tissue remains in situ. Defining treatment success is the aims of the primary assessment during follow-up by determining whether ablation of the entire target tissue has occurred. Treatment success can be assessed with intravenous contrast radiographic imaging such as computed tomography (CT) or magnetic resonance imaging (MRI). No enhancement of the target area indicates that there is no blood flow, which is considered as a non-vital sign. Focal enhancement is considered as an incomplete ablation and needs to be followed up with a repeat CT or MRI after 3 months. Also, imageguided target biopsies can be performed. To date, only standard 6-month follow-up biopsy data are available. Aron et al. found in 2 out of 63 patients

that vital RCC at 6-month CT-guided percutaneous biopsy also had positive 38 findings on the preceding MRI . The chance for T1 renal malignancy to metastasize is approximately 7%. The risk for metastasis after cryoablation is not clearly determined since reports are biased on short follow-up periods. Risk factors are high nuclear grade and vascular invasion. However, determining vascular invasion and the accuracy of grading based on biopsy specimen is under discussion. Pulmonary metastases are the most common metastasis of renal cancer. Therefore, the check-up should include a yearly chest X-ray. One should aim to detect local recurrence or new developing renal masses (multifocality) at the follow-up of nephron-sparing surgery. Klatte et al. reported that the chance for local progression or recurrence is 8.5 % after 32 cryosurgery . They also found that the risk for local progression increased with 85% per 1cm increase of tumor size. The mean follow-up for cryosurgery in this study was 29 months. Although this follow-up time is 38,39 short, local recurrence is rarely reported after 2 years . The follow-up algorithms of EAU and AUA guidelines do not distinguish between histological subtypes. However, Leibovich et al. reported that in patients diagnosed with renal cancer, the histological subtype of clear cell carcinoma is an independent predictor of progression of distant metastasis 40 and cancer-specific survival . Tsivian et al. reported that lesion size was 41 independently associated with occult multifocality . Also, histologic subtypes other than clear cell had an increased risk for occult multifocality of which the papillary subtype had the strongest association which conferred a seven-fold odds increase. Rare subtypes such as medullary and collecting duct RCC increased the odds by >10-fold. Table 5 shows a proposed algorithm for follow-up of cryoablated RCC from the EAU guidelines. However, it is unclear how intermediate and high risk can be assessed for tumors that are cryotherapeutically. Risk factors for small renal masses that underwent cryoablation still need further investigation. Long term follow-up and histological diagnosis are necessary in order to identify risk factors for local and distant progression of cryoablated RCC.

Table 5 Follow-up algorithm for surveillance after cryosurgical treatment of renal cell carcinoma with combined patient risk profile and treatment efficacy (EAU guidelines). CT= computed tomography, XR= x-ray, US= ultrasonography.

6 months 1 year 2 year 3 year 4 year 5 year Following

Intermediate-risk CT Chest XR and US CT Chest XR and US Chest XR and US CT Yearly Chest XR and US

High-risk CT CT CT CT CT CT Chest-XR / CT in alternate years

Scope of the thesis

Cryobiology and cryotechnology are essential for understanding the surgical process of cryoablation. Hereby, the procedure can be customized to the required setting, monitored and adjusted if needed. The implications of vascular injury for possible adjuvant sensitizing therapy before cryosurgery and post-procedural radiological imaging are discussed. In chapter 2, we describe the combinational use of a casting technique, cryomicrotomical imaging and three-dimensional computer analysis as a method for visualization and reconstruction of the arterial vascular tree in a porcine model. The same experimental model was used for studying the acute vascular injury due to cryosurgical freezing. In chapter 3, we evaluate how a microvascular diameter threshold for acute vascular damage can be established. The time course of vascular damage and its related perfusion disturbances after cryoablation in the same experimental settings is quantified and discussed in chapter 4. The introduction of the R.E.N.A.L. and the Preoperative Aspects and Dimensions Used for Anatomical Classification (PADUA) nephrometry scores for renal masses has led to its widespread use in the literature of small renal masses. It is developed to quantify the anatomical characteristics of renal masses in order to be helpful in decision-making and as essential for effective comparisons of extirpative surgery. In chapter 5, we describe the use of the nephrometry scores for small renal masses treated with laparoscopic cryoablation. Risk factors for the efficacy and complications at laparoscopic cryoablation are assessed using nephrometry score, Charlson comorbidity classification and Dindo-Clavien complication index.

Today, there is no consensus on the follow up of cryoablated small renal malignancies, although many agree that contrast enhanced CT or MRI are the best methods to define cryosurgical treatment success. However, intravenously administered contrast can jeopardize renal function. Nevertheless, patients with impaired renal function are often thought to be candidates for cryosurgery. In chapter 6 and 7 we discuss possible alternative imaging methods of small renal masses treated with laparoscopic cryoablation. In chapter 8, the presented data is discussed and describes future perspectives for these subjects.

References

Arnott J. Practical illustrations of the remedial efficacy of a very low or anaesthetic temperature in cancer. I. In cancer. Lancet. 1850; 2: 257 - 259.

Cooper IS, Lee AS. Cryostatic congelation: a system for producing a limited controlled region of cooling or freezing of biologic tissues. J Nerv Ment Dis. 1961; 133: 259 - 263.

Onik G, Cobb C, Cohen J, Zabkar J, Porterfield B. US characteristics of frozen prostate. Radiology. 1988; 168: 629 - 631.

Saliken JC, McKinnon JG, Gray R. CT for monitoring cryotherapy. AJR Am J Roentgenol. 1996; 166: 853 - 855.

Tacke J, Speetzen R, Heschel I, Hunter DW, Rau G, Günther RW. Imaging of interstitial cryotherapy -- an in vitro comparison of ultrasound, computed tomography and magnetic resonance imaging. Cryobiology. 1999; 38: 250 - 259.

Le Pivert P. Basic considerations of the cryolesion. In “Handbook of Cryosurgery” (R. Ablin, Ed.), Dekker, New York 1980, pp. 15 - 68.

Gage AA, Baust J. Mechanisms of tissue injury in cryosurgery. Cryobiology. 1998; 37: 171 - 186.

Hoffmann NE, Bischof JC. The cryobiology of cryosurgical injury. Urology. 2002; 60: 40 - 49.

Adams-Ray JA, Bellman S. Vascular reactions after experimental cold injury: a microangiographic study of rabbit ears. Angiology. 1956; 7: 339 - 367.

Lewis T, Love WS. Vascular reactions of the skin to injury. III: Some effects of freezing, of cooling, and of warming. Heart. 1926; 13: 27 - 60.

Rotnes PL, Kreyberg L. Eine methode zum experimentelen Nachweis von stase mittels spezieller praeparate. Acta Pathol Microbiol Scand Suppl. 1932; 11: 162 165.

Giampapa VC, Oh C, Aufses AH. The vascular effect of cold injury. Cryobiology. 1981; 18(1): 49 - 54.

Rabb JM, Renaud ML, Brandt A, Witt CW. Effect of freezing and thawing on the microcirculation and capillary endothelium of hamster cheek pouch. Cryobiology. 1974; 11: 508 - 518.

Rothenborg H. Cutaneous circulation in rabbits and humans before, during and after cryosurgical procedures, measured by Xenon-133 clearance. Cryobiology. 1970; 6: 507 - 511.

Zacarian S, Stone D, Clater M. Effects cryogenic temperatures on microcirculation in the golden hamster cheek pouch. Cryobiology. 1970; 7: 27 - 39.

Russo P. Localized renal cell carcinoma. Curr Treat Options Oncol. 2001; 2(5): 447 - 455.

Remzi M, Katzenbeisser D, Waldert M, Klingler HC, Susani M, Memarsadeghi M, Heinz-Peer G, Haitel A, Herwig R, Marberger M. Renal tumour size measured radiographically before surgery is an unreliable variable for predicting histopathological features: benign tumours are not necessarily small. BJU Int. 2007; 99: 1002 - 1006.

International Union against Cancer (UICC). TNM classification of malignant th tumors, 7 ed. Sobin L, Gospodarowicz M, Wittekind C (eds). Chichester, WileyBlackwell, 2010.

Ficarra V, Righetti R, Piloni S, Dâ&#x20AC;&#x2122;Amico A, Maffei N, Novella G, Zanolla L, Malossini G, Mobilio G. Prognostic factors in patients with renal cell carcinoma: retrospective analysis of 675 cases. Eur Urol. 2002; 41(2); 190 - 198.

Lau WK, Cheville JC, Blute ML, Weaver AL, Zincke H. Prognostic features of pathologic stage T1 renal cell carcinoma after radical nephrectomy. Urology. 2002; 59(4): 532 - 537.

Lindblad P. Epidemiology of renal cell carcinoma. Scand J Surg. 2004; 93(2): 88 96.

Murai M, Oya M. Renal cell carcinoma: etiology, incidence and epidemiology. Curr Opin Urol. 2004; 14: 229 - 233.

Bergstrom A, Hsieh CC, Lindblad P, Lu CM, Cook NR, Wolk A. Obesity and renal cell cancer -- a quantitative review. Br J Cancer. 2002; 85: 984 - 990.

Mellemgaard A, Engholm G, McLaughlin JK, Olsen JH. Risk factors for renal cell carcinoma in Denmark. I. Role of socioeconomic status, tobacco use, beverages, and family history. Cancer Causes Control. 1994; 5(2): 105 - 113.

Wolk A, Gridley G, Niwa S, Lindblad P, McCredie M, Mellemgaard A, Mandel JS, Wahrendorf F, McLaughlin JK, Adami HO. International renal cancer study. VII. Role of diet. Int J Cancer. 1998; 65: 67 - 73.

Algaba F, Akaza H, Lopez-Beltran A, Martignoni G, Moch H, Montironi R, Reuter V. Current pathology keys of renal cell carcinoma. Eur Urol. 2011; 60(4): 634 - 643.

Signaleringscommissie Kanker, KWF Kankerbestrijding. Trends en prognoses van incidentie, sterfte, overleving en prevalentie van kanker: Nierkanker. In: Kanker in Nederland: trends, prognoses en implicaties voor zorgvraag. 2004; pp 155 - 161.

Eble JN, Sayter G, Esptein JI, Sesterhenn IA, editors. World Health Organization classification of tumours. Pathology and genetics tumours of the urinary system and male genital organs. Lyon, France: IARC press; 2004. p 10.

Cheville JC, Lohse CM, Zincke H, Weaver Al, Blute ML. Comparisons of outcome and prognostic features among histologic subtypes of renal cell carcinoma. Am J Surg Pathol. 2003; 27(5): 612 - 624.

Dutch Guideline Committee for renal cancer. Niercelcarcinoom. Versie 2.0, evidenced based 2010-12-01. Integraal Kankercentrum Nederland, http://oncoline.nl.

Campbell SC, Novick AC, Belldegrun A, Blute ML, Chow GK, Derweesh IH, Faraday MM, Kaouk JH, Leveillee RJ, Matin SF, Russo P, Uzzo RG. Practice Guidelines Committee of the American Urological Association. Guideline for management of the clinical T1 renal mass. J Urol. 2009; 182: 1271 - 1279.

Klatte T, GrubmĂźller B, Waldert M, Weibl P, Remzi M. Laparoscopic cryoablation versus partial nephrectomy for the treatment of small renal masses: systematic review and cumulative analysis of observational studies. Eur Urol. 2011; 60: 435 - 443.

Kunkle DA, Uzzo RG. Cryoablation or radiofrequency ablation of small renal mass: a meta-analysis. Cancer. 2008; 113(10): 2671 - 2680.

Hui GC, Tuncali K, Tatli S, Morisson PR, Silverman SG. Comparison of percutaneous and surgical approaches to renal tumor ablation: metaanalysis of effectiveness and complication rates. J Vasc Interv Radiol. 2008; 19: 1311 - 1320.

Gill IS, Novick AC, Soble JJ, Sung GT, Remer EM, Hale J, Oâ&#x20AC;&#x2122;Malley CM. Laparoscopic renal cryoablation: initial clinical series. Urology. 1998; 52: 543 551.

Goel RK, Kaouk JH. Single port access renal cryoablation (SPARC: a new approach. Eur Urol. 2008; 53: 1204 - 1209.

Ljungberg B, Cowan NC, Hanbury DC, Hora M, Kuczyk MA, Merseburger AS, Patard JJ, Mulders PF, Sinescu IC; European Association of Urology Guideline Group. EAU guidelines on renal cell carcinoma: the 2010 update. Eur Urol. 2010; 58: 398 406.

Aron M, Kamoi K, Remer E, Berger A, Desai M, Gill I. Laparoscopic renal cryoablation: 8-year, single surgeon outcomes. J Urol. 2010; 183: 889 - 895.

Klatte T, Mauermann J, Heinz-Peer G, Waldert M, Weibl P, Klingler HC, Remzi M. Perioperative, oncologic, and functional outcomes of laparoscopic renal cryoablation and open partial nephrectomy: a matched pair analysis. J Endourol. 2011; 25: 991 - 997.

Leibovich BC, Lohse CM, Crispen PL, Boorjian SA, Thompson RH, Blute ML, Cheville JC. Histological subtype is an independent predictor of outcome for patients with renal cell carcinoma. J Urol. 2010; 183: 1309 - 1315.

Tsivian M, Moreira DM, Caso JR, Mouraviev V, Madden JF, Brataslavsky G, Robertson CN, Albala DM, Polascik TJ. Predicting occult multifocality of renal cell carcinoma. Eur Urol. 2010; 58: 118 - 126.

CHAPTER 2 Computerized three-dimensional renal arterial reconstruction with a vascular casting and imaging cryomicrotome technique in a porcine model

This chapter is published as: Lagerveld BW, ter Wee RD, de la Rosette JJ, Spaan JA, Wijkstra H. Vascular fluorescence casting and imaging cryomicrotomy for computerized three dimensional renal arterial reconstruction. BJU Int. 2007; 100(2): 387-391.

Abstract

Introduction We studied the combined use of a casting technique, cryomicrotomy imaging, and three-dimensional (3-D) computer analysis as a method for visualizing and reconstructing the arterial vascular tree in a porcine renal model. Material and methods The arterial branches of two porcine kidneys were filled with a fluorescent cast, after which they were cut in slices of 50µm in an imaging cryomicrotome. From each section, digital images of the cutting plane of the sample were taken and stored in the computer, after which stacks of images were rendered in 3-D. Results A 3D computerized reconstruction of the arterial vascular tree was constructed and showed the complete arterial anatomy up to arterioles of 50µm. Conclusion With visualization by fluorescence imaging cryomicrotomy, the anatomical reconstruction and 3-D reconstruction of the renal arterial blood supply in a pig kidney is possible up to a resolution of 50µm.

Introduction

The human kidney has multipapillary architecture with several calyces and an elaborate system of segmental and interlobar vessels supplying each 1 lobe of the kidney separately . The segmental arteries are so-called â&#x20AC;&#x153;end arteriesâ&#x20AC;? that nourish a defined region of the kidney, which implies that there 2 are no anastomoses between each of these vessels . The anatomical structure and function of the pig kidney is known to be similar to that of the human kidney and is therefore a useful model for surgical and radiological 1 research . In the last two decades, surgical and radiological techniques have increasingly developed towards minimally invasive and nephron-sparing methods, e.g. highly selective embolization, partial nephrectomy, renal tumor 3-8 ablation, and percutaneous nephroscopy . Also, the vascular effects of noninvasive medical therapies for renal cancer, such as anti-angiogenetic 9 drugs, are under study . Therefore, there is an increasing need for specific understanding of the functional anatomy, in particular of the vascular anatomical structure. The vascular anatomy and the functional vascular pattern can be studied in general by different methods of vascular reconstruction, by static or dynamic methods, or a combination of both. Radiological imaging, e.g. IVU, angiography, contrast-CT or MRI, give an impression of the anatomy that is expressed statically or dynamically. Some of these methods have the advantage of providing a computerized three-dimensional (3-D) reconstruction. However, a clinical situation the resolution of these radiological methods is too limited for identifying micro-vascularization. This can be overcome by using micro-computed tomography (micro-CT), which can produce high-resolution detailed images, from which information on the micro-vascular structures can be collected. However, the limitation is that 10 only small volumes of tissues can be studied . Currently, in clinical situations, the more commonly used 64-slice CT provides improved resolution, of up to 100Âľm. When more detailed images are required of the renal microvascularity, or a higher resolution, static 3D anatomic models can be obtained using casting and corrosive techniques. The cast and exposed luminal structures can than be studied macroscopically, but also in fine detail using scanning electron microscope technique (SEM), which is capable of showing the finest 11, 12 ramifications, with details as endothelial cell imprint patterns . However, 13 the limitation of the SEM method is stability and vulnerability . Furthermore, with this technique any 3-D orientation is difficult without compromising total specimen integrity and geometrical configuration.

A recent study showed that vessels as small as 40µm in diameter of the intramural arterial vasculature of an entire goat heart can be clearly identified 14 by a fluorescent imaging cryomicrotomy . Digital storage of the data gives the opportunity to reconstruct the anatomy without losing material. In this pilot study, we assessed the combined use of a fluorescent casting technique, cryomicrotome imaging, and 3-D computer analysis as a new static method for visualizing and reconstructing the vascular tree in a porcine renal model. Material and methods

Unaffected kidneys (volume ≈100 mL) of domestic farm pigs (40 kg), which were used in our laparoscopic training programmed for urology residents, were used in the present pilot study of anatomical vascular reconstruction with an imaging cryomicrotome technique. Before blocking the renal blood supply, 10.000 units of heparin were administrated intravenously. After ligating the artery the complete specimen was dissected and removed via a laparotomy. On the bench, the main renal arterial branch was cannulated while preventing ingress of air in. The kidney was flushed under 80 mmHg pressure with 100 – 200 mL saline containing 10 µmol/L adenosine to maximally dilate the vascular bed. Thereafter, a cast of Batson no.17 plastic replica material (Polysciences Inc, Eppelheim, Germany) consisting of a monomer base solution (methyl methacrylate), a catalyst and a promoter, were introduced under a steady pressure of 80 mmHg (compressed air) and allowed to harden for 24 hours (figure 1). The cast is made fluorescent by 30 µg/mL base solution of Potomac yellow (Radiant Colour nv, Belgium). The entire kidney was then submerged in a cylindrical container filled with 0 5% carboxymethylcellulose sodium solvent, and frozen to -20 C for at least 2 days. In an imaging cryomicrotome, built and developed in the Academic Medical Centre, Amsterdam, and based on an existing cryomicrotome, the kidney was cut in slices of 50µm from anterior to posterior side. After each cut, a 2000 x 2000 pixel digital camera (Kodak) equipped with a 70-180 mm lens (Nikon) was used to take images of the entire cutting plane of the remaining sample; each pixel represented 50 x 50 µm. The fluorescence was measured using, an excitation filter (D440/20x, Chroma Technology Corp, Rockingham, USA) in the light path towards the cutting plane, and an emission filter (D505/ 30 m, Chroma Technology Corp, Rockingham, USA) in the light path from the cutting plane towards the camera (figure 2). All images were processed using custom-made software written in Delphi Pascal (Borland, vs. 7.0) using the Toolkit of MCM (Birkerød, Denmark) and were stored in the computer. The stacks of images were then rendered in 315 D using proprietary image registration and visualization software .

Figure 1. The fluorescent cast is injected via a continuous propulsion system with 80 mmHg pressures for 24 hours.

Figure 2. A simplified schematic set-up of the fluorescent imaging cryomicrotome.

Results

Two domestic farm pigs (40 kg) were selected for the additional study of the renal blood supply. In the first, a left kidney was dissected without affecting its arterial blood flow. The kidney was removed via a laparotomy and prepared for casting. It was sectioned in the cryomicrotome in 550 slices. An image with normal white light of a single dissection surface shows no cast vessels (figure 3a), whereas, the cast can be clearly distinguished when the fluorescence is visualized via the excitation and emission filter (figure 3b). However, one image alone gives no clear impression of the vascular

structure; this becomes more recognizable when a stack of consecutive images is selected (figure 4). Vessels of <50 Âľm cannot be visualized due to the limit of the optical resolution, but the fluorescence in the smaller vessels results in a higher intensity of background noise of the image, which results in an unfocused grey background. If all images are selected, a 3-D computerized reconstruction of the arterial branches shows the complete arterial anatomy up to small arterioles of 50Âľm (figure 5). In the second pig using the right kidney, the arterial blood supply was insufficiently blocked during laparoscopy, but the congestion of the kidney that occurred after ligating the renal vein turned did not influence for the results of the imaging method. After laparotomy the artery was completely ligated and the specimen removed. The kidney was cut in the cryomicrotome in 750 slices of 50Âľm thick; the same computerized reconstruction was used, which resulted in similar images to the first kidney. Also in this kidney, stacks of consecutive images were selected (figure 6) and a 3-D image of the arterial vascular tree constructed.

Figure 3a,b. Two digital images of the same surface (kidney 1). The image on the left is taken with white light (A), the image on the right with fluorescent light (B).

Figure 4. Maximum intensity projection (MIP) of 50 consecutive surfaces representing a thickness of 2.5 mm (kidney 1). The contrast brightness values of the insert have been adjusted to visualize the small vessels.

Figure 5. 3D-rendered model seen from three projections (kidney 1).

Figure 6. Maximum intensity projection (MIP) of 50 consecutive surfaces, representing a thickness of 2.5 mm. The contrast brightness values of the insert have been adjusted to visualize the small vessels (kidney 2).

Discussion

Several casting methods have been used previously to create a static reconstruction of the anatomical microvascular distribution in organs. The 7, 16, waxes used previously have been replaced with silicon-like compounds 17 . Most often these vascular casts are used in combination with a corrosive preparation technique. All organic tissue around the hardened cast is removed using corrosive or digestive substance, with or without detergents, and by several mechanical means. The casts can be created in different

colors, allowing differentiating among several luminal systems, e.g. arterial, venous, and urine-collecting system. However, the loss of integrity after corroding the connective and supporting tissues among the remaining cast 18 lumina is to be expected . This leads to changes in the anatomical distances and thus relationships among the different cast structures, which invalidates the 3-D orientation. The use of different colored casts in separate luminal structures is also possible with the cryomicrotomy method. The advantage is that, by leaving the connective tissues in place, the dimensional distances between these structures remains intact, giving a realistic image of the static anatomical relationships. A reliable reconstruction these relationships among vascular branches and the collecting system of a kidney can be important, e.g. when studying percutaneous access to the collecting system or for taking renal biopsies. The use of intravenously contrast materials in radiological imaging gives a static and dynamic impression of the vascular anatomy, but 3-D reconstructions are only possible with CT and MRI. Another possible way of measuring vascular flow is with the fluorescent imaging cryomicrotomy, by 19 using microspheres . These polystyrene particles, if administrated in the arterial stream proximal to the region of interest, are carried by the flow and entrapped in the vessels within the organ of interest. If the microspheres are fluorescent, they can be detected with the cryomicrotome imaging method and digitally counted. Combining the casting method with the use of microspheres and the digital imaging cryomicrotome provides a model for both static and dynamic anatomical reconstruction. In this pilot study, the fluorescence imaging cryomicrotome technique provides an uncorroded 3-D static model, which shows cast structures up to 50µm. However, when using the same casting technique as described here, microscopy of the microvascular branches of goat hearts showed complete 14 filling, with replicas of vessels <15µm . Thus, vessels with a diameter <50µm can only be visualized using a digital camera with higher quantum efficiency, combined with a lens of higher aperture. A higher resolution can, within limits, also be obtained by minimizing the sample-imaging surface. For example, with our model, a 40µm diameter resolution can be obtained with 2 an imaging surface of 64 cm . Although the resolution will never be as high as with the micro-CT or SEM, it is higher than the standard radiological imaging used for tissues or organs of larger volume. Because the anatomical structure and function of the pig kidney is known to be similar to that of the human kidney, the porcine kidney is a good anatomical model for understanding surgical and radiological methods used 1 in the human kidney . To evaluate kidney vascular anatomy and function a technique is required that can assess a volume as large as the entire kidney. Furthermore, if the geometric relationships are not to be disturbed,

techniques using corrosive preparation and casting, and micro-CT, seem to be less appropriate. Vascular anatomical models can be of benefit for research into renal cancer treatment. The vascular role in this cancer, e.g. neo-angiogenesis, is 9, 20 becoming clearer . It is possible to visualize neo-angiogenic vessels in tumors using corrosive casting and SEM, but the resolution of the cryomicrotome technique is not yet sufficient to visualize the newly formed 20 vessels of 8 Âľm . Ablative surgery for renal tumors, e.g. cryotherapy and radiofrequency ablation, could also be studied with this method. Especially in cryoablation, the role of vascular damage plays is important in the final lethal 21 zone . The vascular effects of freezing are known to be dynamic over time, starting with acute microvascular damage and later followed by 21-23 thromboembolic ischemia of arterioles . Also, nephron-sparing minimal invasive surgery, like laparoscopic partial nephrectomy, can be studied using the imaging cryomicrotome technique although the best method is a dynamic imaging model with a high sensitivity for vascular flow, combined with a high resolution of the microvasculature; this is possible using microspheres combined with the cryomicrotome method. Conclusion

3-D visualization and reconstruction of the renal arterial blood supply in a pig kidney is possible using a fluorescence imaging cryomicrotome. The arterial geometry of the complete organ can be visualized at a resolution of up to 50Âľm of the complete.

References

Evan AP, Connors BA, Lingeman JE, Blomgren P, Willis LR. Branching patterns of the renal artery of the pig. Anat Rec. 1996; 246(2): 217 - 223.

Graves FT. Anatomy of intrarenal arteries and its application to segmental resection of the kidney. Br J Surg. 1954; 42: 132 - 137.

Heye S, Maleux G, Van Poppel H, Oyen R, Wilms G. Hemorrhagic complications after nephron-sparing surgery: angiographic diagnosis and management by transcatheter embolization. AJR Am J Roentgenol. 2005; 184(5): 1661 - 1664.

Russo P, Goetzl M, Simmons R, Katz J, Motzer R, Reuter V. Partial nephrectomy: the rationale for expanding the indications. Ann Surg Oncol. 2002; 9(7): 680 - 687.

Weld KJ, Bhayani SB, Belani J, Ames CD, Hruby G, Landman J. Extrarenal vascular anatomy of kidney: assessment of variations and their relevance to partial nephrectomy. Urology. 2005; 66(5): 985 - 989.

Mabjeesh NJ, Avidor Y, Matzkin H. Emerging nephron sparing treatments for kidney tumors: a continuum of modalities from energy ablation to laparoscopic partial nephrectomy. J Urol. 2004; 171(2 Pt 1): 553 - 560.

Sampaio FJ, Pereira-Sampaio MA, Favorito LA. The pig kidney as an endourological model: anatomic contribution. J Endourol. 1998; 12(1): 45 - 50.

Thiruchelvam N, Mostafid H, Ubhayakar G. Planning percutaneous nephrolithotomy using multidetector computed tomography urography, multiplanar reconstruction and three-dimensional reformatting. BJU Int. 2005; 95(9): 1280 - 1284.

Marx J. Cancer. Encouraging results for second-generation antiangiogenesis drugs. Science. 2005; 308(5726): 1248 - 1249.

Nordsletten DA, Blackett S, Bentley MD, Ritman EL, Smith NP. Structural morphology of renal vasculature. Am J Physiol Heart Circ Physiol. 2006; 291(1): H296 - 309.

Hodde KC, Steeber DA, Albrecht RM. Advances in corrosion casting methods. Scanning Microsc. 1990; 4(3): 693 - 704.

Hodde KC, Miodonski A, Bakker C, Veltman WA. SEM of microcorrosion casts with special attention on arterio-venous differences and application to the ratâ&#x20AC;&#x2122;s cochlea. Scanning Electron Microsc. 1977; 11: 477 - 484.

Lametschwandtner A, Lametschwandtner U, Weiger T. Scanning electron microscopy of vascular corrosion casts: technique and applications: updated review. Scanning Microsc. 1990; 4(4): 889 - 940.

Spaan JAE, ter Wee R, van Teeffelen JWGE, Streekstra G, Siebes M, Kolyva C, Vink H, Fokkema DS, VanBavel E. Visualisation of intramural coronary vasculature by an imaging cryomicrotome suggests compartmentalisation of myocardial perfusion areas. Med Biol Eng Comput. 2005; 43(4): 1 - 4.

Van Herk M, de Jaeger K, de Munck J, Hoogeman M, Lebesque J. A delineation system for N modalities: software aspects. Proceedings of the 13th ICCR, th Heidelberg, Germany, 22-25 May, 2000.

Batson OV. The venous network of the nasal mucosa. Trans Am Laryngal Rhinol Otol Soc. 1954; 75: 54 - 63.

Nopanitaya W, Aghajanian JG, Gray LD. An improved plastic mixture for corrosion casting of the gastrointestinal microvascular system. Scan Electron Microsc. 1979; (3): 751 - 755.

Pereira-Sampaio MA, Favorito LA, Sampaio FJ. Pig kidney: anatomical relationships between the intrarenal arteries and the kidney collecting system. Applied study for urological research and surgical training. J Urol. 2004; 172(5 Pt 1): 2077 - 2081.

Bernard SL, Ewen JR, Barlow CH, Kelly JJ, McKinney S, Frazer DA, Glenny RW. High spatial resolution measurements of organ blood flow in small laboratory animals. Am J Physiol Heart Circ Physiol. 2000; 279(5): H2034 - 2052.

Konerding MA, Miodonski AJ, Lametschwandtner A. Microvascular corrosion casting in the study of tumor vascularity: a review. Scanning Microsc. 1995; 9(4): 1233 - 1243.

Hoffmann NE, Bischof JC. The cryobiology of cryosurgical injury. Urology. 2002; 60(2 Suppl 1): 40 - 49.

Bourne MH, Piepkorn MW, Clayton F, Leonard LG. Analysis of microvascular changes in frostbite injury. J Surg Res. 1986; 40(1): 26 - 35.

Daum PS, Bowers WD, Tejada J, Hamlet MP. Vascular casts demonstrate microcirculatory insufficiency in acute frostbite. Cryobiology. 1987; 24(1): 65 - 73.

CHAPTER 3 Cryosurgical injury: Acute vascular changes after renal cryosurgery in a porcine model

This chapter is published as: Lagerveld BW, van Horssen P, Laguna Pes MP, van den Wijngaard JPHM, Streekstra GJ, de la Rosette JJMCH, Wijkstra H, Spaan JAE. Immediate effect of kidney cryoablation on renal arterial structure in a porcine model studied by imaging cryomicrotome. J Urol. 2010; 183(3): 1221 â&#x20AC;&#x201C; 1226.

Abstract

Introduction Injury to blood microvessels has a crucial role in effective cryoablation for renal masses. We visualized the vascular injury induced by a clinically applied cryoablation instrument and established a microvascular diameter threshold for vascular damage. Material and methods In five anesthetized pigs one kidney each was exposed and three 17-gauge cryoneedles were inserted in one pole. Tissue was exposed to freezing in for 2 x 10 minutes with 10-minute thaw between freezes. After nephrectomy the arteries were injected with fluorescence dyed casting material and the o kidney was frozen to – 20 C and cut in 40 to 60 µ slices in the imaging cryomicrotome, where fluorescent images of the cutting plane of the bulk were obtained. This resulted in a 3-dimensional image of the arterial tree that was segmented, resulting in unbranched vessel segments. Histograms were constructed with the total segment length per diameter bin was plotted as function of diameter. Results The ablated zone was sharply demarcated on fluorescent and normal light images. Mean + SD diameter at the peak of the histogram from the control areas was 152.4 + 5.3 µ. Compared to control areas the peak diameter of ablated areas was shifted to a larger diameter by an average of 25.4 + 2.6 µ. Conclusion Immediate renal cryoablation injury destroys arteries smaller than 180 µ. Branching structures of larger arteries remain anatomically intact and connected to vascular structures in surrounding tissue.

Introduction

Cryosurgery is gaining interest as a curative or palliative treatment for urological cancer, including renal tumors, prostate cancer, and bone metastasis. Thus, it is important to understand the involved pathophysiological mechanisms that eventually lead to complete, reliable 1,2 eradication of viable cancerous cells . Freezing induced arterial vascular 3 injury provides an important contribution to tissue destruction . Apart from direct effects of freezing of cells in general, vascular injury induces a cascade of processes that eventually leads to blood flow cessation, causing ischemia, which is considered a therapeutic effect because of further tissue 1, 2, 4 destruction . The vascular effect of freezing tissue has been especially 5, 6 studied in relation to frostbite but not to tumor ablation . In recent studies the focus was on the effect of freezing and thawing on the structure and 7-10 function of the microcirculation of superficial tissues . Daum et al. reported a corrosion cast study of the acute effects of freezing on circulation of the rat 11 hind limb . They noted that especially microcirculation was destructed without specifying a threshold for the diameter of affected vessels. More detailed insight into the vascular effects of cryoablation could be useful. Obviously improved insight into the processes involved may result in improved technique but it may also aid in advancing the clinical assessment of cryoablation intervention for cancers. Clinical renal tumor cryoablation success is assessed by intravascular contrast enhanced imaging, such as 12 computerized tomography and magnetic resonance imaging . Thus, it is important to understand the acute changes in the arterial vascular bed induced by cryoablation since these vascular alterations are the basis of 13 what is visualized . We previously reported that the vascular pattern of normal porcine kidneys could be reconstructed by fluorescent 14 cryomicrotome imaging . We applied that method in this study to detect acute changes in the renal circulation induced by ablation and establish a threshold value for vessel diameter sensitive to cryoablation destruction. Methods

Six domestic farm pigs weighing 35 to 40 kg were used for a laparoscopic training procedure approved by the Animal Research Institute at the Academic Medical Centre of Amsterdam. The pigs were fully anesthetized during the procedure and sacrificed after the procedure was completed. Ablation was done at the anterior side of 1 kidney at 1 pole with a double freeze-thaw cycle cryoablation using a triple needle probe configuration TM Oncura cryoneedle in 5 pigs. In a triangular configuration all needles were placed parallel at a perpendicular angle to the renal capsule using a

template with a 7 mm interneedle distance. Total freezing time was 20 minutes. Before kidney harvesting it was ensured that all frozen tissue had thawed a minimum of 20 minutes after probe removal. Probing kidney consistency by touching it with a finger was sufficient to test thawing. After kidney removal the arteries were cannulated and cast with a fluorescent dyed, Batson no. 17 plastic replica, consisting of a monomer base solution, a catalyst and a promoter. After the cast hardened the kidneys were entirely frozen again to 0 14 20 C . In our novel developed imaging cryomicrotome the kidneys were 0 15, cut in slices from anterior to posterior under standard conditions at -20 C 16 TM . A Megaplus 4.2i digital camera equipped with a 70 to 80 mm NikonÂŽ lens was used to image the renal surface cutting plane after each slice. Lens settings were adjusted so that the maximal specimen cross-section was imaged, resulting in a pixel size of between 40 and 60 Âľm. Slice thickness was chosen accordingly so that cubic voxels resulted. Images of the fluorescent cast were obtained by applying a D440/20x excitation filter and a D505/ 30 m emission filter (Chroma Technology Corp, Vermont, USA). All images were processed using custom developed software written in Delphi (Borland, version 2005). Image analyses resulted in 3-D representations of the branching arterial system of ablated and nonablated renal tissue. Arterial tree segmentation and segment diameter estimation Vessel diameters were measured from 3-D vascular bed data. A peeling algorithm was used to extract vessel center lines while keeping the topology 17 intact . Spatial location and number of connected center points were determined for all center points along these center lines. When a center point had more than 2 adjacent center points, this was defined as a node and with only 1 adjacent center point it was defined as an end point. Adjacent center points were grouped into segments, resulting in a mathematical vascular tree representation divided into segments spanning between nodes or a node and an end point. Segment length followed from a polynomial fit through the segment points. Segment diameter was determined. At each segment mid point the local vessel orientation is determined from the 2 adjacent skeletal points. In a plane perpendicular to the local segment direction intensity distributions curves as a function of distance to the center point were calculated over 256 radial lines evenly distributed in the plane. Intensity along a radial line was determined by linear 3-D interpolation over 0.1-pixel intervals, i.e. approximately 5 Âľ. The vessel border was defined as the full width half maximum of the intensity curve reduced with the estimated background 18 value of the image data .

Maximal intensity projection Two-dimensional images of the branching nature of the arterial tree were obtained by the technique of Maximal Intensity Projection (MIP). In this technique a stack of data of arbitrary size is compressed to a single image, taking only maximum pixel values in the longitudinal direction of the stack. Generalized data All vessel segments were classified in bins by diameter. In a 3-D tissue region of interest the length values of all segments of a certain diameter range were added and histograms of total summed vessel length as function of diameter were plotted. These histograms were quantified by the diameter at the histogram peak. In this way the effect of cryoablation was quantified as a function of diameter range. Results

All six pigs were hemodynamically stable while under anesthesia. Kidneys in this study appeared to be macroscopically normal without any congenital abnormalities. From pig P1 a kidney was harvested without cryoablation and processed for arterial reconstruction to test the algorithms needed for histogram construction. Figure 1-A shows a MIP from 554 slices, indicating how the arteries branch from center to periphery and end in vessels of similar diameter distributed in the renal parenchyma. Figure 2-A shows the distribution of total segment length as a function of diameter for the lower pole, the upper pole and the interpolar. These distributions had a similar shape with peak values at the same diameter but peak values were different and, thus, they were not interpreted further. The decrease in total vascular length with decreasing diameter may express in part an anatomical reality but it also resulted from the limitation by which small diameters can be estimated by this technique with its current limitations. Figure 1-B shows a MIP from more than 300 images, representing slices in which ablation in the lower pole was noticeable from the outline images. A ring of almost small, vessel-free tissue was noted around the frozen area. Some contrast leakage was recognizable as dots of fluorescent material in the ablation zone. After thawing and before harvesting the kidney a typical mark of the ablation is visible as a circular area surrounded by an outer band, appearing as a halo of colour resembling hematoma. In the cutting planes of the cryomicrotome this concentric nature of the ablated area was also noted in the outline image.

Figure 2-B shows histograms of total segment length obtained from ablated and nonablated control poles. In the control area the histogram peak was at 156 µ but in the ablated area the total length of vessels at that diameter was decreased to 30% of the total length at peak diameter in the ablated area. The table shows overall peak diameter results in ablated and reference histograms. The mean + SD peak value was 152.5 + 5.3 µ in reference histograms and 177.9. + 3.8 µ in ablated areas for a mean 25.4 + SD 2.6 µ shift to the right in ablated tissue, which was significant (1-tailed paired t test p=<0.0001). Figure 1. MIP’s. A, entire stack of images from pig P1 untreated kidney shows no normal vessel distribution. B, images 1 to 300 of lower pole cryoablation in pig P2 kidney. Ablated area (arrow) is particularly noticeable due to surrounding ring or halo in ablated areas. Because of image selection we could not draw conclusions based on visual appearance of vascular structures only. C, 60 images representing 3.3 mm deep layer of pig P2 kidney containing ablation crater cross section (black area). Larger vascular structure is intact in crater and anatomically connected to microcirculation in surrounding tissue.

With the centre of the ablation area as centre line concentric tubes were created with a 6 to 48 mm outer diameter in 6-mm steps. For each 3 mm thick tube wall a histogram was constructed (figure 3). The tube with the largest diameter contained unaffected tissue and was considered the control histogram. Histograms for smaller tube diameters demonstrate revealed a shift of the peak to the right when more ablated tissue was included. When outer tube diameter was smaller than 30 mm, peak diameter did not shift further.

Figure 2. Filled plot lines of summed length of segments of a class of diameters as function of diameter. Histograms were made for cylindrical regions of 500 pixels or 26.2 mm in diameter. A, lower pole (black area), upper pole (dark grey area) and interpolar region of pig P1 kidney. Histograms cover 250 images in the total image stack. Peak diameters of histograms are rather close and independent of polar region but magnitude of summed lengths at each diameter, including peak, may differ. B, ablated and control area in pig P2 kidney. Histogram covers images 1 to 300 in stack. Two curves can only be compared by peak diameter shape and position. Black area indicates cryoablation. Gray area indicates pole.

Table 1. Peak values of total segment length as function of diameter in healthy and cryoablated tissue in 5 pigs. Peak (vessel diameter Âľ) Kidney No. P2 P3 P4 P5 P6

Healthy renal tissue

Ablated area

Shift

159.5 156 147 148 152

181.5 180 172.2 176 180

22 24 25.2 28 28

Figure 1-C shows an MIP representing a 3.3 mm thick ablated tissue layer in pig P2, which contains as much intact structure as possible. This image confirmed visually that especially smaller vessels are affected by cryoablation. The remaining larger arterial structures traversed the cryoablated area and connected with vascular units in the surrounding tissue. Figure 3. Filled line plot of summed length of segments of class of diameters as function of diameter for concentric tube wall areas around ablation area centre in pig P3 kidney. Two largest tube diameters contained nonablated tissue, serving as control for histograms of ablated area. Peaks of walls of the smaller tubes were about same diameter. To correct for measured volume difference summed segment lengths rings were normalized with respect to largest ring volume.

Discussion

In this investigation of the immediate effects of cryoablation on vascular injury we used histograms of total summed vessel length as a function of diameter to quantify cryoablation effects. The peak diameter of a histogram of total segment length may shift to a higher value by the disappearance of segments with a diameter less than the original peak diameter or by the addition of segments with a larger diameter. However, the creation of more segments with larger diameters by ablation is mechanistically unlikely. The additional possibility that the ablated area had a larger peak diameter because this diameter was already larger to begin with was excluded since all peak diameter values in the ablated area were larger than in controls. Hence, this study shows that the vessels acutely damaged by freezing were less than about 180 Âľ in diameter but anatomically intact vessels smaller than this threshold were still found. The position of the peak diameter of the remaining patent vessels in the ablated area was not related to any particular ablation zone and, thus, it was independent of distance to the freezing centre. Intact structures of larger vessels in the ablated area were still connected to the intact microcirculation of the tissue surrounding the ablated area. The power of the current imaging cryomicrotome technique is that structures of the intact vasculature after ablation may be studied in detail up to a resolution of 40 Âľ resolution in relation to other images, providing structural information such as outline images. The resolution limitation was the result of the choice to image the kidney as a whole rather than zooming in on only parts of it. Daum et al. used a corrosion cast technique applied to a rat hind limb o o 11 submerged in cooled alcohol at temperatures of -10 C and -20 C . In that study the freezing front entered the tissue from outside the tissue while we studied the situation relevant for cryosurgery, for which needles for injecting cold are used. Hence, in our study the freezing front spread from 3 needles 0 had a much lower source temperature (-110 C) outward over an area that dependent on the balance between the supply of cold from the center and the removal of cold at the outside ablation ring. Also, in the hind limb study Daum et al. concluded that especially smaller vessels disappeared from the cast with freezing but no threshold value for microvascular diameter was provided. The similarity between those results and especially figures 1, C and 3 shows that the effect of freezing is not so much related to the degree o o of freezing, that is -10 C is as effective as â&#x20AC;&#x201C; 100 C with respect to vessel obstruction. However, with the needle approach the extensiveness of the frozen area depends on the temperature at the needles. The lower the core temperature, the larger the area.

We used the 3 smaller needles rather than 1 larger, thick needle because the 3-needle technique is used at our clinic and a thicker needle may compromise the vascular structures more than the smaller ones. However, because of the circle symmetry of our freezing lesions, apart from extension of the freezing area, results may not be different using a single but larger cryoprobe. Without cryoprotection cells are killed in the acute phase after freezing, 1 including endothelial cells . Thus, the structures of a vessel as a tube may still exist after thawing the interface but flowing blood may be damaged and 10 thrombi may form, which contributes to subsequent tissue necrosis . Damage in the venous system may be similar to that in the arterial system but we did not analyze this. In our earlier analysis of the coronary circulation the casting material filled 15 vessels as small as 10 Âľm . Hence, filling microvessels was not a limitation of this study. Diameter measurement is also influenced by the point spread function, describing how a small fluorescent object in tissue is blurred in the image because of the optical system. This effect was studied before, showing that diameters may have an error of 15% around 150 Âľm, which 18 decreases to zero at 250 Âľm . Hence, the point spread function has no influence on the estimated shift in peak diameter in control versus ablated regions. By penetrating the vascular lumina the casting material had to push saline out of the vessels that would have passed obstructions in the microvessels, halting the high viscosity casting material. Also, saline may have extruded through the vessel walls because of the long duration of the arterial pressure of the saline solution. There are a few other study limitations. It is not possible to use the histogram of the ablated area before freezing as a control for the situation after freezing. This limits the possibility of interpreting absolute values of the magnitude of the summed length as a function of diameter. Also, cryoablation is done with to destroy cancerous tissue while we studied the effect of freezing on the arterial vasculature of normal tissue. The effects of destruction via thrombus formation in the smallest vessels may also exist in cancerous tissue but the structure of the vascular bed may be different. Our study does not provide information on the process that further evolves with time after cryoablation and it is likely that the remaining structures disappear completely with time. A follow up study is needed to determine these effects. Our method is also limited since it restricts the possibility of additional morphological and histological measurement of areas identifiable on the 3-D

reconstruction. Tissue slices are collected normally as waste and not identifiable with respect to images. Thus, using the cryomicrotome technique more detailed correlation among cryolesion size, tissue necrosis and arterial vessel wall destruction requires further development of the technique and cannot currently be provided. It is not well documented in the literature but after clinical cryoablation we observe an outer ring or halo, which in appearance is different from the frozen and surrounding tissue on the kidney capsule surface. The halo is also visible on the epi-illumination images and on cryomicrotome MIPâ&#x20AC;&#x2122;s. It is unlikely that this halo is the result of post-ablation hyperemia since it coincides with the disappearance of microvessels on MIPâ&#x20AC;&#x2122;s. To our knowledge the cause and implication of the structural difference in this halo region in regard to the clinical outcome of cryoablation remains to be established. The remaining arterial structures in the ablated area warrant further critical 12, 19 analysis in immediate postoperative perfusion studies . Residual vascular structures that conduct contrast medium through the ablated area may remain unnoticed on images because of resolution issues. According to the distribution pattern of the cast material contrast medium passes through the residual larger vessels in the ablated region rapidly but is not distribute in the ablated area, although it is distributed in bordering nonablated tissue. Conclusion

Cryoablation with three 17-gauge needle probes of normal parenchyma in a pig kidney resulted in an ablated area in which especially vessels smaller than 180 Âľ in diameter are blocked flow in the acute phase. Some anatomically patent arteries remained traversing the ablated area and connecting with nonablated tissue vasculature. The role of these transport vessels in post-ablation distribution of blood flow and malicious cells in a temporal manner warrant further attention.

References

Gage AA, Baust J. Mechanisms of tissue injury in cryosurgery. Cryobiology. 1998; 37(3): 171 – 186.

Hoffmann NE, Bischof JC. The cryobiology of cryosurgical injury. Urology. 2002; 60(2 Suppl 1): 40 – 49.

Le Pivert P. Basic considerations of the cryolesion. In “Handbook of Cryosurgery” (R. Ablin, Ed.), Dekker, New York 1980, pp. 15 - 68.

Adams-Ray JA, Bellman S. Vascular reactions after experimental cold injury: a microangiographic study of rabbit ears. Angiology 1956; 7: 339 – 367.

Lewis T, Love WS. Vascular reactions of the skin to injury. III: Some effects of freezing, of cooling, and of warming. Heart. 1926; 13: 27 – 60.

Rotnes PL, Kreyberg L. Eine methode zum experimentelen Nachweis von stase mittels spezieller praeparate. Acta Pathol Microbiol Scand Suppl. 1932; 11: 162 – 165.

Giampapa VC, Oh C, Aufses AH. The vascular effect of cold injury. Cryobiology. 1981; 18(1): 49 – 54.

Rabb JM, Renaud ML, Brandt A, Witt CW. Effect of freezing and thawing on the microcirculation and capillary endothelium of hamster cheek pouch. Cryobiology. 1974; 11: 508 – 518.

Rothenborg H. Cutaneous circulation in rabbits and humans before, during and after cryosurgical procedures, measured by Xenon-133 clearance. Cryobiology. 1970; 6: 507 – 511.

Zacarian S, Stone D, Clater M. Effects cryogenic temperatures on microcirculation in the golden hamster cheek pouch. Cryobiology. 1970; 7: 27 – 39.

Daum PS, Bowers WD, Tejada J, Hamlet MP. Vascular casts demonstrate microcirculatory insufficiency in acute frostbite. Cryobiology. 1987; 24(1): 65 – 73.

Anderson JK, Shingleton WB, Cadeddu JA. Imaging associated with percutaneous and intraoperative management of renal tumors. Urol Clin N Am. 2006; 33: 339 – 352.

Weight CJ, Kaouk JH, Hegarty NJ, Remer EM, O’Malley CM, Lane BR, Gill IS, Novick AC. Correlation of radiographic imaging and histopathology following cryoablation and radio frequency ablation for renal tumors. J Urol. 2008; 179(4): 1277 - 1281.

Lagerveld BW, ter Wee RD, de la Rosette JJ, Spaan JA, Wijkstra H. Vascular fluorescence casting and imaging cryomicrotomy for computerized threedimensional renal arterial reconstruction. BJU Int. 2007; 100(2): 387 – 391.

Dankelman J, Cornelissen AJ, Lagro J, VanBavel E, Spaan JA. Relation between branching patterns and perfusion in stochastic generated coronary trees. Med Biol Eng Comput. 2007; 45(1): 25 – 34.

Palagyi K, Kuba A. A 3D 6-subiteration thinning algorithm for extracting medial lines. Pattern Recognition Letters. 1998; 19: 613 – 627.

Rolf MP, ter Wee R, van Leeuwen TG, Spaan JA, Streekstra G. Diameter measurement from images of fluorescent cylinders embedded in tissue. Med Biol Eng Comput. 2008; 46: 589 – 596.

Wink MH, Laguna MP, Lagerveld BW, de la Rosette JJ, Wijkstra H. Contrastenhanced ultrasonography in the follow-up of cryoablation of renal tumours: a feasibility study. BJU Int. 2007; 99(6): 1371 – 1375.

CHAPTER 4 Cryosurgical injury: Change of vascular anatomy and blood flow in a porcine kidney

This chapter is published as: Lagerveld BW, van Horssen P, Laguna MP, van den Wijngaard JPHM, Siebes M, Wijkstra H, de la Rosette JJMCH, Spaan JAE. The gradient change of arterial vascular anatomy after cryoablation. J Urol. 2011; 186(2): 681 - 686.

Abstract

Purpose We quantified temporal changes in vascular structure and blood flow after cryosurgery of porcine kidney in vivo. Materials and Methods We studied 5 groups of 4 kidneys each with a survival time of 20 minutes, 4 hours, 2 days, 1 and 2 weeks after cryoablation. Before harvesting the kidneys, fluorescently labelled microspheres were administrated in the descending aorta. After harvest the kidney and its vasculature was casted with fluorescently dyed elastomere, frozen and processed in an imaging cryomicrotome to reveal the 3-dimensional arterial branching structure and microspheres distribution. In regions of interest vessels were segmented by image analysis software and histograms were constructed to reveal the total summed vessel length as function of diameter. A characteristic diameter of the ablated area was measured. Results The 20-minute survival group histograms showed a significant shift of the peak to larger diameters (p=<0,002), indicating that smaller vessels were destroyed. Microsphere density was decreased to 2% in the ablated region but not in the nonablated border zone, depending on the remaining crater crossing larger vessels. After 2 weeks neither vessels nor microspheres were left in the ablated area, which had shrunk by about 40% in diameter. Study limitations are the lack of histological confirmation and the use of normal rather than cancerous tissue. Conclusion Larger vessels remain patent just after ablation and transport blood to the border of the ablation crater but perfusion within the crater is halted instantly. Characteristic crater diameter increases initially but decreases thereafter. Destruction of vessels and tissue is complete 2 weeks after cryoablation.

Introduction

Cryoablation is a valid, alternative minimally invasive treatment for small renal tumors, especially in the elderly and fragile populations who sometimes have compromised renal function. Radical nephrectomy in these patients often results in renal function loss. Also, partial nephrectomy can impact the renal function when prolonged clamping of the renal vasculature is a prerequisite to perform the surgery under good conditions. Renal cryoablation overcomes some shortcomings while maintaining good 1 oncological outcomes . Vascular stasis after cryosurgery is considered to have a significant role in 2 final cell death . Thus, it is important to understand when vascular stasis is complete. Contrast enhanced imaging, in which local absence of contrast medium in the treated area is considered a sign of a successful ablation, can 3-5 assess clinical success of renal tumor ablation . However, interpretation of these images must consider that although microvessels are affected by 6 cryoablation immediately, larger vessels can remain patent . Therefore, it was postulated that at the border of the cryosurgical lesion some cells die while others survive and adjunct therapy should be considered to influence 2 the outcome of surviving cells . Several theories have been put forward of the mechanism of tissue injury in cryosurgery, such as direct cellular damage provoked by the freeze-thaw intervals as well as cryogenic induced apoptosis and vascular injury resulting in a coagulative necrosis. However, understanding the advancement of tissue damage after cryoablation requires a better definition of the time course of vascular damage and related perfusion disturbances. In our earlier study on porcine kidneys particular vessels smaller than 180 Âľm in diameter 7 were destroyed in the acute phase after cryoablation but not all of them . We also observed that some anatomically patent arteries remained, traversing the ablated area and connecting with the microvasculature of nonablated tissue to allow continuing perfusion of that region. Thus, we quantified the immediate and transient effects of cryoablation on the perfusion and structure of the renal arterial circulation. Materials and methods

Study design Ten domestic farm pigs weighing 35 to 40 kg were used in accordance with an approved protocol of the National Institutes of Health Guide for the Care and Use of Laboratory Animals that was registered with the Animal

Research Institute, Academic Medical Center, Amsterdam, the Netherlands. In each pig simultaneous open bilateral renal ablation was performed at the anterior side of the lower or upper pole. Experiments were divided into five groups of 4 kidneys each with different survival times, including group 1 - 20 minutes, group 2 - 4 hours, group 3 - 2 days, group 4 - 1 week and group 5 2 weeks. In groups 3-5 the pigs were awakened after abdominal closure and later anesthetized again for nephrectomy. Intervention and preparation Cryoablation was done using a CryoNeedle triple needle probe configuration (Galil Medical, Yokneam, Israel). The probes were placed in a coaxial and triangular configuration with a 7 mm distance between them. Total freezing time was 20 minutes using a double freeze-thaw cycle. In preparation for TM nephrectomy 200,000 red fluorescence dyed microspheres (Invitrogen ) with a diameter of 15 µ were suspended in a syringe and rinsed with Tween in 2 cc autologeous heparinized blood. Ten minutes before kidney harvest the solution was injected in the aortic artery at suprarenal level. After nephrectomy the renal arteries were cast with a Batson no. 17 green fluorescence dyed polymer cast (Polysciences, Warrington, Pennsylvania). Subsequently the kidneys were submerged in a container filled with 5% carboxymethylcellulose sodium solvent (Brunschwig chemie, Amsterdam, the Netherlands) and 5% Indian ink (Royal Talens, Apeldoorn, the o 8,9 Netherlands) and frozen to -20 C . Preparations were sliced in our custom-made imaging cryomicrotome. Using a Megaplus® 4.2i camera equipped with a 70-180 mm lens (Nikon®) digital images were taken from the cutting plane of each slice. The normal white light reflection images were indicated as outline images. Images of the fluorescent cast and microspheres were obtained by applying D440/20x and D560/20x excitation filters, and D505/30m and D635/30m emission filters 10 (Chroma Technology, Bellow Falls, Vermont, USA) . Generalized data The green fluorescent images resulted in a 3-D image of the arterial vasculature of a kidney down to 20 µm vessels. In this stack of images arterial vessels were segmented and vessel diameters were estimated using 11, 12 a combination of filtering and peeling algorithms . Thereafter all vessel segments could be classified by diameter, which was divided by bins of 4 µ wide starting from 40 to 500 µm. The length of all segments of a specific bin diameter was added and histograms of total summed vessel length were plotted as function of diameter. These histograms were quantified by the diameter at the peak of the histogram. In all groups the graphically reported histograms were averaged. Histograms for the ablated area were compared to histograms from a control area in a different pole of the same kidney.

Thus, the effect of cryoablation was quantified as function of the diameter range. Microsphere data Microsphere positions in the kidney were determined from the appropriated 10 stack of images by dedicated self-developed software . Microsphere density in N/ml in the center of the ablated area, the border zone of the ablated area and a control area were compared to quantify the differences in perfusion through these areas. In group 1 the border zone was determined from the microvascular images, which clearly showed the part perfused by vessels traversing the ablated area. In later groups the border of the ablated area was determined by the intact microvasculature surrounding the ablation area and the outline images showing adjacent scar tissue. The control area was chosen in the same pole but away from the ablated region. Ablation zone diameter Ablation zone diameter was assessed by selecting the image with the largest ablated area from the stack of outline images of each kidney. A circle was projected over the image to best match the ablated area contours. The diameter of the circle was considered as the characteristic diameter and used to calculate tissue retraction with time. Statistical analyses Segment length histograms were affected by cryoablation in 2 ways, that is 1) total vessel segment length at certain diameter decreases and 2) the diameter at which the peak occurred shifted to larger diameters. The difference between the sum of the lengths of vessel segments at the peak under control and after acute ablation conditions did not always attain significance in our earlier study, although the shift in the peak diameter did 7 achieve significance . In the current study we determined the histogram of each individual tissue region. In experimental groups 1 and 2 the shift in peak diameter was determined by the 2-tailed paired t-test. In groups 2 and 3 the difference between peak values was tested by the 2-tailed paired t-test. For groups 4 and 5 no further statistical analysis was done. A one-way ANOVA was used to quantify the retraction of ablation zone diameter. Groups were compared to each other using the Bonferroni multiple comparison test.

Results

In each pig the survival time of the 2 kidneys was equal. No hemodynamic disturbances were noted during the surgical procedures. Kidneys were processed in the imaging cryomicrotome by applying different types of optical filters, resulting in a set of 3 images after each cut of 40 µm. The 3 stacks of images resulted in 1) a 3-D representation of the vascular bed, 2) outline images of the tissue showing ablated area contours and 3) the positions of injected, fluorescently labeled microspheres. All kidneys had a clearly demarcated cryoablation zone at the capsular surface. One group 1 kidney was not fit for analysis and was excluded due to underexposure of fluorescence images due to a technical problem. Figure 1 shows the development of the ablation zone and the vasculature retained in it in groups 1 to 5. Groups were based on survival time after ablation, including group 1 – 20 minutes, group 2 – 4 hours, group 3 – 2 days, group 4 – 1 week and group 5 – 2 weeks. Ablation zones were clearly visible in all groups due to absent tissue auto-fluorescence, which are seen as red areas. The green fluorescence of the post mortem injected casting agent was already strongly decreased in group 1 but it decreased in the 2 subsequent groups and was practically absent after a week in group 4. Figure 1 shows outline images of groups 1 and 2 in which the transition between the ablated area and its surrounding is sharp while a circular fluorescent band had developed by day 2 and week 1 in groups 3 and 4, respectively, ending in encapsulation after 2 weeks in group 5. The ablated area retracted with time. Vascular damage in the ablated areas was quantified and compared by the averaged histograms of total segment length, which was determined from 3D reconstructions of the vascular bed from the casting material, as function 7 of bin diameter (figure 2). In agreement with our earlier observations ablation acutely affected the distribution of total segment length by shifting the diameter of at which the peak occurred significantly to a higher value (p=0.002), indicating the disappearance of vessels with diameter smaller than the peak diameter in the ablated area. In group 2 at 4 hours after cryoablation peak diameter had shifted to a larger diameter and the peak value of total segment length was significantly decreased (p=0.012 and p=0.016, respectively). The strong decrease in total segment length at all diameters at later time points was obvious in subsequent histograms. Some small vessel segments were left after 2 days but after 1 week no detectable structures remained (figure 2, D and E).

Figure 1. Representative digital images of group 1- acute vascular injury (A), and 2vascular injury after 4 hours (B), 3- after 2 days (C), 4- after 1 week (D) and 5- after 2 weeks (E) reveal fluorescence cast (green areas) in cryoablation zone vascular structures and autofluorescence of direct surrounding tissue (red areas) achieved by applying D440/20x excitation filter and D505/30m emission filters to single slice cryomicrotome specimen.

Figure 2. Histograms of average total segment length in groups 1 (A), 2 (B), 3 (C), 4 (D) and 5 (E) with total segment length per group based on area used for calculation. Black areas indicate cryoablated zone. Gray areas indicate noncryoablated zone.

Figure 3 shows a typical ablation crater reconstructed from the autofluorescence images and within it the remaining vasculature in 3 dimensions in group 1. The crater wall was determined by applying a threshold for reflective light, resulting in isoreflectance curves. Figure 3 supports the conclusions from the histograms that smaller vessels in the crater disappeared but a structure of larger vessels survived ablation. Fluorescently labeled microspheres were injected in the descending aorta before kidney removal. The regional density distribution of microspheres was representative for perfusion distribution. In group 1 the average relative microsphere density in the ablated crater decreased to 2 % that of the control value and it was practically zero in the other groups. Figure 4 shows microsphere positions with respect to the cast in group 1 in a 3-D visualization in which the surviving vasculature in the ablated area is clearly visible. Microspheres were transported to the perimeter of the vascular structures into nonablated renal parenchyma but were practically absent in

the ablated core. Hence, in group 1 the larger vessels in the ablation zone that were visible by casting remained patent but hardly transported any blood to the ablated tissue. However, by 1 week after ablation the vascular structures in the crater had disappeared completely and microvessels in the border zone were only connected to vascular structures approaching the crater wall from outside. Figure 3. Three-D reconstruction from outline images (gray areas) reveal cryoablation crater at lower pole of group 1 kidney with 20-minute survival time. Note cast of surviving vasculature (orange areas) in crater.

Figure 4. Three-D visualization shows reconstructed center lines (white areas) of remaining vascular structures of group 1 kidney traversing the cryoablation area and connecting to surrounding noncryoablated tissue. Vascular structures outside ablation zone are not shown. Color of the originally red dyed microspheres was digitally adjusted. Microspheres (green dots) distributed by traversing vessels were noted particularly in border (arrows) outside the ablation crater (gray areas) (A). Gray dots represent microspheres distributed by vessels outside ablation area. Note detailed side view of same cryoablation area (B).

Figure 5 shows a quantification of the retraction of the ablated area with time (figure 1). Circles were fitted to the borders of the ablated areas by eye and the mean diameter of each group was calculated (figure 5). Diameters at all time points differed from each other (p=<0.0001). Hence, 4 hours after ablation the characteristic diameter was larger than at the acute phase but it decreased thereafter by 50%. The increase in lesion diameter from the acute phase to 4 hours probably represents tissue swelling due to the development of edema as the initial response to cellular and vascular tissue 2 injury .

Figure 5. Mean diameter of each cryoablated area per group, as measured on outline images at maximum crater size.

Discussion

This study demonstrates that in the target area cryoablation immediately destroys vessels with a diameter of less than 180 Âľm with a practically complete decrease in local perfusion. In the acute phase the microcirculation in the border zone of the target area remains connected and perfused by the larger remaining structures in the ablated region. With time these larger vascular structures also disappear and the process of secondary vascular stasis is completed within 2 weeks. The filling with casting material is obviously only possible with an intact structure of the remaining vascular tree in the ablated area. Although occasionally some leakage of casting material was found in the ablated area, this was not the rule. Already in group 1 perfusion in the ablated area was practically decreased to zero. Therefore, filling of the remaining vascular structures with casting material was the result of displacing perfusate into

the surrounding tissue by intact vessels structures or by leakage into damaged tissue. One may assume that each microsphere passed through the smallest, most proximal detectable vessel in its neighborhood. The result in group 1 clearly showed perfusion of the rim surrounding the ablated area that was perfused by the remaining vascular structures. In our earlier study we reported a halo7 like structure surrounding the ablated area . A suggested mechanism was hyperemia in this tissue. However, this hyperemia could not be confirmed by the current measurements since the microsphere density at the rim did not differ from nonaffected tissue in either group. Some of the larger vessels that originally remained patent in the ablated area survived the direct cryoablative conditions. However, their vascular cells were exposed to indirect cell kill mechanisms, such as cryogenically 13 imposed apoptosis and ischemia in surrounding tissue . Flow in the remaining vascular structures must have been strongly decreased, as follows from the very low microsphere density in the ablated area and the decreased amount of intact tissue that depended on these vascular structures. Normally decreased flow in a vessel segment results in inward 14 remodeling of these vessels but this requires days . Thus, it is unlikely that remodeling alone explains the difference between groups 1 and 2, which had duration difference of only 3.5 hours. Hence, most likely the patency of these remaining vessels was severely affected by acute ablation effects. Our study method currently restricts the possibility for additional morphological and histological measurements from areas identifiable from the 3-D reconstruction due to the destructive nature or our cryomicrotome method. However, Ames et al. described the histopathological findings of 15 cryoablated porcine normal renal parenchyma after 2 weeks of survival . Ablated lesions showed a peripheral rim of fibrosis with chronic inflammation, calcification and foreign body-type giant cells surrounding the central complete coagulative necrosis. These findings correspond to our results showing no vessels and no microspheres in a sharply demarcated zone after 2 weeks. During the last decades several pathways of cryogenic cell death have been identified that involve direct and indirect mechanisms. Therefore, some investigators have assessed the effects of cellular injury for a period of days to weeks after cryoablation. Our study reveals that the characteristic diameter of the ablation area shrinks from 36mm to 30mm during the period from 20 minutes to 1 week post cryoablation. Consequently this effect can bias results when late cryogenic tissue response is studied in relation to temperature measurements assessed at cryoablation.

A limitation of this study is that it was done in normal porcine renal parenchyma. However, when cryoablation is used to treat cancer, we can only suggest that these cancer tissues will respond to ischemia in the same way. In the clinical setting perfusion measurements are made soon after ablation to determine its success. However, we noted that flow through the ablated area is still possible for a few days and early perfusion studies should be interpreted with care. However, 2 weeks after ablation secondary vessel destruction should be fully complete and, thus, intravenous contrast measurement can be considered a true indicator of ablation success. Conclusion

In the 2 weeks after cryoablation of porcine normal renal parenchyma gradual vascular anatomical disruption of the arterial architecture develops. At the end of this period without exception anatomical vascular branches and blood flow have fully disappeared from a well-demarcated cryoablation zone.

References

Campbell SC, Novick AC, Belldegrun A, Blute ML, Chow GK, Derweesh IH, Faraday MM, Kaouk JH, Leveillee RJ, Matin SF, Russo P, Uzzo RG; Practice Guidelines Committee of the American Urological Association. Guideline for management of the clinical T1 renal mass. J Urol. 2009; 182(4): 1271 - 1279.

Gage AA, Baust JG. Mechanisms of tissue injury in cryosurgery. Cryobiology. 1998; 37(3): 171 - 186.

Anderson JK, Shingleton WB, Caddedu JA. Imaging associated with percutaneous and intraoperative management of renal tumors. Urol Clin N Am. 2006; 33(3): 339 - 352.

Beemster P, Phoa S, Wijkstra H, de la Rosette J, Laguna P. Follow-up of renal masses after cryosurgery using computed tomography; enhancement patterns and cryolesion size. BJU Int. 2008; 101(10): 1237 - 1242.

Weight CJ, Kaouk JH, Hegarty NJ, Remer EM, Oâ&#x20AC;&#x2122;Malley CM, Lane BR, Gill IS, Novick AC. Correlation of radiographic imaging and histopathology following cryoablation and radio frequency ablation for renal tumors. J Urol. 2008; 179(4): 1277 - 1281.

Daum PS, Bowers WD, Tejada J, Hamlet MP. Vascular casts demonstrate microcirculatory insufficiency in acute frostbite. Cryobiology. 1987; 24(1): 65 - 73.

Lagerveld BW, van Horssen P, Laguna Pes MP, van den Wijngaard JPHM, Streekstra GJ, de la Rosette JJMCH, Wijkstra H, Spaan JAE. The immediate effect of kidney cryoablation on renal arterial structure in a porcine model studied by imaging cryomicrotome. J Urol. 2010; 183(3): 1221 - 1226.

Spaan JA, ter Wee R, van Teeffelen JW, Streekstra G, Siebes M, Kolyva C, Vink H, Fokkema DS, VanBavel E. Visualisation of intramural coronary vasculature by an imaging cryomicrotome suggests compartmentalization of myocardial perfusion areas. Med Biol Eng Comput. 2005; 43(4): 431 - 435. van Horssen P, Siebes M, Hoefer I, Spaan JA, van den Wijngaard JPHM. Improved detection of fluorescently labeled microspheres and vessel architecture with an imaging cryomicrotome. Med Biol Eng Comput. 2010; 48(8): 735 - 745.

Bennink HE, van Assen HC, Streekstra GJ, ter Wee R, Spaan JAE, ter Haar Romeny BM. A Novel 3D Multi-scale Lineness Filter for Vessel Detection. Med Image Comput Assist Interv Int. 2007; 10(Pt 2): 436 - 443.

Palagyi K, Kuba A. A 3D 6-subiteration thinning algorithm for extracting medial lines. Pattern Recognition Letters. 1998; 19: 613 - 627.

Hoffmann NE, Bischof JC. The cryobiology of cryosurgical injury. Urology. 2002; 60(2 Suppl 1): 40 - 49.

van den Akker J, Schoorl MJ, Bakker EN, Vanbavel E. Small artery remodeling: current concepts and questions. J Vasc Res. 2010; 47(3): 183 - 202.

Ames CD, Vanlangendonck R, Venkatesh R, Gonzales FC, Quayle S, Yan Y, Humphrey PA, Landman J. Enhanced renal parenchymal cryoablation with novel 17-gauge cryoprobes. Urology. 2004; 64(1): 173 - 175.

CHAPTER 5 The utility of RENAL and PADUA nephrometry indices to predict for complications of laparoscopic cryoablation for small renal tumors

This chapter is accepted for publication as: Lagerveld BW, Brenninkmeijer M, van der Zee JA, van Haarst EP. Can RENAL and PADUA nephrometry indices predict for complications of laparoscopic cryoablation for clinical stage T1 renal tumors? J Endourol 2013.

Abstract

Objective Assessment of anatomical complexity with the RENAL and PADUA nephrometry indices is used to predict complications related to surgical extirpation treatment for patients with clinical T1a/b renal mass (RM). This single centre study aims to investigate the value of these indices in order to predict complications in a cohort of patients treated with laparoscopic cryoablation (LCA) for cT1 RM. Materials and Methods Single institution data from consecutive LCA procedures were prospectively collected from December 2006 to April 2013. RM anatomical complexity was categorized according to RENAL and PADUA indices. Comorbidity was assessed by the Charlson-index. Intraoperative complications (IOC) were reviewed and categorized in: blood loss >100ml, conversion, tumor fracture, and incomplete ablation. Postoperative complications (POC) were graded using the modified Clavien-index. Univariate and multivariate logistic regression models addressed the risk for complications. Results 99 LCA procedures were included. The median RENAL-score was 7.0 (SD 1.7), and the median PADUA-score was 8.0 (SD 1.6). IOC occurred in 19 procedures (19%). The risk for IOC was significantly correlated (p<0.05) with tumor diameter (mm), surface, volume, the RENAL domains “R-size”, “Nnearness to collecting system”, “RENAL score”, and the PADUA domain “diameter”. In multivariate analysis with surgical complication as the independent variable, tumor diameter, surface and volume were a determining factor. A threshold was set for 35mm tumor diameter, it being predictive for an increased risk for IOC performing LCA. 23 POC which occurred in 20 patients. On univariate analysis the RENAL domain “nearness to collecting system”, and no PADUA domains, had a significant association with POC. Conclusion The RENAL score, and not the PADUA score, is associated with a higher risk for IOC. A non-categorized method of scoring tumor diameter showed a more significant correlation with the risk for IOC than the categorized method of the nephrometry indices. As a result a threshold diameter of 35mm was established.

Introduction

Principal guidelines advise that treatment methods for small renal masses 1, 2 must aim to preserve renal function and provide oncologic control . Partial nephrectomy (PN) is considered a therapeutic standard for the conduct of clinical T1 renal masses. Currently, in selected cases, the thermal ablative modality of cryoablation (CA) is regarded as an alternative treatment option to PN or radical nephrectomy (RN). Anatomic classification systems (ACS) such as the Preoperative Aspects and Dimensions Used for Anatomical Classification (PADUA) and RENAL nephrometry indices, were originally designed to aid in the decision-making process in order to best determine which type of surgery, PN or RN is most 3, 4 desirable . However, in the case of a clinical stage T1 renal mass, it would be most convenient if nephrometry indices can be used to provide advice for extirpative as well as for ablative therapy. Today, minimally invasive surgical techniques for the treatment of small renal masses reduce hospital stays and treatment related morbidity, thus 1, 2 permitting the rapid return to everyday life . Laparoscopic or robotic PN 5 requires high-level surgical skills and has a challenging learning curve . However, in experienced hands, the complication rate of laparoscopic or 6 robotic PN is equivalent to that of open PN . Clearly, the preference for a particular surgical technique can be influenced by its accompanying complication rate. Despite selection bias, radiofrequency ablation (RFA) and 1, 2, 7 CA are recognized for their lower complication rates compared to PN . Therefore, principal guidelines regarding RFA and CA as alternative treatment options are needed especially for high-surgical risk patients desiring active treatment. Several study groups investigated the predictive value of the PADUA and RENAL nephrometry indices for the risk of complications in extirpative 3, 8-10 surgery . Hence, if the standardization of renal mass anatomical complexity and its correlation with risk for complications is considered as relevant in extirpative surgery, then this should ideally also apply to ablative therapies. Currently, series of renal masses treated with percutaneous image guided or laparoscopic CA or RFA have studied the relation between anatomical complexity using the RENAL nephrometry score and the risk of 11-15 complications . No such studies have been performed using the PADUA index. To date, patients selected for LCA are informed about the risk for complication based on the surgeonâ&#x20AC;&#x2122;s experience and intelligence gathered from published series. We emphasize that a standardized scoring system for the complexity of renal mass is desired when the benefits and risks,

including oncological and renal functional outcome, as well as possible morbidities of nephron-sparing surgical treatments such as PN (open, laparoscopic, robotic) and ablation modalities (CA, RFA; percutaneous, laparoscopic), are compared. The principal objective of this study is to evaluate the value of RENAL and PADUA indices for predicting the risk of intraoperative (IOC) and postoperative (POC) complications in a cohort of patients that underwent LCA for a small renal mass. The additional objective of this study is to identify whether tumor upper pole location or medial/hilar location are risk factors for complications of LCA. The final objective of this study is to establish a threshold value for tumor diameter that indicates the sensitivity for the increased risk of IOC performing LCA. Materials and methods

Patient characteristics and data collection In this non-randomized retrospective cohort study, consecutive cases of primary renal tumors treated with LCA between December 2006 and April 2013 were evaluated. Patients were identified in our institutional database. The local internal review board approved the study design and the submission for publication. Patient records were examined and data of age, body mass index (BMI), 14 Charlson Comorbidity Index (CCI) , age-adjusted Charlson Comorbidity 15 Index (a-CCI) , pre- and postoperative renal function (GFR-MDRD), laparoscopic approach, side of surgery, histology, and hospital stay were collected. The anatomical complexity of each renal mass was determined using tumor size, location, and closeness of the renal mass to the urinary collecting system. These parameters, as well as their categorized equivalents according to the PADUA and RENAL indices for classification of the 3, 4 anatomical complexity of renal masses were examined . Intravenous contrast-enhanced coronal and axial Computed Tomography (CT) scanning or Magnetic Resonance (MR) imaging were used to assess each category of these scores. The interpretation of the imaging and the scoring of the anatomical complexity were performed by a single staff urologist. Tumor surface and volume were calculated using the diameter of the tumor in millimeters (mm), assuming round symmetry. Tumors located at the renal upper pole or located at the medial side near to the hilum are considered less accessible for LCA than tumors located lateral

and below the upper polar or sinus lines. Therefore, the domain “longitudinal location” of the PADUA nephrometry index and the domain “location relative to the polar line” of the RENAL nephrometry index were analyzed for true upper pole location. Laparoscopic cryoablation According to principal guidelines, all patients were informed about optional treatment methods for small renal tumors. Patient, surgeon or multidisciplinary uro-oncology committee preferences set indication for LCA. All patients consented with LCA. The same surgeon performed all procedures. A retroperitoneal approach was preferred whenever it was considered technically feasible. Tumor location and identification was confirmed with endoscopic ultrasonography (Hitachi Medical Systems, Tokyo, Japan). All renal masses were biopsied intraoperatively before cryoprobe placement. A standard of 3-5 biopsies were performed using a TM ® Magnum biopsy gun (Bard , Covington, Louisiana, USA) loaded with a disposable 16-gauge automatic side-cutting biopsy needle with a sample notch range of 19 mm. For cryoablation, multiple 17-gauge cryoprobes (Galil Medical, Yokneam, Israel) were used. The selection of the number and type of cryoprobes depended on the tumor volume. Temperature probes were used at indication. A standard of two freeze cycles of 10 minutes were performed. After each freeze cycle a passive thaw cycle for approximately 6 minutes was initiated and thereafter switched to an active thaw for 2 minutes. The endoscopic ultrasonography was also used for guiding probe placement and monitoring the freeze process. At indication probes are replaced, freeze cycles adjusted, or additional probes are introduced. After completing the cryoablation and the removal of the cryoprobes a compress ® of collagen sponge (TachoSil ) was draped over the lesion as a method for hemostasis. Complication outcomes Intraoperative complications were scored and categorized in technical complication (conversion), blood loss > 100 ml, and tumor fracture. Also, an incomplete ablation of the renal tumor accessed at first time postoperative imaging (range 2-14 weeks) was considered as an intraoperative complication. It can be argued that incomplete ablation should be considered as an IOC, however, we defined that a clearly incomplete ablation is a technical or procedural failure related to the surgery itself and therefore should be considered as a complication of surgery. Postoperative complications, with a minimum of 1-month follow-up were scored according to the modified Clavien complication classification (CCS). They were graded 18 as minor (CCS: 1-2) or major (CCS: 3-5) . For the report of complications, 19 the Martin criteria were followed .

Statistical analysis Data of the reviewed patients were anonymously entered in an anonymous database and analyzed with Statistical Package for Social Sciences software, v.18.0 (IBM Corporation, SPSS Statistics). For comparison of continuous variables, the t-test was used. Pearsonâ&#x20AC;&#x2122;s correlation coefficient r was used to test the relation of several parameters. Multivariate logistic regression analysis using stepwise selection was used to identify significant predictors of complications, controlling for age and sex. Univariate analysis was used to select variables. A p value <0.05 was considered as statistically significant. Results

General results Patient characteristics and general surgical information of the cohort is demonstrated in table 1. The median follow-up was 37.7 months (standard deviation (SD) 20.1 months). Two patients underwent a second laparoscopic cryoablation for a newly developed renal mass or a synchronous renal mass at the contralateral kidney. Out of the 99 primary tumors that were treated with cryoablation, 84 were histologically diagnosed as malignant (clear cell RCC n=54; chromophobe RCC n=12; papillary RCC n=15; undefined carcinoma n=2; B-cell lymphoma n=1) and 15 were diagnosed benign (oncocytoma n=11; angiomyolipoma n=4). In 3 patients, the diagnosis was assessed by preoperative image guided biopsies. A biopsy specimen alone is not eligible for studying capsular or perirenal fat invasion, therefore, in 39 procedures, in addition to intraoperative renal mass biopsy, the peritumoral fat tissue covering a renal cancer was sampled for histological examination for staging purposes. None of these 39 specimens showed malignant invasion into the peritumoral fat. The treatment success of cryoablation is determined at the time of the first postoperative contrast CT-imaging a minimum of 2 weeks after surgery and is defined by the appearance of (focal) enhancement of the ablated tumor 20 area . In three procedures, the ablation was found incomplete at first time imaging, which was considered as an intraoperative complication. Therefore, the surgical success rate for primary cryoablation assessment was 97%. After initial treatment success, a later focal recurrence at the primary tumor ablation site is most likely to be expected in those cases that revealed renal cancer (RCC). In the group of 84 proven RCC tumors treated by LCA, three late recurrences were detected at a consecutive follow-up. The median follow-up of this RCC cohort was 36 months (SD 20.3 months). The three

recurrences were successfully retreated with percutaneous CT-guided cryoablation. Three patients, all with proven RCC, died. One patient died of metastasized RCC; 1 died of metastasized transitional cell carcinoma of the bladder, and 1 died of metastasized rectal cancer. The overall oncological failure rate in this series of primary LCA for RCC, assessed by the number of incomplete ablation plus the number of later occurred recurrences, was 7.1%. The mean postoperative hospital stay was 3 days (range 1-12 days). There was no significant difference in hospital stay (p=0.193) among patients who were approached by retroperitoneal or transperitoneal surgery. Within the time frame of 1 month after LCA, one patient was readmitted because of fever due to a perirenal abscess that required intravenous treatment with antibiotics. 2

The mean preoperative GFR-MDRD was 73.0 ml/min/1.73m . The 2 postoperative mean GFR-MDRD was 72.2 ml/min/1.73m . This difference was not significant (p= 0.439). Anatomic classification systems The renal tumors were classified according to their anatomical complexity. Using the PADUA-index, a the score of low-, intermediate-, and high-grade complexity was assessed for 42 (score 6-7), 38 (score 8-9) and 19 (score ≥10) tumors, respectively, whereas 48 tumors scored low- (score 4-6), 48 intermediate- (score 7-9) and 3 high-grade (score 10-12) complexities using the RENAL-index. As shown in figure 1, there seems a trend towards increased anatomical complexity using the PADUA-index compared to the RENAL-index. However, this was not significant (p=0.5). The median RENAL-score was 7.0 (SD 1.7), and the median PADUA-score was 8.0 (SD 1.6). In the domain “longitudinal location” of the PADUA-index, 26 tumors were found entirely above the upper sinus line. In the domain “location relative to the polar line” of the RENAL-index, 27 tumors were located entirely above the upper polar line.

Table 1. Patient and surgical characteristics; (N= number; SD= standard deviation; GFR-MDRD= Glomerular Filtration Rate â&#x20AC;&#x201C; Modification of Diet in Renal Disease; ASA= American Society of Anesthesiologists).

N Patients Male Female Age (years)

97 67 30

LCA procedures Tumor diameter (mm) Benign histology Malignant histology

99 99 15 84 2

99 25 8

Body Mass Index (kg/cm ) ASA Charlson Comorbidity Index Age-adjusted Charlson Comorbidity Index

99 50 99 99

Laparoscopic retroperitoneal approach Laparoscopic transperitoneal approach Right side Left side

52 47 55 44

Preoperative GFR-MDRD (ml/min/1.73m ) 2 Preoperative GFR-MDRD<60 (ml/min/1.73m ) Solitary kidney 2

Mean

Range

66.4

36 - 84

10.6

29.9

15 - 50

7.6

73.0 43.5

19 - 149 19 - 59

23.8 13.3

27.1 2.0 1.9 4.0

18.7 - 41 1-3 0-8 0 - 11

4.7 0.5 2.0 2.5

Figure 1. Distribution patterns of anatomical complexity categories according to the RENAL and PADUA indices for the same cohort of renal masses, each treated with laparoscopic cryoablation (n=99).

Complications A total of 23 intraoperative complications occurred in 19 out of the 99 procedures. The complications for the categories blood loss >100ml, tumor fracture, incomplete ablation and conversion were scored 12, 5, 3, and 3 times respectively. In 4 patients, more than 1 intraoperative complication occurred. Following the 99 LCA-procedures, in the postoperative period up to 30 days, a total of 23 complications occurred in 20 patients. Two major complications (CCS: 3a) were scored. In this case, a subphrenic infected hematoma was drained under local anesthesia and using ultrasound guidance. Also, a pleural effusion needed to be drained under local anesthesia in the same patient. The remaining complications were categorized as minor, scoring CCS: 1 in 15 patients and CCS: 2 in 6 patients. The minor complications were: band-aid allergy (n=1), hematoma (n=2), anemia (n=1), cardiac chest pain (n=1), dyspnea (n=2), fever of unknown cause (n=5), complicated urinary tract infection (n=5), neuropathy upper leg (n=1), hypotonic abdominal muscle (n=2), reversible sensibility loss of the arm (n=1), and embolism of an ocular artery (n=1). In two patients more than one postoperative complication was recorded. In those two patients, the complication

that scored the highest Clavien score was used for statistical analysis. Table 2 shows how the IO and PO complications are divided over the grade of anatomical complexity for both nephrometry indices. Both indices show that an increase of anatomical complexity is related to an increased risk for IO complications. Table 2. The number and percentages of intraoperative (IOC) and postoperative (POC) complications for each grade of anatomical complexity of the PADUA and RENAL nephrometry indices. Four patients were excluded for whom intraoperative complications were not scored (except for conversion). POC and IOC are dichotomously scored for each procedure (yes vs. no).

Tumor anatomical complexity

PADUA

RENAL

Tumor

IOC

POC

Tumor

IOC

POC

Low

6/42 14.3%

8/42 19.1%

6/48 12.5%

7/48 14.6%

Moderate

6/38 18.8%

6/38 15.8%

12/48 25.0%

High

7/19 36.8%

6/19 31.6%

1/3 33.3%

Statistical results Table 3 shows the results of the univariate analysis. For the risk of IO complications, there was a significant correlation (p<0.05) with the RENAL domains “R-size”, “N-nearness to the collecting system”, and “RENAL score”. The correlation with the domain “diameter” of the PADUA-index was also significant. The additional studied parameters studied tumor diameter 2 3 (mm), surface (mm ) and volume (mm ) and were found significantly correlated with IOC as well. For the risk of PO complications, there was a significant correlation found for the RENAL domain ”N-nearness to the collecting system”. Other RENAL domains, such as score and grade, and the PADUA domains, also score and grade, showed no significant relation with PO complications.

The additionally studied parameters (PADUA medial location, PADUA upper pole location, RENAL upper pole location) were not correlated with a significant risk for IO complications nor for PO complications. At multivariate analysis with IO complication as the independent variable, only tumor volume was a determining factor (p=0.005, predictive value of the model R=0.286, R∧2=0.082). That is, tumor diameter (in mm), categorized diameters (PADUA and RENAL), and surface were outweighed by tumor volume. However, when we used tumor diameter alone, in agreement with clinical practice, the predictive value of the model was R=0.271 (R∧2=0.074, p=0.008). For PO complications, no multivariate analysis was performed, since the RENAL domain “N-nearness to the collecting system” was the only significant predicting factor. The tumor diameter in millimeters, was used to establish a threshold diameter that predicted the occurrence of IO complications. The odds ratio with a 95% confidence interval for the occurrence of IOC at the cut-off diameters 30mm (≤30 or >30), 35mm (≤35 or >35), and 40mm (≤40 or >40), was 4.54 (1.479 to 13.923), 5.32 (1.763 to 16.004), and 4.80 (1.078 to 21.370), respectively. Therefore, the best discerning threshold of tumor diameter above which an increased risk for IO complications performing LCA exists, was set at 35mm.

Table 3. Pearsonâ&#x20AC;&#x2122;s correlations (r) of parameters using univariate and if appropiate multivariate analysis for intraoperative and postoperative complications of laparoscopic renal mass cryoablation. * = Significant p values <0.05; ** = # Significant p values <0.01; = Values of all diameter related parameters were obtained by using only one of these at the time of multivariate analysis; GFR-MDRD= Glomerular Filtration Rate â&#x20AC;&#x201C; Modification of Diet in Renal Disease; BMI = Body Mass Index; CCI = Charlson Comorbidity Index; a-CCI = age-adjusted Charlson Comorbidity Index; UCS = urinary collecting system. IOC Univariate

multivariate

p-value

0,054 -0,060 -0,028 0,002 0,105 -0,162 0,060

0,601 0,562 0,786 0,983 0,309 0,118 0,561

0,227 0,200 0,218

0,027 0,052 * 0,034

0,000 0,127 0,204 0,152

1,000 0,221 * 0,047 0,142

PADUA Diameter % of tumor deepening Relation UCS Longitudinal location Rim location PADUA score PADUA grade

0,227 0,158 0,140 -0,011 0,129 0,198 0,175

0,027 0,126 0,175 0,919 0,212 0,054 0,090

ADDITIONAL Tumor diameter (mm) 2 Tumor surface (mm ) 3 Tumor volume (mm ) Medial location (PADUA) Upper pole (PADUA) Upper pole RENAL

0,271 0,271 0,286 0,127 -0,120 -0,120

0,008 ** 0,008 ** 0,005 0,221 0,249 0,249

Age BMI CCI a-CCI Approach trans/retro Preoperative GFR-MDRD Solitary kidney RENAL R (size) E (exophytic / endophytic) N (nearness to the collecting system) L (lines) Hilar suffix RENAL score RENAL complexity grade

POC

p-value

0,027

univariate r

p-value

0,068 0,192 0,149 0,170 -0,075 0,035 -0,057

0,503 0,057 0,141 0,092 0,459 0,733 0,576

0,035 -0,015 0,230

0,728 0,885 * 0,022

0,102 0,044 0,165 0,185

0,315 0,663 0,103 0,066

0,035 -0,005 0,183 0,065 0,108 0,175 0,122

0,728 0,964 0,069 0,521 0,287 0,083 0,228

0,178 0,178 0,133 0,044 -0,129 -0,129

0,079 0,079 0,191 0,663 0,204 0,204

0,386

0,505

0,027

0,008 # 0,008 # 0,005

Discussion

The first objective of this retrospective study was to evaluate the utility of the RENAL and PADUA nephrometry indices for predicting the risk for IO and PO complications in a cohort of patients that underwent LCA for a small renal mass. It showed a significant correlation (p=0.041) between the RENAL score and the risk for IO complications. However, the PADUA score did not (p=0.054). Although, there was a clear increase of IO complications in relation with increased complexity grade of both indices, this was not significant. Additionally, this study showed no significant correlation between PO complications and the score and grade of both indices. However, the indices are meant to describe tumor anatomical complexity in a standardized and categorical manner, but are not primarily designed to predict the surgical 3, 4 efficacy, IO and PO complication . Some domains of the indices showed a significant correlation that allows one to predict IO and PO complications risk using univariate analysis, hence, all were not determining factors for multivariate analysis. Because factors other than the components of the RENAL and PADUA indices may influence the risk for IOC, we assessed the relation between additional parameters and the risk for IOC. First, the approach and subsequent presentation for perpendicular cryoprobe introduction of a tumor located at the renal upper pole can be demanding and, therefore, poses the risk of incorrect probe placement. However, in the present study, the parameter “upper pole location” for tumors located at the upper renal pole scoring PADUA-Longitudinal line: 1 or RENAL-Lines: 1 showed no significant correlation with the risk for IOC. Secondly, medial and hilar tumors can require longer operative dissection time due to the proximity of hilar vessels or pyelum. Earlier reports have shown that hilar tumors 21, 22 increased the risk of complications performing LCA . In the RENAL index, a hilar tumor is addressed as a suffix to the nephrometry sum and is considered as an additional anatomical complexity factor. From the domain PADUA-Rim location, the tumors were selected with a score of 2, which reflects a medial location. However, no significant correlation was found between IOC and these medial/hilar parameters. Also, since the surgical approach was transperitoneal or retroperitoneal, it showed no significant correlation for the risk of IOC. It should to be noted that in this series the number of retroperitoneal approached LCA procedures annually decreased as a result of the introduction of computed tomography guided percutaneous cryoablation. Using multivariate analysis, the tumor diameter, measured in millimeters, was a determining factor for the prediction of surgical complications of LCA. The domains “diameter” of the RENAL and PADUA indices were both significantly correlated with the risk for IOC but were not a determining factor

using multivariate analysis. A limitation of the two nephrometry indices is that they are conducted by categorically scoring anatomical parameters reflecting the event of complicated surgery. However, the reference, or cut-off, for the categorical index per parameter is arbitrary. Renal tumor diameter scores 04 cm, 4-7 cm and > 7 cm are categorized (1, 2, and 3 points, respectively) 23 and seem to follow the latest TNM-classification in the same fashion . In a head to head comparison for scoring tumor diameter, we showed that the non-categorized method has a stronger relation with the risk for IOC than the categorized method of each of the nephrometry indices. Earlier studies clarified that tumor diameter is an important metric for complications related to LCA. Lehman et al reported that tumor size appeared to be a key metric for incomplete ablations and the risk for complications following LCA (44 22 patients; 51 tumors) . All 13 complications (21 patients) were found at tumors >3 cm diameter. This present study confirms that patients with a larger tumor diameter have an increased risk for IOC. The threshold diameter of 35mm (odds ratio 5.32; 95% confidence interval) was found to be predictive for an increased risk for IOC performing LCA. The RENAL and PADUA indices are similar in their methodology and describe tumor location categorically. Despite the variations of categories between the indices, they both are used to assess the risk for complications for renal tumor surgery. However, a head to head comparison between the two indices is somewhat confusing because of differences such as the total amount of domains used, the definition of the sinus lines, and the evaluation of the anatomical relationship between the tumor and the urinary collecting system (or renal sinus). The PADUA “score” (6-14 points) is the total sum of 6 anatomical components, whereas, the RENAL “score” (4-12 points) is the total sum of 4 anatomical components. In the current study, this is reflected in the total sum of each scoring method for the same tumor, which results in the grading into different risk-groups for each complexity index. We clearly noticed a shift towards increased complexity using the PADUA-index compared to the RENAL-index. This difference may have made the comparison of the predictive outcome of both indices in this series more difficult. Another concern is that both indices score equally for tumor diameter (1-3 points); however, the weight of this domain is different for the total sum of each index. Therefore, the score or complexity grade does not reflect the contribution of each parameter. This has to be taken into account when the two indices are compared. For a comparison between the PADUA and RENAL nephrometry this study population was too limited. Before starting this study no power analysis was performed. However, the results reported may support the study groups during the design of new studies. A single staff urologist assessed the PADUA and RENAL nephrometry scores in this study. This might be considered as a limitation of the study. However, several studies reported that the RENAL nephrometry index has 25-27 good interobserver reliability . However, it should be pointed out that

these studies showed that the interobserver agreement was moderate for each of the components of the indices. It can be questioned whether all postoperative complications in this study are related to the anatomical complexity of the tumor or to general surgical procedure and patient comorbidity factors. Simhan et al reported the incidence of complications of partial nephrectomy categorized by organ 8 system . They noted that genitourinary complication rates are significantly different between the complexity groups (p<0001), whereas no clear trends corresponding to the complexity and other organ system complications were found. Using a population based sample, Joudi et al reported that the risk for complications following partial and radical nephrectomy is significantly higher 28 in patients with a CCI greater than 2 . The present study shows independently that, CCI, age, or combined in the age-adjusted CCI had no significant correlation with complications performing or following LCA. Accordingly, current principal guidelines consider LCA as a viable alternative to partial nephrectomy, especially for patients with an increased comorbidity 1, 2 and/or high-surgical risk . The data of this study show that increased comorbidity in elderly patients does not significantly affect the risk for IOC or POC at LCA. Clearly, comorbidity and age are always taken into account when counseling patients. However, this study design was not adjusted for this selection bias. It can be suggested that IOCâ&#x20AC;&#x2122;s performing LCA relates to a surgeonâ&#x20AC;&#x2122;s experience. However, at the start of this study, the surgeon was well trained in laparoscopy and had performed >50 LCA procedures in other institutions since 2004. Therefore, the complications as scored in this study seem not to be affected by the learning curve of LCA surgical skills. Currently, anatomical complexity systems are increasingly implemented in the decision-making process for patients with clinical stage T1 renal mass. However, the majority of studies reported the relationship between ACS with partial or radical nephrectomy. Canter et al concluded that the RENAL-index could be used for standardizing the reporting of solid renal masses and 29 effectively stratifying the extirpative treatment type . Today, active surveillance and ablative therapies are also recognized as viable treatment 1, 2 strategies . In addition to surgical treatment decision-making, the ACSâ&#x20AC;&#x2122;s are used to objectify the morbidity, functional and pathologic outcomes of nephron-sparing surgery. Therefore, the ACS needs to be validated for all viable treatments and needs to allow for effective comparisons. Also, a threshold needs to be established and validated for each component of the PADUA and RENAL indices. The design of this study is a major limitation because of the probability of selection bias. However, the cohort of the study is considered representative compared to other series of LCA. Furthermore, the study is relevant because

histology is attained in all cases. This enables one to interpret the impact of IOC and POC in relation to the anatomical complexity of renal cancer treated with LCA. To our knowledge, this single centre study is the first study to assess the PADUA and RENAL indices for patients that underwent LCA. In a multiinstitutional study, Okhunov et al report that the RENAL index predicts the 12 complications at LCA . They identified 77 patients with a mean tumor size of 2.6 cm diameter with available preoperative imaging. On multivariate analysis, the RENAL nephrometry score was independently associated with a higher risk of postoperative complications (Odds ratio 2.23, 95% Confidence interval 1.05-2.11, p=0.008). The authors propose a cut-off RENAL score of 8 for identifying those patients that are most likely to benefit from LCA. After a univariate analysis of the present study sample size of 99 LCA-procedures, we noticed a significant correlation between the occurrence of intraoperative complications and the RENAL nephrometry score. However, the sample size of these retrospective studies is limited and this hypothesis will have to be tested in future studies. So far, no other study has reported the PADUA nephrometry score in association with LCA. Conclusions

For the occurrence of complications after laparoscopic renal tumor cryoablation, this single centre study demonstrated that the score and gradecategory assessed by the RENAL and PADUA indices are not determining factors using multivariate analysis. However, using univariate analysis, the RENAL nephrometry score is associated with a higher risk for intraoperative complications but not for postoperative complications. The categorizing method of the nephrometry indices limits its use for the evaluation of laparoscopic cryoablation. This study showed that tumor size is a significant predictor for the risk of intraoperative complications. The non-categorized method of scoring tumor diameter showed a more significant correlation than the categorized method of the nephrometry indices. A threshold diameter of 35mm was thereby established. Further evaluation in larger cohorts of laparoscopic renal tumor cryoablation is needed in order to validate the anatomical classification systems.

References

Ljunberg B, Cowan NC, Hanbury DC, Hora M, Kuczyk MA, Merseburger AS, Patard JJ, Mulders PF, Sinescu IC. European Association of Urology Guideline Group. EAU guidelines on renal cell carcinoma: the 2010 update. Eur Urol. 2010; 58(3): 398 406.

Ficarra V, Novara G, Secco S, Macchi V, Porzionato A, De Caro R, Artibani W. Preoperative aspects and dimensions used for an anatomical (PADUA) classification of renal tumours in patients who are candidates for nephronsparing surgery. Eur Urol. 2009; 56(5): 786 - 793.

Kutikov A, Uzzo RG. The R.E.N.A.L. nephrometry score: a comprehensive standardized system for quantitating renal tumor size, location and depth. J Urol. 2009; 182(3): 844 â&#x20AC;&#x201C; 853.

Pierorazio PM, Patel HD, Feng T, Yohannan J, Hyams ES, Allaf ME. Roboticassisted versus traditional laparoscopic partial nephrectomy: comparison of outcomes and evaluation of learning curve. Urology. 2011; 78:813-819.

Aboumarzouk OM, Stein RJ, Eyraud R, Haber GP, Chlosta PL, Somani BK, Kaouk JH. Robotic versus laparoscopic partial nephrectomy: a systematic review and meta-analysis. Eur Urol. 2012; 62:1023-1033.

Simhan J, Smaldone MC, Tsai KJ, Canter DJ, Li T, Kutikov A, Viterbo R, Chen DYT, Greenberg RE, Uzzo RG. Objective measures of renal mass anatomic complexity predict rates of major complications following partial nephrectomy. Eur Urol. 2011; 60(4): 724 - 730.

Mottrie A, Schatterman P, De Wil P, De Troyer B, Novarra G, Ficarra V. Validation of the preoperative aspects and dimensions for an anatomical (PADUA) score in a robot-assisted partial nephrectomy series. World J Urol. 2011; DOI: 10.1007/s00345-010-0639-y.

Hew MN, Baseskioglu B, Barwari K, Axwijk PH, Can C, Horenblas S, Bex A, de la Rosette JJMCH, Laguna Pes MP. Critical appraisal of the PADUA classification and assessment of the R.E.N.A.L. nephrometry score in patients undergoing partial nephrectomy. J Urol. 2011; 186(1): 42 - 46.

Reyes J, Canter D, Putnam S, Simhan J, Smaldone MC, Kutikov A, Viterbo R, Chen DY, Uzzo RG. Thermal ablation of the small renal mass: case selection using the R.E.N.A.L-nephrometry score. Urol Oncol. 2012; Doi: 10.1016/j.urolonc.2011.09.006.

Okhunov Z, Shapiro EY, Moreira DM, Lipsky MJ, Hillelsohn J, Badani K, Landman J, Kavoussi LR. R.E.N.A.L. nephrometry score accurately predicts complications following laparoscopic renal cryoablation. J Urol. 2012; 188(5): 1796 - 1800.

Lian H, Guo H, Zhang G, Yang R, Gan W, Li X, Ji C, Liu J. Single-center comparison of complications in laparoscopic and percutaneous radiofrequency ablation with ultrasound guidance for renal tumors. Urology. 2012; 80: 119 â&#x20AC;&#x201C; 124.

Sisul DM, Liss MA, Palazzi KL, Brilles K, Mehrazin R, Gold RE, Masterson JH, Mirheydar HS, Jabaji R, Stroup SP, Lâ&#x20AC;&#x2122;Esperance JO, Wake RW, Rivera-Sanfeliz G, Derweesh IH. RENAL nephrometry score is associated with complications after renal cryoablation: a multicenter analysis. Urology. 2013; 81(4): 775-880. Doi: 10.1016/j.urology.2012.11.037.

Schmit GD, Thompson RH, Kurup AN, Weisbrod AJ, Boorjian SA, Carter RE, Geske JR, Callstrom MR, Atwell TD. Usefulness of R.E.N.A.L. nephrometry scoring system for predicting outcomes and complications of percutaneous ablation of 751 tumors. J Urol. 2013; 189(1): 30-35. Doi: 10.1016/j.uro.2012.08.180.

Charlson ME, Pompei P, Ales KL, Mackenzie CR. A new method of classifying prognostic comorbidity in longitudinal studies: development and validation. J Chronic Dis. 1987; 40(5): 373 - 383.

Charlson ME, Szatrowski TP, Peterson J, Gold J. Validation of a combined comorbidity index. J Clin Epidemiol. 1994; 47(11): 1245 - 1251.

Dindo D, Demartines N, Clavien PA. Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg. 2004; 240(2): 205 - 213.

Martin RCG, Brennan MF, Jaques DP. Quality of complication reporting in the surgical literature. Ann Surg. 2002; 235(6): 803 - 813.

Goldberg SN, Grassi CJ, Cardella JF, Charboneau JW, Dodd GD 3 , Dupuy DE, Gervais D, Gillams AR, Kane RA, Lee FT Jr, Livraghi T, McGahan J, Phillips DA, Rhim H, Silverman SG; Society of Interventional Radiology Technology Assessment Committee; International Working Group on Image-Guided Tumor Ablation. Imageguided tumor ablation: standardization of terminology and reporting criteria. Radiology 2005; 235: 728-739.

Hruby G, Reisiger K, Venkatesh R, Yan Y, Landman J. Comparison of laparoscopic partial nephrectomy and laparoscopic cryoablation for renal hilar tumors. Urology 2006; 67(1): 50-54.

Hruby G, Edelstein A, Karpf J, Durak E, Phillips C, Lehman D, Landman J. Risk factors associated with renal parenchymal fracture during laparoscopic cryoablation. BJU Int 2008; 102: 723-726.

Sobin L, Gospodarowicz M, Wittekind C. International Union against Cancer th (UICC). TNM classification of malignant tumors, 7 ed. Chichester, WileyBlackwell, 2010.

Lehman DS, Hruby GW, Phillips CK, Mckiernan JM, Benson MC, Landman J. Laparoscopic renal cryoablation: efficacy and complications for larger renal masses. J Endourol. 2008; 22(6): 1123 - 1128.

Montag S, Waingankar N, Sadek MA, Reis-Bahrami S, Kavoussi LR, Vira MA. Reproducibility and fidelity of the R.E.N.A.L. nephrometry score. J Endourol. 2011; 25(12): 1925 â&#x20AC;&#x201C; 1928.

Weight CJ, Atwell TD, Fazzio RT, Kim SP, Kenny M, Lohse CM, Boorjian SA, Leibovich BC, Thompson RH. A multidisciplinary evaluation of inter-reviewer agreement of the nephrometry score and the prediction of long-term outcomes. J Urol. 2011; 186(4): 1223 - 1228.

Kolla SB, Spiess PE, Sexton WJ. Interobserver reliability of the RENAL nephrometry scoring system. Urology. 2011; 78(3): 592 - 594.

Joudi FN, Allareddy V, Kane CJ, Konety BR. Analysis of complications following partial and total nephrectomy for renal cancer in a population based sample. J Urol. 2007; 177(5): 1709 - 1714.

Canter D, Kutikov A, Manley B, Egleston B, Simhan J, Smaldone M, Teper E, Viterbo R, Chen DYT, Greenberg RE, Uzzo RG. Utility of the R.E.N.A.L. nephrometry scoring system in objectifying treatment decision-making of the enhancing renal mass. Urology. 2011; 78(5): 1089 - 1094.

CHAPTER 6 Interpretations of alternative imaging methods for the post-cryosurgical renal mass: Contrast-pulse Sequence Ultrasonography

Parts of this chapter are published in:

Wink MH, Lagerveld BW, Laguna MP, de la Rosette JJ, Wijkstra H. Cryotherapy for renal-cell cancer: diagnosis, treatment, and contrastenhanced ultrasonography for follow-up. J Endourol. 2006; 20(7): 456 - 458: discussion 458 - 459. Wink MH, Laguna MP, Lagerveld BW, de la Rosette JJMCH, Wijkstra H. Contrast-enhanced ultrasonography in the follow-up of cryoablation or renal tumours: a feasibility study. BJU Int. 2007; 99(6): 1371 - 1375.

Abstract

Cryotherapy is a curative treatment option for patients with small (<4cm) renal cell cancers. For the follow-up of ablated lesions, imaging is the current standard, but the best imaging tool has yet not been determined. The method selected should be able to determine the presence of viable tissue in the area and measure the size of the lesion. The aim of this chapter is to evaluate the characteristics and perfusion patterns of contrast-enhanced ultrasonography using contrast-pulse sequence imaging (CPS) in renal masses treated with cryoablation (CA). A Siemens Acuson Sequoia device with contrast pulse sequence imaging and Sonovue (Bracco) as a contrast agent was used. The perfusion characteristics in the lesions were described and scored. The first experiences with CPS-imaging for the follow-up of renal mass CA show that this technique can be used to characterize local perfusion defects at various times after CA.

Introduction

Nephron-sparing procedures for the management of small renal tumors have become increasingly accepted during the last decade, including partial 1, 2 nephrectomy, and thermal ablation procedures . Currently, the most widely used energy modalities for thermal ablative techniques are radiofrequency (RF) ablation and cryoablation (CA). They can be performed percutaneously assisted by image-guidance or under direct visualization at laparoscopic (LCA) or open surgery. Image-guided percutaneous thermal ablation techniques have potential advantages over surgical ablation, including a decreased convalescence with reduced morbidity and appropriate oncological efficacy. However, the availability of large-cohort long-term results is limited. For the most part, the patients who can benefit from thermal ablation procedures are those who are poor surgical candidates because of compromised renal function and/or comorbid disease. Because the tumor remains in situ after ablative treatment of a proven malignant renal mass, the efficacy can be evaluated only by means of imaging or biopsies. Histological evaluation of the ablated tissue at follow-up 3, 4 would be an option for assessing the efficacy of the treatment . However, this method is not routinely used because of its invasive character, sampling error, failure to detect small recurrences, risk for complications when carried out regularly, and the non-confirmed histological accuracy. Vascular damage and consequently ischemic injury is significant in CA. The current mainstay of follow up is the assessment of vascular flow in and around the ablated area. Therefore, the recommendation for follow up of CA 5 for small renal tumors is based on the imaging of blood flow . The imaging method selected should be able to evaluate the presence or absence of perfusion in the ablated area and measure the size of the lesion. Routinely, contrast-enhanced Computed Tomography (CT) or Magnetic Resonance 6 (MR) imaging is used . However, allergic reactions and renal toxicity are the disadvantages of the contrast-agent used. Therefore, complementary adjustments need to be arranged in order to avoid these risks. In practice, according current guidelines, there is no consensus on which set of diagnostic tools, time and frequency of follow up of the cryoablated renal 1, 2 mass is recommended . The Society of Interventional Radiology Technology Assessment Committee and the International Working Group on Image-Guided Tumor Ablation published a framework for standardizing the 5 method and reporting of follow up findings and complications . Alternative imaging methods can focus either on the vascular or the molecular changes in the cryoablated zone. Vascular patterns can be

recognized by contrast-enhanced ultrasonography (CEUS) in kidneys . CEUS depicts perfusion in a real-time setting in which all phases of enhancement can be evaluated. Various CEUS techniques have been developed during the last decades using the linear and the non-linear 8 reflections of the microbubble contrast agent in an ultrasound wave . The technique used offers a highly sensitive and selective depiction of the contrast agent. Because the microbubbles remain inside the vasculature, selective perfusion imaging is possible. This method has the advantage of easy access and low costs. Furthermore, the contrast-agent used has a low risk for adverse events. However, the limitation of this modality at follow up is that it is only used for the assessment of the local ablation status. In this chapter CEUS-imaging characteristics and perfusion patterns of the contrast-agent in renal mass before and after LCA are evaluated and discussed. The feasibility study of Wink et al is used to describe these 9 phenomena in more detail . Materials and methods

Patient preparation for CEUS Patients are prepared with intravenous access before the investigation. Because of small number of cardiac events after the use of Sonovue (Bracco, Milan, Italy) in patients with serious cardiac morbidity, patients with unstable cardiac conditions cannot be investigated with this microbubble agent. Otherwise, adverse events are rare. CEUS technique For these investigations, contrast pulse sequence (CPS) imaging was used. All harmonic imaging techniques exploit the fact that microbubbles react in a nonlinear way to an ultrasound wave, whereas tissue mainly reflects linear signals. This CEUS technique separates the signal from the tissue from that reflected by the microbubbles by applying multipulse sequences, each pulse having a different amplitude and phase. Phillips and Gardner describe the 8 technique in detail . For ultrasonography, the Acuson Sequoia (Siemens Medical, Mountain View, CA, USA) and a 4C1 transducer, equipped with CPS, was used. The two images that are created, a tissue-only and a contrast-only image, can be viewed simultaneously, which offers selective perfusion imaging making it possible to localize the abnormality on the Bmode image. Target areas are localized using the B-mode ultrasonography. A bolus of 2.4 mL of Sonovue is administered intravenously. While the target area is kept within the ultrasound range of imaging, the microbubbles arrive in the renal blood flow about 15 seconds after administration.

Cryoablation Cryoablation was carried out under transperitoneal or retroperitoneal laparoscopic assistance using 1.47 mm diameter (Seednet Gold®, Oncura, Plymouth Meeting, PA, USA) sharp and closed-tip cryoprobes and thermosensors. Depending on the tumor size, several cryoprobes were placed percutaneously and 2 freezing cycles were used. The freezing process was monitored by laparoscopic ultrasound (EUB-6500, Hitachi, Tokyo, Japan), temperature registration, and the endoscopic camera overview. Study group In the study of Wink et al, 7 patients treated with LCA for small solid renal 9 tumors were included . A researcher with experience in CPS did all the investigations. Renal mass CPS-CEUS imaging was performed in the follow up of LCA. Data were stored for off-line analysis. Two independent readers measured perfusion defects three times each. Perfusion characteristics were described using a scale from 0 to 4: • • • • •

0 - no perfusion; 1 - increased enhancement in the rim of the lesion; 2 - diffuse enhancement throughout the lesion; 3 - localized enhancement; 4 - no perfusion defect visible.

Inter- and intraobserver variability of the measurements was determined. The results of the CEUS imaging were compared to the most recent standard follow up imaging consisting of contrast-CT or MRI every three months during the first year after cryoablation and every six months thereafter. Results

CEUS of renal mass Regular grey-scale ultrasonography performs the identification and localization of the renal mass. After switching to the contrast-only image, the same lesion is without any signal. About 15 seconds after intravenously administrating 2.4 mL Sonovue, the micro bubbles arrive in the renal parenchyma. Enhancing of the normal renal tissue will appear as a regular pattern. However, using CEUS, a renal mass can be identified by its inhomogeneous enhancement pattern. Panels a, b, and c of figure 1 show examples of three types of imaging of a small renal mass in patient number

100

3. Grey-scale US (panel A) demonstrates a solid exophytic renal mass. The same lesion at contrast-CT imaging (panel B) shows only a slight enhancement. However, performing CEUS (panel C), this lesion is clearly demarcated by the enhancement of an inhomogeneous vascular pattern imbedded in normal renal parenchyma appearing as a regular vascular pattern. At grey-scale US, the perirenal fat will appear as hyper-echoic as compared to the renal parenchyma. However, using CEUS the same fat will demonstrate a lower vascular density, or at least a different pattern than the renal parenchyma and the renal mass. CEUS in the follow up of LCA 9

The patients, studied by Wink et al , were investigated at different times after ablation (29-527 days). In all but one patient the cryoablation lesion could be detected and described. Figure 1 shows all the relevant follow up imaging data from patient number 3 (panels D, E, and F). At one month after cryoablation, a new CPS investigation was performed and the cryoablation lesion could be visualized as a complete perfusion defect (perfusion score 0). Figure 1, panel F shows the complete absence of perfusion in the contrast-only image. The first CT-scan three months after cryoablation shows the same lesion with no enhancement after contrast administration. Patient number 6 was investigated with CPS imaging twice, once at 29 days and again 8 months after cryoablation; Figure 2 shows the result of both investigations. Whereas the first CPS shows no enhancement in this area (perfusion score 0), during the second investigation some reflections inside this area were visible, suggesting reperfusion in the cryoablation lesion (perfusion score 2-3). The size of the defect had not changed over time. The latest contrast-CT, 9 months after cryoablation, showed no enhancement. During the investigations, the grey-scale US images (at the right of panel A and B) demonstrated that the ablated zone could not be clearly defined. Another example of this phenomenon is seen in figure 1, panel D. Patient number 5 was examined 18 months after cryoablation. MR imaging was performed because of renal insufficiency. The latest MR imaging before CPS imaging visualized the cryoablation lesion as a retracted parenchyma and interpreted as an extrarenal fibrotic area of 31mm. During CPS imaging the lesion could not be identified and thus no obvious perfusion defect was objectified in the kidney (perfusion score 4). The other 4 patients showed comparable perfusion defects with those found in patient number 3. A summary of the findings is presented in table 1. In the patients who were investigated within 5 weeks after the ablation (patients numbers 2, 3 and 6), the perfusion defect was larger than the initial tumor as measured in the pre-treatment CT in two of the three patients (see

101

table 1). In the third patient, the lesion was 2mm smaller than the initial tumor. In patients numbers 1, 6a and 7, the measured dimension of the lesion without perfusion at CPS imaging was still larger than the dimension of the tumor before cryoablation, despite the fact that imaging was performed between 196 and 266 days after treatment. In patient number 4, the lesion was smaller than the tumor at 71 days after ablation. The lesion in patient number 5 was not visualized. The mean standard deviation (SD) of the sizes in the CPS imaging was 1.1mm. The mean (SD) difference between the two investigators was 0.7mm (1.4mm). Figure 1. Patient 3; Panels A+B: US and CT images of a small renal tumor (21 mm, panel B) before cryoablation. Panel C: CEUS image of this tumor before cryoablation. Neo-vascularization in renal tumors causes alterations in perfusion patterns that were demonstrated in this tumor using CEUS: the perfusion patterns in the area of the tumor are clearly different and less homogeneous than the perfusion pattern in the normal renal parenchyma. Panel D: Unenhanced ultrasound image one month after cryoablation. The lesion has a solid aspect and measures 17 mm. Panel E: Contrast enhanced CT scan after three months. A clear perfusion defect is visible. Panel F: The same perfusion defect as seen during CEUS investigation 36 days after surgery.

102

Figure 2. Patient 6; Panel A: CEUS investigation of a renal tumor 29 days post cryoablation. No perfusion can be seen in the ablated area (Score 0). Panel B: The same patient, the same tumor location, 8 months after surgery. Some signs of perfusion (Score 2-3) can be observed in the tumor area.

103

Table 1. The patientâ&#x20AC;&#x2122;s tumor and lesion characteristics using CT/MR and CPS-CEUS imaging after cryoablation. *) The CT/MR results closest to the CPS investigation; **) CEUS perfusion scores: 0, no perfusion; 1, increased enhancement in the rim of the lesion; 2, diffuse enhancement throughout the lesion; 3, localized enhancement; 4, no perfusion defect visible.

Patient number

Age (years)

Gender (M/F)

Tumor diameter (mm)

Post-ablation Contrast-CT/MR * Lesion diameter (mm) Time since ablation (days)

168

111

485

105

205

270

Lesion size (mm) (SD)

22.4 (0.6)

30.0 (2.1)

25.5 (1.2)

22.4 (1.0)

21.6 (0.8)

20.9 (1.4)

27.9 (0.7)

Time since ablation (days)

196

527

233

266

Investigator 1

Investigator 2

CEUS

Investigator perfusion ** score

104

Discussion

The advantage of CPS over other CEUS techniques is its greater specificity for microbubble detection. Signals in the contrast-only image correspond to perfusion. Without the gray-scale signal of the tissue interfering, enhancement patterns can be evaluated selectively. Correlation of these patterns with the histological findings is necessary to evaluate the diagnostic accuracy. The major benefits of CEUS are its easy applicability, low cost, and safe contrast medium. Furthermore, the high sensitivity to micro-vascular perfusion might enable CEUS to image the short-term dynamic process of vascular injury after cryoablation. Clearly, CEUS can detect blood flow in tissue. After cryoablation, blood follow is expected to have ceased in the cryoablative area, and therefore CEUS can be performed to demonstrate absence of blood flow in certain areas. The study of Wink et al shows that this technique is capable of doing 9 so . However, the resolution and the interpretation of the images is the key issue to the success of this method. So far, this study proved that perfusion defects could be detected and discriminated and this is compatible with the findings at standard contrast-CT imaging. Simply put, both methods are capable of detecting and showing the existence of blood flow. Two investigators measured the size of the lesions three times. The mean SD of all six measurements was 1.1mm, and the mean (SD) difference between the investigators was 0.7mm (1.4mm). To the best of our knowledge, the variation in lesion measurements after cryoablation using CT or MRI has not been investigated. However, information is available on the size of the kidney tumors measured in CT/MRI and pathological evaluations 10, and this fails to correspond in approximately 10 % of cases 11 .Furthermore, Hopper et al describe an inter- and intra-observer variation 12 for CT tumor measurements of 15% and 6%, respectively . Therefore, we conclude that the inter- and intra-observer variability of CPS imaging measurements is acceptable. Due to the design of this feasibility study, CPS and CT/MRI were not performed on the same day. Therefore, an accurate comparison between the measurements was not possible. Comparing the measurements of the CT/MRI closest to the CEUS date to CPS measurements shows differences of approximately 10%. Barwari et al have studied the concordance between the detection of enhancement assessed 13 by contrast CT/MR and CEUS . At three months follow-up assessed in 32 patients, they found a specificity and negative predictive value of 92% and 77% respectively. At one-year follow-up assessed in 21 patients, the specificity found was 90% with a negative predictive value of 100%. However, the absence of focal recurrences in this series limits the ability to

105

draw conclusions on the sensitivity and positive predictive value of CEUS used as method for follow up. Perfusion defects after renal mass cryoablation were identified and scored in 6 out of 7 patients investigated (perfusion score 0-4). In most patients (n=5), no CPS signals were detected inside the lesion (perfusion score 0), and thus according to CPS imaging, no perfusion was present. In all these five patients these findings corresponded with the contrast CT/MRI results. In patient number 6, one month after cryoablation, no perfusion was detected using either CPS or CT imaging. However, 8 months after the ablation using CPS imaging, perfusion was observed inside the lesion (perfusion score 3 and 2), whereas no enhancement was found performing contrast CT. The implication of this finding could not be that CPS imaging is a more sensitive technique for detecting reperfusion than CT. However, the other option is a false-positive CPS result. Haendl et al studied the perfusion patterns of suspected malignant renal 14 masses at CT-scan using CPS-CEUS imaging . They compared the findings of vascularisation patterns in the early phase (<30 seconds) and the late phase (60-120 seconds) to the histopathological results. All 25 proven renal cell carcinomas showed a chaotic vascularisation pattern at both phases. In the early phase, compared to the perfusion of surrounding normal renal parenchyma, 12 tumors showed hyperperfusion, 3 showed isoperfusion, and 9 showed hypoperfusion. During the late phase, 5 tumors showed hyperperfusion, 9 showed isoperfusion, and 10 showed hypoperfusion. One cystic lesion did not indicate contrast enhancement at any time. Since hypoperfusion of malignant tumors was found at the two phases, it can be questioned how the perfusion patterns of focal recurrences of ablated tumors would appear. Currently, in order to define cryoablative treatment success, the follow up contains contrast-enhanced CT or MR imaging. However, ionizing radiation, allergic reactions and the renal toxicity are the downside of the contrastagent used. Therefore, complementary adjustments need to be arranged in order to avoid these risks. Descriptive evaluations of the use of contrast enhanced CT and MR imaging for follow up of renal mass cryoablation have 15, 16 been published . A successful ablation has been described as one in which the lesion showed less than 10 Hounsefield Units of contrast medium enhancement on CT or no qualitative evidence of enhancement after 17 gadolinium contrast enhanced MR imaging . Cryoablated renal tumors usually decrease in size over time. In a study of 56 patients treated with laparoscopic cryoablation for solid renal masses, Gill et al observed a gradual involution in the size of the ablation zone by an average of 75% 3 years after cryoablation, and 38% of ablation zones were undetectable on 18 MR imaging . CT and MRI can reveal a thin rim of peripheral enhancement 3, 19, around the ablation zone in 17-30% of cases at 1 month after treatment

106

. This peri-ablational enhancement is considered benign. It is suggested that this is a benign physiologic response to thermal injury and it appears as a relatively concentric, symmetric, and uniform process with smooth inner 15 margins . It seems that during the first months after ablation this finding is of no consequence, however, if it occurred later in the follow-up, that can be interpreted as a sign of recurrence and will typically appear as irregular peripheral enhancement. This peri-ablational enhancement has not been described as such when performing CEUS. However, at three-month followup, Barwari et al reported in 2 cases signs of enhancement performing CEUS, whereas no enhancement was found at the corresponding CT-scan 13 . In one of the two cases it was described as rim enhancement. The search for the best method to assess the presence of recurrent and persistent vital tumor tissue after therapy is ongoing. However, this first investigational study using CPS imaging for follow up of cryoablation has, as have most feasibility studies, many limitations. Only a few patients, at different times after cryoablation, were studied to examine the possibility of using CPS imaging in order to characterize lesions. No longitudinal data were collected and no direct comparison with CT/MRI or histology was intended. However, the results of the present study showed that CPS imaging could be used to describe the perfusion characteristics of the cryoablation lesions. This justifies, therefore, the implementation of larger prospective studies with long term follow-up in order to determine the exact value of this technique for detecting remnant or recurrent tumors or reperfusion of the scar tissue. In one patient, 1.5 years after cryoablation, the lesion was not recognized. This might be due to the fact that the initial tumor was mainly exophytic and the cryoablation lesion was thus mostly located outside the contour of the kidney. Over 1.5 years, the lesion shrank and the contour of the kidney was reinstated. The MRI of this patient showed a slightly retracted parenchyma and extra-renal scar tissue with no enhancement. As minimal enhancement is seen in the perirenal fatty tissue during CPS imaging, this is probably the reason that the lesion had not been recognized as such. Therefore, long term follow up with CEUS needs to be studied as well. However, the risk for local recurrence at the ablation site is relatively low (8.5%) and it has still not been determined what the best imaging method is for detecting a local 21 recurrence . So far, for long term follow up, still contrast CT and MR imaging are used to assess treatment success of the renal mass. However, since most recurrence appear at the first years of follow up, it can be debated whether CT or MR is the preferred imaging mode for the long term 22, 23 follow up . A limitation of this CEUS technique for the follow up of renal mass cryoablation is that it only assesses the local situation. The risk for the development of distant or seeding metastases after clinical T1a renal cancer

107

cryoablation is not high (1%) . However, if CEUS investigation is only performed in the local region bearing the ablated tumor, it will miss possible metastases. CEUS scanning range and scanning time is limited and is dependent on microbubble dosage and stability. To assess a larger scanning area than only the ablation area, more than one dose of microbubbles is needed. The scanning area covered by CT or MR imaging is dependent on the range it is set for. Even though the scanning range is covering a larger area the dose of intravenous contrast medium does not have to be increased or repeated. Only the ionizing radiation dose will increase when the scanning range is enlarged. CEUS is not likely to replace standard CT or MR imaging but can be a valuable alternative in selected cases. For patients with a small renal mass treated with CA and with relative or imperative contra-indications for the use of contrast-agents at CT or MRI imaging, CEUS may be a reasonable alternative technique for imaging at follow-up. Conclusion

This study of the CEUS technique for the follow up of renal mass cryoablation shows that CPS imaging can be used to characterize perfusion defects at different times after cryoablation. A longitudinal prospective study comparing CEUS results to those of CT/MRI and histology are needed in order to establish the exact diagnostic value at long-term follow-up of renal mass cryoablation.

108

References

Ljunberg B, Cowan NC, Hanbury DC, Hora M, Kuczyk MA, Merseburger AS, Patard JJ, Mulders PF, Sinescu IC; European Association of Urology Guideline Group. EAU guidelines on renal cell carcinoma: the 2010 update. Eur Urol. 2010; 58(3): 398 406.

Remer EM, Weinberg EJ, Oto A, O’Malley CM, Gill IS. MR imaging of the kidneys after laparoscopic cryoablation. AJR Am J Roentgenol. 2000; 174(3): 635 - 640.

Gill IS, Remer EM, Hasan WA, Strzempkowski B, Spaliviero M, Steinberg AP, Kaouk JH, Desai MM, Novick AC. Renal cryoablation: outcome at 3 years. J Urol. 2005; 173: 1903 - 1907.

Matsumoto ED, Watumull L, Johnson DB, Ogan K, Taylor GD, Josephs S, Cadeddu JA. The radiographic evaluation of radio frequency ablated renal tumors. J Urol. 2004; 172(1): 45 - 48.

Nilsson A. Contrast-enhanced ultrasound of the kidneys. Eur Radiol. 2004; 14(suppl 8): 104 – 109.

Phillips P, Garner E. Contrast-agent detection and quantification. Eur Radiol. 2004; 14(suppl 8): 4 - 10.

Wink MH, Laguna MP, Lagerveld BW, de la Rosette JJMCH, Wijkstra H. Contrastenhanced ultrasonography in the follow-up of cryoablation or renal tumours: a feasibility study. BJU Int. 2007; 99(6): 1371 - 1375.

Hallscheidt PJ, Bock M, Riedasch G, Zuna I, Schoenberg SO, Autschbach F, Soder M, Noeldge G. Diagnostic accuracy of staging renal cell carcinomas using multidetector-row computed tomography and magnetic resonance imaging: a prospective study with histopathologic correlation. J Comput Assist Tomogr. 2004; 28(3): 333 - 339.

Yaycioglu O, Rutman MP, Balasubramaniam M, Peters KM, Gonzalez JA. Clinical and pathologic tumor size in renal cell carcinoma: difference, correlation, and analysis of the influencing factors. Urology. 2002; 60: 33 - 38.

Hopper KD, Kasales CJ, Van Slyke MA, Schwartz TA, TenHave TR, Jozefiak JA. Analysis of interobserver and intraobserver variability in CT tumor measurements. AJR Am J Roentgenol. 1996; 167: 851 – 854.

109

Barwari K, Wijkstra H, van Delden OM, de la Rosette JJ, Laguna MP. Contrastenhanced ultrasound for the evaluation of the cryolesion after laparoscopic renal cryoablation: an initial report. J Endourol. 2013; 27(4): 402-407. Doi: 10.1089/end.2012.0400.

Haendl T, Strobel D, Legal W, Frieser M, Hahn EG, Bernatik T. Renal cell cancer does not show a typical perfusion in contrast-enhances ultrasound. Ultraschall Med. 2009; 30(1): 58 - 63.

Kawamoto S, Solomon SB, Bluemke DA, Fishman EK. CT and MR imaging appearance of renal neoplasms after radiofrequency ablation and cryoablation. Semin Ultrasound CT MR. 2009; 30(2): 67 - 77.

Wile GE, Leyendecker JR, Krehbiel KA, Dyer RB, Zagoria RJ. CT and MR imaging after imaging-guided thermal ablation of renal neoplasms. Radiographics. 2007; 27(2): 325 - 339; discussion 339 - 340.

Farrell MA, Charboneau WJ, DiMarco DS, Chow GK, Zincke H, Callstrom MR, Lewis BD, Lee RA, Reading CC. Imaging-guided radiofrequency ablation of solid renal tumors. AJR Am J Roentgenol. 2003; 180(6): 1509 - 1513.

Gill IS, Remer EM, Hasan WA, Strzempkowski B, Spaliviero M, Steinberg AP, Kaouk JH, Desai MM, Novick AC. Renal cryoablation: outcome at 3 years. J Urol. 2005; 173(6): 1903 - 1907. Bolte SL, Ankem MK, Moon TD Hedican SP, Lee FT, Sadowski EA, Nakada SY. Magnetic resonance imaging findings after laparoscopic renal cryoablation. Urology. 2006; 67: 485 â&#x20AC;&#x201C; 489.

Rutherford EE, Cast JE, Breen DJ. Immediate and long-term CT appearances following radiofrequency ablation of renal tumours. Clin Radiol. 2008; 63(2): 220 230.

Aron M, Kamoi K, Remer E, Berger A, Desai M, Gill I. Laparoscopic renal cryoablation: 8-year, single surgeon outcomes. J Urol. 2010; 183: 885 - 889.

Klatte T, Mauermann J, Heinz-Peer G, et al. Perioperative, oncologic, and functional outcomes of laparoscopic renal cryoablation and open partial nephrectomy: a matched pair analysis. J Endourol. 2011; 25: 991 - 997.

Kunkle DA, Uzzo RG. Cryoablation or radiofrequency ablation of the small renal mass: a meta-analysis. Cancer. 2008; 113(10): 2671 - 2680. Doi: 10.1002/cncr.23896.

110

CHAPTER 7 Interpretations of alternative imaging methods for the post-cryosurgical renal mass: 18

F- fluorodeoxyglucose positron emission tomography in combination with low-dose computed tomography

This chapter is submitted as: Lagerveld BW, Sandkuyl R, Sivro F, van der Zee JA, Baars PC. 18 F-FDG PET-CT findings before and after laparoscopic renal cryoablation; an initial report. BJU Int 2014.

112

Abstract

Objectives The success of cryosurgical treatment of small renal mass (SRM) is defined by the absence of contrast-enhancement at computed tomography (CT). The use of a contrast agent is relatively contra-indicated in patients with renal function impairment which mandates alternative follow-up strategies. The aim of this study is to describe the characteristics of molecular imaging with positron emission tomography (PET) in combination with low-dose CT in SRM treated with cryoablation (CA). Materials and Methods In selected patients set up for SRM cryoablation, several reasons were identified as criteria for performing PET-CT before and/or after cryoablation. Between July 2007 and January 2012, 9 patients (men:8, women:1; mean age 72 years) treated with cryosurgery and in case pre-operative work-up 18 and/or follow-up with F-FDG PET-CT were retrospectively studied. Results Patients were investigated at different times before and after ablation. The histology revealed renal cell carcinoma in 7 patients and oncocytoma in 2 patients. In 6 patients a PET-CT was performed before and after cryoablation. In one patient the PET-CT was performed before cryoablation and in 2 patients after cryoablation. Before cryoablation, there was clearly 18 metabolic uptake of F-FDG in the SRM in all patients. Following 18 cryoablation, the absence of F-FDG uptakes in the SRM could clearly be noticed. However, the tracer cannot always be distinguished from focal recurrence or reactive inflammatory tissue. In one patient, asymptomatic metastatic bone lesions were noticed performing PET-CT at follow-up. Conclusions 18

This pilot study with F-FDG PET-CT for the follow up of SRM cryosurgery 18 showed that F-FDG PET-CT imaging could be used to characterize cryoablative tissue injury at different times after CA. A longitudinal 18 prospective study comparing F-FDG PET-CT imaging to CT/MRI and histology is needed in order to establish its exact value in the follow-up of SRM cryoablation.

113

Introduction

The number of new renal cancer cases in the Netherlands was estimated to be 2000 in the year 2007 and is expected to increase to 2300 new cases by 2020. This increase may be due in part to an increase in the discovery of 1 small incidental solid renal masses using cross-sectional imaging . Recommended by principal guidelines nephron-sparing procedures for the management of small renal tumors have become the standard. Recently, cryoablation has been added as a viable treatment option for patients with 2, small renal cell carcinomas (RCC) and those who are at high-surgical risk 3 . It can be performed using image-guided percutaneous approaches or under direct visualization during laparoscopic (LCA) or open surgery. Image guided percutaneous cryoablation has potential advantages over surgical resection, including a decreased convalescence with reduced morbidity and 4 appropriate oncological efficacy . Patients who can particularly benefit from thermal ablation procedures are those who are poor surgical candidates because of compromised renal function and/or comorbid disease. However, patients with impaired renal function who are candidates for ablation can be poor subjects for the typical investigational methods used at follow-up. According to principal guidelines, in practice, there is no consensus on which the set of diagnostic tools, time frame and frequency of follow-up of the 2, 3 cryoablated renal mass is recommended . Vascular damage and consequently ischemic injury is one of the mechanisms of action in 5 cryoablation . The mainstay of follow-up is the assessment of perfusion in and around the ablated area. Therefore, the current recommendation for follow-up of CA for small renal tumors is based on imaging of blood flow. The imaging method selected should be able to evaluate the presence or absence of vital tissue in the ablated area and measure the size of the lesion. Routinely, contrast-enhanced Computed Tomography (CT) or 6 Magnetic Resonance (MR) imaging is used . Intravenously administered contrast agents are used to identify contrast enhancement in the target lesion. The observation that the standard follow-up of cryoablated small renal masses in patients with declined renal function is not without risk of jeopardizing the remaining renal function led to the study of alternative methods of follow-up. The development of contrast-induced nephropathy is a significant complication of intravascular contrast medium use that is related 7, 8 to excess morbidity and mortality . The most important risk factor is preexisting renal impairment which increases the risk for contrast-induced 9 nephropathy by more than 20 times . Therefore, alternative imaging not requiring radiographic contrast medium should be considered if the alternate imaging adequately addresses the diagnostic questions. Alternative methods of imaging can focus either on the vascular or the molecular changes in the cryoablated zone. A method for studying the

114

molecular status of renal masses is the positron emission tomography (PET) in combination with low-dose CT, which assesses both renal mass anatomy 18 and metabolic activity. Fluorodeoxyglucose ( F-FDG) is the most frequently used radiopharmaceutical tracer in PET-CT imaging. However, it is not very accurate in distinguishing RCC from benign solid renal neoplasm. Relative 18 differentiated cancers such as RCC show faintly or no F-FDG uptakes that consequently result in negative PET-CT scans. As a result, it is not routinely 10 used in the initial diagnostic work up of solid renal mass . It does appear to be moderately useful in the detection of distant metastatic disease of RCC. There are nuclear tracers that better distinguish for RCC, but the availability 11 is limited by their short half-life . For example, carbonic anhydrase IX (CAIX), a transmembrane enzyme, plays a role in the tumor adaption to hypoxic conditions by regulating the pH of the intracellular and extracellular compartment. It is over-expressed in >95% of the clear cell subtype of RCC 12 18 and is rarely expressed in the other known subtypes . However, F-FDG PET-CT can possibly be a reasonable alternative to the routine mode of imaging in those patients with relative or imperative contra-indications for the 18 use of contrast-agents used in CT or MR imaging. This factor makes FFDG PET-CT of interest for the follow-up of cryoablated tumors. The 18 hypothesis of this study was that if F-FDG metabolisms could be detected in renal mass before cryoablation, it should not be detectable in the target zone after cryoablation. The purpose of this study is to describe the spectrum of pre- and post18 ablation F-FDG PET-CT findings and define their value during follow-up for renal tumors treated with cryosurgery. Materials and methods

All patients were identified with a solid renal mass suspected for malignancy. According to guidelines, patients were informed about optional treatment techniques and each typical follow-up method. All patients consented with CA. The same surgeon performed all laparoscopic or percutaneously CTguided procedures. For cryoablation, multiple 17-gauge cryoprobes (Galil Medical, Yokneam, Israel) were used. Histological biopsies were obtained before cryoablation or intraoperatively. Several reasons were identified as inclusion criteria to prefer PET-CT instead of CT or MR imaging: renal function impairment, contrast allergy, contra-indication for use of intravenous contrast medium, claustrophobia, metal implants, PET-CT already performed in a referring center, staging renal cancer, and the identification of a viable tissue metabolism in case of suspected local recurrence of renal cancer after initial ablative treatment.

115

Those patients that underwent F-FDG PET-CT before and/or after renal mass cryoablation were retrospectively studied. Patients were identified in our institutional database. The local internal review board approved this study and its submission for publication. PET-CT technique 18

F-FDG is a glucose analog with a positron-emitting radioactive isotope nd fluorine-18 substituted for the hydroxyl group at the 2 position in the glucose molecule. PET imaging of tumors with the FDG-tracer is based on the observation that the tumor cells have an enhanced rate of glycolysis compared to most normal tissue. The uptake of glucose and analog FDG into malignant cells is facilitated by the increased expression of glucose 13 transporter molecules at the tumor cell surface . After intracellular transport, FDG is phosphorylated by hexokinase to FDG-6-phosphate, does not proceed further in the metabolic pathway and remains trapped in cells. PET identifies this selective focal accumulation of positron emitting FDG in malignant tumors. The glucose metabolic status can be analyzed quantitatively as the differential uptake ratio and distribution absorption ratio known as the Standardized Uptake Value (SUV) index. 2

Patients with renal function impairment (GFR-MDRD <60 ml/min/1.73m ) 18 before LCA treatment were offered F-FDG PET-CT imaging. In case the target lesion showed metabolic activity before cryoablation, the PET-CT was repeated after LCA in order to assess therapy success. Post-cryoablation PET-CT imaging was performed within a minimum of three months after surgery. It was postulated that shortly following ablation within and around 18 the ablation zone metabolic activity was to be expected at F-FDG PET-CT. This would make it difficult to discriminate between inflammatory/reactive tissue and viable tumor tissue. According to the local protocol, the treatment success of cryoablation is determined the first time postoperative contrast CT-imaging is performed within a minimum of 2 weeks after surgery and is defined by the absence of 14 (focal) enhancement within the ablated tumor area . However, under the circumstances of case-specific indications and awareness of preoperative PET-CT findings, a postoperative contrast CT or MR-imaging was not performed in all cases. In several cases, the PET-CT was combined with abdominal ultrasonography. A contrast-CT was only performed in those cases where PET-CT and or ultrasonography showed evidence for recurrence or metastatic disease. Blood glucose level was measured prior to administrating FDG. The plasma 18 glucose level was used to correct SUV measurements. A bolus of F-FDG (range 141 - 233 MBq) was intravenously administrated 60-90 minutes prior to imaging. A PET-scan was performed from the skull base to the groin and

116

combined with a low-dose CT for attenuation correction and anatomic correlation. At preoperative PET-CT imaging, the metabolic uptake (SUV) was measured in the centre of the tumor and the liver for reference. At postoperative PET-CT, the metabolic uptake was calculated in the tumor center, the peripheral rim of the tumor, suspect lesions, and the liver for reference. Results

General results Between July 2007 and January 2012, a total of nine patients (mean age of 72 years) with small renal tumors treated with cryosurgery were retrospectively studied in case pre-operative work up and/or follow-up 18 consisted of an F-FDG PET-CT. Patient characteristics and general surgical information of this study are demonstrated in table 1. The median follow-up was 38 months (standard deviation Âą12 months). Out of the 9 primary tumors that were treated with cryoablation, 7 were histological diagnosed as malignant and 2 as benign. Indications for PET-CT before and or after LCA were: renal function impairment (n=6), suspicion for local recurrence following LCA (n=1), contraindication for intravenous contrast medium use (n=1), and in one patient a PET-CT was performed at the referring centre for staging purposes. Table 1. Patient characteristics and demographics. CA=cryoablation; RCC=renal cell carcinoma; GFR-MDRD=Glomerular Filtration Rate â&#x20AC;&#x201C; Modification of Diet in Renal Disease. Study number 1 2 3 4 5 6 7 8 9

Age at time CA (years) 76 80 72 68 64 79 64 70 75

Gender

Tumor size (mm)

M M M M F

45 44 19 36 30

M M M M

40 43 38 48

117

Histology clear cell RCC clear cell RCC clear cell RCC clear cell RCC chromophobe RCC clear cell RCC clear cell RCC oncocytoma oncocytoma

GFRMDRD

Solitary kidney

31 32 55 61 52

no yes yes no yes

65 33 99 21

no no no no

During follow-up of the 7 patients that were diagnosed with RCC, one patient was diagnosed with metastatic disease, one patient was diagnosed with a focal recurrence of RCC in the ablated area, and in one case a metachronous renal tumor was diagnosed in the previously treated solitary kidney. Retrospectively, the SUV scores were calculated in the liver, the centre of the renal tumor and the surrounding rim of the ablation zone. Before 18 cryoablation, there was clearly metabolic uptake of F-FDG in the renal tumor in all patients (n=8). In this series we performed a PET-CT before and after ablative surgery in 6 cases [table 2]. It was clearly noticed that 18 metabolic uptake of F-FDG can be detected in solid renal lesions and, therefore, this could be used as a reference to discriminate for metabolic activity after renal mass cryoablation. In all those cases we found a significant decrease of metabolic FDG-uptake in the centre of the cryoablated tumors. However, in the surrounding rim of the ablation area in 18 some patients, clear absence of the F-FDG tracer could not always be distinguished from focal recurrence or reactive inflammatory tissue. Table 2. 18

In 6 patients F-FDG PET-CT imaging was performed before and after cryoablation. SUV scores before CA (a), at first time follow up (b), and consecutive (c, d, e) follow up. Patient number 2a 2b 2c 3a 3b 4a 4b 4c 4d 4e 5a 5b 5c 7a 7b 7c 9a 9b

SUV renal mass centre 1.8 0.8 0.7 3.2 3.2 3.9 1.5 0.6 0.6 0.7 1.95 1.4 1.4 2.5 1.5 1.5 3.1 1.0-1.5

SUV renal mass periphery 1.7 3.1 3.1 2.8 2.6 3.3 2.2 0.9 0.9 0.7 1.95 2.9 2.1 2.6 2.7 2.9 3.5 4.5-4.7

118

SUV liver 2.6 2.4 2.6 2.8 3.2 3.2 3.0 2.3 2.7 3.2 2.95 3.2 3.0 2.8 3.2 3.4 3.7 3.0

Case reports The first patient (number 1) was diagnosed with a 45mm diameter renal mass in the left kidney at contrast-CT and referred for LCA. Histology revealed clear cell RCC. Because of the patientâ&#x20AC;&#x2122;s declined renal function, PET-CT was offered as the method of imaging at initial follow-up. There was no baseline preoperative PET-CT scan performed for comparison. The follow-up PET-CT at 10 months showed no metabolic activity in the centre of the ablation zone. However, there was some uptake of FDG around the border of the initial tumor. This could be due to reactive, inflammation or rest tumor activity. This test was not conclusive in view of the absence of a baseline PET-CT. The patient did have consecutive PET-CT imaging at 2 and 3 years follow-up. Both scans reported no evidence of tumor activity in the ablation zone. The activity as noticed at the first post-cryo PET-CT was no longer visible. The low-dose CT scans showed a decrease in size of the ablated tumor. In this case, there were no contrast CT or MR scan performed as control study. At 67 months follow-up, no recurrence or the novo renal tumor was found using ultrasonography. Patient number 2 was diagnosed with a 44mm diameter RCC of a solitary left kidney. Medical history revealed a right radical nephrectomy for RCC. The renal function impairment was the reason for a PET-CT scan prior to ablative surgery. Hypo-intense (compared to the liver) FDG-uptake (SUV 1.8) was noticed in the renal mass centre [figure 1-A]. Contrast-CT at one month after cryoablation showed a complete ablation of the tumor with some inflammatory reactive tissue in the perirenal fat. Five months postcryoablation, the PET-CT image showed no metabolic activity in the ablation zone (SUV 0.8). However, some activity was noticed in the perirenal fat covering the outer border of the ablated tumor (SUV 3.1). This activity could be due to reaction or inflammation [figure 1-B]. A PET-CT scan, performed at 13 months follow-up, again showed no metabolic activity in the ablation zone (SUV 0.7) and again a slight FDG-uptake (SUV 3.1) was noticed in the perirenal fat covering the outer border of the ablated tumor. At 40 months follow-up, there was no sign of recurrence reported from the referring urological centre. The medical history of patient number 3 revealed a right radical nephrectomy for RCC and transurethral resection of single metachronous metastases in the bladder. Follow up contrast-CT scan showed an enhancing solid lesion of 19mm diameter in the left solitary kidney. Precryoablation PET-CT showed iso-metabolic (compared to surrounding renal parenchyma) FDG-uptake in the lesion (SUV 3.2). During follow-up PET-CT, three months after LCA, there still was irregular FDG-uptake (SUV 3.2) noticed in the ablated tumor. However, the contrast-CT at 6 months after LCA showed a complete ablated tumor without contrast enhancement. No postoperative histological biopsy was performed in order to assess

119

recurrence. At one year follow-up the patient was diagnosed with metastatic prostate cancer without evidence for vital renal RCC at contrast CT. Figure 1. 18

Patient 2: An example of F-FDG-distribution in a renal mass before (A) and after 5 months cryoablation (B) at PET-CT. The light active iso-intense uptake in the tumor in the left kidney before cryoablation (green arrow panel A) has changed to a hypo-metabolic area after ablation (green arrow panel B). The activity around the ablated tumor in the perirenal fat is possibly due to inflammatory tissue reaction. A contrast CT-scan as control showed no enhancement of the ablation zone after treatment.

Patient number 4 was diagnosed with a 36mm diameter RCC of the left kidney. Because there was suspicion for macroglobulinemia (M. Waldestrรถm), there was a contraindication for intravenous contrast medium usage. The pre-cryoablation PET-CT showed a moderate intense uptake of FDG (SUV 3.9) in the renal tumor [figure 2-A]. After LCA, the suspicion of macroglobulinemia was not proven and a control contrast CT was performed which showed a complete ablation of the tumor. Five months following LCA, the PET-CT revealed no FDG-uptake (SUV 1.5) in the ablated tumor as shown in figure 2-B. Also at 15 and 24 months follow-up the PET-CT scans showed no sign of recurrence [figure 2-C and D]. However, at 34 months follow-up, the PET-CT revealed FDG-uptake at the dorsolateral side of the left kidney in the perirenal fat that covered the ablation area [figure 2-E]. This uptake was considered suspect for RCC recurrence. And contrast-CT confirmed the diagnosis. However, the patient refused biopsy or retreatment. Contrast-CT at 40 months follow-up revealed the same enhancing tissue in the perirenal fat. However, no other suspected lesions and no enhancement

120

of the ablated zone was found. The suspected lesion did not increase in size. A repeat contrast-CT at 47 months follow-up this lesion has almost disappeared. Patient number 5 had a right radical nephrectomy for RCC in the past. Now the patient was diagnosed with a suspected malignancy in the left solitary kidney. The pre-cryoablation PET-CT revealed a tumor with low FDG-uptake (SUV 1.95). Following cryoablation at 7 months, there was no accumulation of FDG in the ablated tumor (SUV 1.4). The border of the ablation site showed some metabolic activity that was not suspected to be focal recurrence. The consecutive PET-CT at 16 months follow-up again revealed no metabolic activity in the ablated tumor (SUV 1.4). Additionally, a renal ultrasound was performed. It showed a decrease in size of the ablated tumor (from 30 to 24 mm). However, there was suspicion of a new solid lesion superior from the ablation zone. At control contrast-CT, there was no enhancement of the ablated tumor. However, near to the abated tumor a new contrast-enhanced solid mass (21mm) was noticed [figure 3-A], conform the findings at ultrasound. It was considered a metachronous metastases and not evidence for focal recurrence after LCA. This tumor was almost completely endophytic and initially not recognized at follow-up PETCT. The patient underwent a successful CT-guided percutaneous cryoablation for this second lesion. The histology confirmed RCC. There was no PET-CT performed prior to LCA in patient number 6. A 40mm RCC was treated with LCA. The first follow-up CT at three weeks after surgery showed a complete ablation of the tumor at contrast-CT. However, the consecutive scan at 7 months showed new focal enhancement in the dorsal-central rim of the initially cryoablated tumor suspect for recurrence [figure 4-X]. At PET-CT, the centre part of the tumor showed no FDG-uptake (SUV 1.2-1.5) compared to the liver as reference (SUV 2.8). However, the dorsal-central area of the initial ablated tumor showed metabolic activity (SUV 2.7-2.8). This FDG-uptake could be related to an inflammatory reaction, focal recurrence or the accumulation of FDG in the collecting system [figure 4-Y]. The finding of the contrast-CT was decisive for offering consecutive treatment. This focal recurrence was successfully treated with CT-guided percutaneous cryoablation. Histological biopsy revealed recurrence of vital RCC tissue.

121

Figure 2. 18

Consecutive F-FDG PET-CT imaging of a renal cancer before and after cryoablation. Green arrows indicate the tumor or the ablation site in the left kidney of patient number 4. Before surgery (A) there was metabolic activity noticed in the tumor. The low dose CT-scans showed consecutive decrease of the tumor volume and no metabolic activity was measured in the tumor 18 using F-FDG PET at 5 months (B), 15 months (C), and 24 months (D) follow-up after LCA. However, at 34 months new developed metabolic activity is noticed in the perirenal fat that covered the ablated tumor (green arrow panel E).

122

Figure 3. Imaging at 15 months follow-up of patient 5. Panel A: Intravenous contrastenhanced CT-scan in patient number 5 showing a non-enhancing zone (white arrow) at the side of the renal lesion treated with LCA. A second solid lesion with clear contrast-enhancement (red arrow) was noticed. Panel B: At 18 F-FDG PET-CT the ablation zone showed no metabolic FDG-uptake (white arrow). The blue arrow indicates the region of the metachronous RCC with FDG-uptake (SUV 3.1).

Patient 7 was offered a PET-CT because of poor renal function before treatment of a suspected renal lesion (43mm) on the upper pole of the right kidney. At the centre of this tumor a low FDG-uptake (SUV 2.5) was found at the pre-cryoablation PET-CT [figure 5-A]. The patient underwent LCA via a retroperitoneal approach. Histology revealed a clear cell carcinoma. At contrast-CT, performed 2 weeks following surgery, the tumor showed consistent enhancement of the superior-anterior part that was considered as an incomplete ablation with persistent vital tumor as a result. Consequently the LCA was repeated by intraperitoneal approach. The PET-CT at six months post-LCA showed the now two times cryoablated renal mass without clues for vital tumor tissue (SUV at the tumor centre was 1.5) [figure 5-B]. However, now there were two FDG avid bone localizations that were strongly suspected to be osteolytic metastases as is shown in figure 6. The contrast-CT as control and for further dissemination study showed no particular enhancement of the ablated tumor. The bone metastases were treated with external beam radiation. During follow-up pulmonary metastases were diagnosed and systemic treatment was administrated.

123

Figure 4. At 8 months following LCA for patient 6, a focal contrast-enhanced area at the posterior side within the ablation zone was noticed on contrast CT-scan (X, blue arrow). Although not as sharply distinctive as the contrast CT-scan 18 we noticed metabolic activation in the same posterior rim area at F-FDG PET-CT-scan (Y, blue arrow).

124

Figure 5. Patient 7; Before treatment (A) the renal tumor in the right kidney shows a 18 low FDG-uptake at F-FDG PET-CT. The PET-CT scan after treatment (B) shows the same tumor without signs of the existence of vital tissue. Green arrows indicate the tumor before (A) and after (B) cryoablation.

125

Figure 6. 18

Panel of F-FDG PET-CT imaging at 6 months follow up of patient 7. There are two FDG avid bone localizations strongly suspected of being metastases (A, B). The low dose CT-scan without contrast shows cystic lesions in the right iliac bone (A, green circle) and in the 5th left costal rib (C, green circle). Both cystic locations correlated with the â&#x20AC;&#x153;hot spotâ&#x20AC;? FDG-uptake as shown in B and D (green arrows).

126

Patient number 8 was referred for laparoscopic cryoablative treatment of an incidentally discovered solid renal mass in the left kidney. The referring 18 centre performed an F-FDG PET-CT for dissemination purposes. The PET-CT showed a metabolic active (SUV 5.7) tumor at the upper pole of the left kidney. The patient underwent LCA for the mass in the upper pole. The histology obtained by intraoperative biopsies revealed oncocytoma. Because of the benign nature of this renal mass, no postoperative PET-CT was performed. The post-operative contrast-CT showed a complete ablation of the renal mass. Patient number 9 was diagnosed with a solid renal mass of 48 mm diameter in the left kidney. This patient was offered PET-CT prior to LCA because of poor renal function. In case of proven malignancy, we could offer follow-up using PET-CT. At pre-cryoablation PET-CT, there was FDG uptake (SUV 3.1) in the tumor located in the left kidney. Even though the intraoperative biopsies revealed a histological diagnosis of oncocytoma, the patient was offered a PET-CT to define treatment success. After cryoablation, in comparison with the PET-CT before cryoablation, there was a changed image of the tumor in the left kidney with a recognizable pattern of isometabolic to no metabolic activity (SUV 1.0-1.5). There was no contrast CT performed as control. Discussion

In 2005, the first report of using fused PET-CT imaging for the follow-up of a 15 renal mass treated with cryoablation was published . This case report showed that a decrease of molecular activity was found using PET-CT in the renal lesion after cryosurgery compared to the PET-CT prior surgery. The purpose of the present study was to determine whether tracer uptakes found 18 at baseline and no-tracer uptake at follow-up in F-FDG PET/CT could be used to assess the response to cryoablation for RCC. The patients reported in this study were in 8 out of the 9 cases referred for laparoscopic cryoablation after assessment of the diagnosis of a solid renal mass suspected to be malignant. In this series, we performed a PET-CT before ablative surgery in 7 cases. We noticed that solid tumors could be 18 18 detected as metabolic active lesions at F-FDG PET-CT imaging. The FFDG PET-CT is not routinely used in the initial diagnostic work up of solid 10 renal masses . Furthermore, it does not discriminate between benign and malignant renal growth. Hence, the present study showed metabolic activity in oncocytoma and RCC. The number of cases was too limited in order to be able to assess a possible difference in the SUV for benign and malignant renal masses. However, the present study showed that metabolic uptake of 18 F-FDG can be detected in solid renal lesions. Therefore, this could be used

127

as a reference to discriminate for metabolic activity after renal mass cryoablation. In all those cases, we found a significant decrease of metabolic FDG-uptake in the centre of the cryoablated tumors. Therefore, we postulate 18 that F-FDG PET-CT imaging can be used as an alternative-imaging method to contrast-CT/MR imaging for the assessment of treatment success of LCA. In all cases, there was a relative or imperative reason for the use of an alternative imaging technique other than intravenous contrast medium imaging to assess the renal tumor viability before and after cryoablation. 18 One of the major benefits of F-FDG PET-CT is that there are no contraindications for patients with decreased renal function. The patientâ&#x20AC;&#x2122;s height, body weight and medical history of diabetes mellitus are essential for 16 the interpretation of SUV measurements . FDG-PET and CT are imaging modalities that have been validated in routine clinical practice. Integrated PET/CT combines PET and CT in a single imaging device and allows morphological and functional imaging to be carried out in a single procedure. This integration has advantages for the assessment of the vitality of a small renal mass (SRM) before and after cryoablation. Vascular damage and 5 consequently ischemic injury are a significant mechanism of action in CA . The current mainstay of follow-up is the assessment of vascular flow in and around the ablated area. Therefore, the recommendations for follow-up of 14 CA for a small renal tumor are based on imaging of blood flow . However, identifying the absence of metabolic activity using PET can also assess the viability of tissue. Another morphologic feature is that cryoablated renal tumors tend to decrease in size over time. At three years follow-up of patients treated with LCA, Gill et al observed a gradual involution of the ablation zone diameter by an average of 75%. Furthermore, 38% of the 17 ablation zones were undetectable on MR imaging after 3 years follow-up . Even though low-dose non-contrast enhanced CT is not intended for radiological diagnosis, it makes it possible to see a decrease in size of most ablated tumors over time. One of the limitations of this study was the resolution achieved by PET-CT. It does not allow for accurate interpretation of metabolic activity at the rim borders of the ablation zone. The measurement of SUV in an area selected smaller than 1 cm diameter becomes inaccurate and, therefore, selection of the region of interest is of the utmost importance. However, at a short distance, a focal recurrence can be surrounded by FDG concentrating morphologic structures as the normal renal parenchyma and/or the urinary collecting system. Artefacts as a result of high FDG concentration in the urine can be minimized by adequate pre-hydration. In patient number 6, FDG-accumulation was found in the rim of the ablation zone at the dorsalcentral border. Initially this was not recognized as a suspect for focal recurrence. Contrast-enhanced CT showed focal enhancement and, therefore, the patient underwent consecutive biopsy and cryosurgery.

128

Histology confirmed the presence of viable RCC tissue. The post-LCA PETCT of patient number 3 revealed irregular FDG accumulations in the ablation zone. However, contrast-CT three months later showed no evidence of enhancement in the ablated tumor. In this case, the limited tumor dimension (19mm diameter) possibly hampered the interpretation of the PET-CT findings. After LCA, in four more patients, metabolic activity was noticed at the rim of 18 the ablation assessed by F-FDG PET-CT. In patient number 4, this was found in the peritumoral fat by the third consecutive PET-CT almost three years post-LCA, whereas previous scans showed no peripheral metabolic activity. Therefore, this must be considered a suspect for metastases. However, this is not confirmed by histology. In patient number 1, 2 and 5, the metabolic activity of the peripheral rim was encircling the metabolic inactive ablation zone as noticed in the first PET-CT following cryoablation. This seems comparable to the literature where findings by CT and MRI of rim enhancement were noticed around the ablation zone in 17-30% of cases at 18-20 1 month after treatment . This peri-ablational enhancement is considered benign. It is suggested that this is a physiologic response to thermal injury, and it appears as a relatively concentric, symmetric, and uniform process 6 with smooth inner margins . It seems that during the first months after ablation this finding is of no consequence; however, if it were to occur later at follow-up it could be interpreted as a sign of recurrence and will typically appear as irregular peripheral enhancement. The PET-CT findings of patient number 4 can possibly be interpreted as such. In this series, the PET-CT cross-sectional area stretched between the skull base and the groin. Therefore, using this technique, more than the local ablation site could be studied. The cross-sectional area of the thorax and abdomen, in combination with integrated method of imaging, enables us to locate metabolic active pulmonary, lymph node, or bone metastases. In patient number 7, two asymptomatic bone metastases were found this way. However, these lesions could also have been noticed during a cross sectional contrast-CT of this area. The search for the best method to assess treatment success after cryosurgery for renal tumors remains subject to new investigations. However, in patients with relative or imperative contra-indications for the use 18 of contrast-agents in CT or MRI imaging, F-FDG PET-CT may be a reasonable alternative in the follow-up in CA of small renal masses. The total accumulated radiation dose of consecutive CT scans performed is considered negative related to the follow-up of LCA. The radiation dose with PET-CT is lower compared to the radiation dose of a full diagnostic cross18 sectional CT scan. The radiation dose of 185 MBq F-FDG is about 3-4 mSv.

129

To our knowledge, this pilot study is the first to assess F-FDG PET-CT imaging reviewing the spectrum of pre- and post-ablation PET-CT findings and to discuss its value in follow-up of renal tumors treated with cryosurgery. However, this study has, as have most pilot studies, many limitations. Only a few patients, at different times after CA, were studied in order to examine the 18 possibility of using F-FDG PET-CT imaging to characterize the lesion. No longitudinal data were collected, and no direct comparison with CT/MRI was intended. Furthermore, the majority of PET-CT findings are not confirmed by histology. Therefore, the accuracy of PET-CT could not be assessed. 18 However, the results of the present study show that F-FDG PET-CT can be used in the follow-up in patients with small renal masses that underwent CA. We assume this justifies a larger prospective and comparative study in order to determine the exact value of this technique for assessing treatment success and detecting recurrent tumors after CA. Conclusion 18

This experience with the F-FDG PET-CT for the follow-up of renal mass 18 CA showed that F-FDG PET-CT imaging could be used to characterize cryoablative tissue injury at different times after CA. A longitudinal 18 prospective study comparing F-FDG PET-CT imaging to CT/MRI and confirmed by histology is needed in order to establish its exact value in the follow-up of renal mass CA.

130

References

Pantuck AJ, Zisman A, Belldegrun AS. The changing natural history of renal cell carcinoma. J Urol. 2001; 166: 1611 - 1623.

Klatte T, Mauermann J, Heinz-Peer G, Waldert M, Weibl P. Klingler HC, Remzi M. Perioperative, oncologic, and functional outcomes of laparoscopic renal cryoablation and open partial nephrectomy: a matched pair analysis. J Endourol. 2011; 25: 991 - 997.

Hoffmann NE, Bischof JC. The cryobiology of cryosurgical injury. Urology. 2002; 60(2 Suppl 1): 40 – 49.

Kawamoto S, Solomon SB, Bluemke DA, Fishman EK. CT and MR imaging appearance of renal neoplasms after radiofrequency ablation and cryoablation. Semin Ultrasound CT MR. 2009; 30(2): 67 - 77.

Levy, EM, Viscoli CM, Horwitz RI. The effect of acute renal failure on mortality. A cohort analysis. JAMA. 1996; 275(19): 1489 - 1494.

Rihal CS, Textor SC, Grill DE, Berger PB, Ting HH, Best PJ, Singh M, Bell MR, Barsness GW, Mathew V, Garratt KN, Holmes DR Jr. Incidence and prognostic importance of acute renal failure after percutaneous coronary intervention. Circulation. 2002; 105(19): 2259 - 2264.

Rudnick MR, Goldfarb S, Wexler L, Ludbrook PA, Murphy MJ, Halpern EF, Hill JA, Winniford M, Cohen MB, VanFossen DB. Nephrotoxicity of ionic and non-ionic contrast media in 1196 patients: a randomized trial. The Iohexol Cooperative Study. Kidney Int. 1995; 47(1): 254 – 261.

Aide N, Cappele O, Bottet P, Bensadoun H, Regeasse A, Comoz F, Sobrio F, Bouvard G, Agostini D. Efficiency of [(18)F] FDG PET in characterising renal cancer and detecting distant metastases: a comparison with CT. Eur J Nucl Med Mol Imaging. 2003; 30(9): 1236 - 1245.

Perini R, Pryma D, Divgi C. Molecular imaging of renal cell carcinoma. Urol Clin North Am. 2008; 35(4): 605 - 611.

Divgi CR, Pandit-Taskar N, Jungbluth AA, Reuter VE, Gönen M, Ruan S, Pierre C, Nagel A, Pryma DA, Humm J, Larsson SM, Old LJ, Russo P. Preoperative characterization of clear-cell carcinoma using iodine-124-labeled antibody chimeric G250 (124I-cG250) and PET in patients with renal masses: a phase I trial. Lancet Oncol. 2007; 8: 304 - 310.

131

Bensinger SJ, Christofk HR. New aspects of the Warburg effect in cancer cell biology. Semin Cell Dev Biol. 2012; 23(4): 352-361. Doi: 10.1016/j/semcdb.2012.02.003.

Wagner AA, Solomon SB, Kavoussi LR. Imaging following cryoablation of a renal lesion. Nat Clin Pract Urol. 2005; 2(1): 52-57.

Boellaard R, O’Doherty MJ, Weber WE, Mottagy FM, Lonsdle MN, Stroobants SG, Oyen WJG, Hoekstra OS, Pruim J, Marsden PK, Tatsch K, Hoekstra CJ, Visser EP, Arends B, Verzijbergen FJ, Zijlstra JM, Comans EFI, Lammertsma AA, Paans AM, Willemsen AT, Beyer T, Bockisch A, Schafer-Prokop C, Delbeke D, Baum RP, Chiti A, Krause BJ. FDG PET and PET/CT: EANM procedure guidelines for tumour PET imaging: version 1.0. Eur J Nucl Med Mol Imaging. 2010; 37(1): 181 - 200.

Gill IS, Remer EM, Hasan WA, Strzempkowski B, Spaliviero M, Steinberg AP, Kaouk JH, Desai MM, Novick AC. Renal cryoablation: outcome at 3 years. J Urol. 2005; 173(6): 1903 - 1907.

Remer EM, Weinberg EJ, Oto A, O’Malley CM, Gill IS. MR imaging of the kidneys after laparoscopic cryoablation. AJR Am J Roentgenol. 2000; 174(3): 635 - 640.

Bolte SL, Ankem MK, Moon TD, Hedican SP, Lee FT, Sadowksi EA, Nakada SY. Magnetic resonance imaging findings after laparoscopic renal cryoablation. Urology. 2006; 67: 485 – 489.

Rutherford EE, Cast JE, Breen DJ. Immediate and long-term CT appearances following radiofrequency ablation of renal tumours. Clin Radiol. 2008; 63(2): 220 230.

132

CHAPTER 8 Future prospects of renal mass cryosurgery

The incidence of small renal masses has increased while the incidence of 1 advance stage renal cancer has decreased over the recent years . Renal masses smaller than 4cm in diameter account for 48-60% of all diagnosed 2 renal cancers . Overall, more than 50% of the patients are asymptomatic at the time of diagnosis. The increased use of body-imaging technologies, performed for a variety of reasons, results in an increased diagnosis of 3 incidentally found renal cancer . When tumors are incidentally diagnosed they tend to be of lower stage and of smaller diameter compared to tumors found symptomatic at the time of diagnosis. Of all small renal masses 4, 5 (<4cm), approximately 20% is benign and 80 % malignant . The majority is diagnosed in elderly patients sometimes bearing a high comorbidity rate 6 that will influence treatment decisions . Clearly, these patients are considered candidates for active surveillance or ablative surgery. However, counseling this group of patients in the context of uncertain tumor pathology and biological potential remains a challenge. To predict whether a radiographically diagnosed small renal lesion is benign or malignant can be difficult. Despite several imaging methods used (gray-scale ultrasound, contrast enhanced ultrasound, contrast CT, and contrast MRI), some lesions have no specific classical discriminating features beside the fact they appear to be solid. Furthermore, tumor size and imaging alone are poor indicators 4 for predicting the biological nature of the lesion . Possible other ways to avoid unnecessary, invasive treatment would be the use of diagnostic markers or to determine histology with image-guided core needle biopsies. However, the use of markers predicting the biological nature of renal masses â&#x2030;¤ 4 cm has not extensively been tested and validated. Principal guideline committees for the treatment of renal cancer advice that in doubt of the indication for surgical intervention one should perform image-guided core 7, 8, 9 needle biopsies for histological examination prior to treatment . The rationale for this recommendation is to guide decision for surgery in indistinct small renal mass on radiological imaging and in patients with increased surgical risk. Despite there is evidence that computed-tomography-guided percutaneous biopsies can help to distinguish between benign and 10 malignant tissue, this method is not routinely used . Hence, arguments such as sampling errors and possible track seeding are still used to favor the risk of surgery in ignorance of histological diagnosis. Although, Volpe et al reported that the complication-risk of image guided renal mass biopsies 11 using coaxial techniques are low and rarely clinical significant . The overall 12 estimated risk for tract seeding due to percutaneous biopsies is <0.01% . Today, laparoscopic and CT-guided renal tumor cryoablation are recognized 7, 8, 9 as a viable alternative treatment to partial nephrectomy . Like all established treatment modalities also cryoablation will keep on undergoing transformations in order to simplify processes and to improve quality control. The two principal future directions of renal mass cryosurgery are focussing at enhancing its efficacy and the assessment of follow-up after treatment. This thesis discussed cryosurgical induced vascular changes in tissues and

134

alternative imaging techniques for the follow-up of cryoablated renal tumors. In order to understand how these findings relate to possible future directions one should critically comprehend the cryobiological processes. Cryobiological principals; rationale for adjuvant therapy

Over the last 40 years, several studies are performed related to cryosurgery. Much of this work addressed the molecular basis of freezing effects in invitro experiments. Pathways of cryogenic cell death are studied and identified as direct and indirect mechanisms of tissue damage in in-vivo cryosurgery. These preliminary investigations formed the basis of today’s therapeutic cryosurgery, especially that of treating malignancies. The biophysical cellular response to freezing and thawing is directly related to the behavior of water molecules in the intracellular and extracellular environment. Tissue response to freezing and thawing resulting in destruction is dependent on cell injury, tissue structure injury and functional impairment of the tissue compounds. The associated different cryobiological responses leading to cell death can be summarized in four pathways: • • • •

13, 14

immediate cell injury , 15-32 cell injury as result of an initiated vascular response , 33-35 apoptosis , 36-43 immunological response .

The primary injury mechanisms are immediate and vascular cell injury. The supplementary mechanisms as apoptosis and the immunological response are only cryobiological relevant in tissue at the ablation border area. There, where insufficiently low temperatures result in limited immediate tissue injury. Adjuvant therapies might be beneficial in achieving complete tissue destruction in this cryobiological sublethal area. Working mechanisms of adjuvant drug therapies will be related to one or more of the basic biological principles of cryosurgical induced cell injury. Immediate cell injury Direct cellular damage is a consequence of freezing. A low temperature leads to destruction of the structural framework of cells and thus results into immediate cell death. Direct cell death can occur in the freezing phase as well as in the thawing phase. The freezing is considered to have a stronger lethal effect on cells than thawing. There are two different cryobiological responses, which are related to 0 freezing rate, that lead to immediate cell death: slow freezing (~ 5 C/min)

135

and fast freezing (~ 25 C/min). A slow freezing rate results in dehydration injury of the cell and fast freezing causes intracellular ice formation. As the temperature gradually falls into the hypothermic parameters, it stresses the structure and function of cells. The solute concentration outside the cells begins to rise, causing dehydration of cells. This injures the cell by damaging the enzymatic processes that destabilizes the cell membrane. Giving sufficient time, this will eliminate most, but not all cells. Fast freezing results in the formation of ice in the intracellular environment. In this process, there is a lack of time for intracellular water to pass the membrane before freezing. The cytoplasm is strongly cooled and ice crystals are formed causing injury to organelles and membranes resulting in cell death. During the thawing phase within the cells, large crystals form through fusing of smaller crystals that are refractory to all cell membranes. This leads to a second prolonged hypothermic situation with continued metabolic derangement also contributing to additional direct destruction of cells. Based on these mechanisms contributing to immediate cell death, four parameters of cryotherapy related to the freezing process can be recognized: 1) cooling rate, 2) minimum temperature (low enough to ensure intracellular ice formation), 3) time held at minimum temperature and 4) thawing rate. Cooling rate is strongly dependent on thermal gradients within the tissue. Most cryosurgical research has been directed to minimumtemperature and aimed at establishing toxic temperatures ensuring killing tumor tissue. These toxic temperatures appeared to be cell-type dependant 13, 14 . Vascular response The cryo-induced vascular response played a central role in this thesis. A variety of earlier conducted studies showed that thermal gradients are strongly dependent not only of cell-type but also of vascular inhomogeneties. Beside the immediate cell injury, the amount of tissue damage is also dependant of the microenvironment in which the cryoablation takes place. This is of interest, especially at the border of the ice ball where the cooling rate is low and the temperature reached is not low enough to provoke 15, 16 immediate cell death . However, our research and other studies show 17-19 that after a few weeks a sharp demarcation zone is noticed . This indicates that besides cryogenic induced immediate cell death the viability of cells must be dependent of adjunctive injury mechanisms such as the vascular pathway. Vascular injury is recognized as the second stage of cryogenic cell dead causing coagulative necrosis. Many investigators 17-23 studied the effect of frostbite on blood vessels , showing that the primary component is circulatory stasis due to cellular anoxia. The vascular response to frostbite of human skin starts with homeostasis in the frozen tissue, surrounded by hyperemia. When the frozen tissue warms up again, a

136

hyperemic state sets in, followed by submission of blood flow and edema in the ablated area after which tissue necrosis and or repair through various 20-22 mechanisms takes place . These intrinsically vascular changes have been demonstrated in experiments with different animal and human soft 0 0 tissues, applying varying temperatures (-20 C to 5 C) and thaw rates. Cooling down of tissue results in vasoconstriction and, therefore, in a decrease of blood flow as a response. Prolonged slow freezing, more than 24 hours, cause vascular stasis starting with capillaries that become inactive. During fast freezing, the formation of ice involves the vascular anatomical structures and causes direct cellular damage to the endothelium. In addition to this cellular injury, ice propagates through the vessel, causing distension of the vessel lumen, thereby tears off the wall and endothelial clefts appear. These events lead to increased vascular permeability, edema formation, hemo-concentration, and cessation of blood flow, hypoxia and 16 consequent tissue necrosis . Many investigators identify the period after thaw as being the most critical in determining the amount of tissue damage induced by vascular stasis. First, as the temperature climbs, circulation returns with vasodilatation. Two hours after the return of circulation defects of endothelial cell junctions give way to increased vascular permeability. It has been proposed that these events are 24 a result of the release of vasoactive factors after thaw. Barker et al theorized that free radicals are formed as a result of the high-oxygen transport in the hyperperfused state. These free radicals cause endothelial 25 damage by peroxidation of lipids in the endothelial membrane. Zook et al described an additional means of endothelial injury after thaw. They demonstrated that neutrophil adhere to the endothelium and release toxic enzymes needed to clean up dead cells. However, these toxic enzymes 23, 26-31 itself can damage the endothelium further . These studies suggest the involvement of endothelial-mediated injury and inflammation as underlying mechanisms in cryoinjury. Multiple (mutual dependent) mechanisms are responsible for the vascular 16 stasis and subsequent necrosis after freeze . In previous reports damage to the endothelium, ischemia-reperfusion injury, inflammation and the resultant loss of microcirculatory support are considered essential in defining the limits of the cryolesion and trigger the induction of apoptosis and cellular 16, 19-23 necrosis . Cessation of blood flow within the ablated area will start with the immediate 31 destruction of microvessels and be complete by the vascular response that ultimately leads to cessation of blood flow in the lager vessels. Our study confirmed that it is expected that blood vessel transport has ceased 18 and vessel destruction should be fully complete after 2 weeks. . This 32 stopped blood flow causes hypoxic stress of the tissue. Kimura et al showed in a cryoablated murine prostate cancer model that microvessel

137

density is decreased in the impact area of freezing and has a negative correlation with hypoxia. At the same site, existing hypoxia had a positive correlation with necrosis and apoptosis. Apoptosis Apoptosis is the process of programmed cell death (PCD) that may occur in multicellular organisms. PCD involves a series of biochemical events that lead to a variety of morphological changes, including blebbing, changes to the cell membrane such as loss of membrane asymmetry and attachment, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation. Research on apoptosis has increased significantly since the early 1990s. In addition to its importance as a biological phenomenon, one can identify defective apoptotic processes in an extensive range of diseases. Excessive apoptosis causes hypotrophy, such as in ischemic damage, whereas an insufficient number of apoptosis results in uncontrolled cell proliferation, such as cancer. Most tumor cell lines have a constitutive expression of anti-apoptotic proteins resulting in inhibition of 33 apoptosis . Freezing induced apoptosis is primarily present in the outer area of the 0 cryogenic lesion. There, temperatures range between -0.5 C and about – 40 0 C, which is not always sufficient to kill all cells and both necrosis and apoptosis take place. An in-vitro study with human renal cancer cell line 34 demonstrated that apoptosis peaked at six hours post-thaw . Even more, neo-adjuvant use of apoptotic initiating agents showed a significant increase in cell death at cryoablation. Therefore, promoting apoptosis with adjunctive therapy might complete destruction of cells and be therapeutically beneficial. In vivo experiments still need to determine to what extent apoptosis is a direct consequence of the freeze/thaw circle or is a secondary effect of 35 dehydration and/or inflammation after and during cryosurgery . Immunological response Freezing induced immunological injury can be considered as the fourth mechanism causing cell death. Earlier data of clinical practice, report the regression of metastatic lesions after cryoablation of the primary tumor that 36, 37 suggests a potential systemic response to the local cryoinjury . However, the existence of a cryo-immunological response has been controversial since results from preclinical studies have been mixed. A review of the 38 literature by Sabel , describes the existing evidence of both stimulatory and suppressive, immune responses after cryoablation. Cryosurgical induced tissue damage induces a pro-inflammatory cytokine synthesis. Cytokines can be released from stromal cells and immune cells (IL-1β, IL-6, TNF-α) or tumor cells (IL-10, TGF-β) within the cryoablated

138

area. Antigen presenting cells, such as macrophages and dendritic cells, will start the acquired immune response by taking up antigen from apoptotic cells, necrotic cells, immune complexes, opsonized tumor cells, and heat shock proteins. These exogenous antigens are processed into peptides that bind with molecules of the major histocompatibility complex (MHC) class I and II, which than will be presented at the cell surface. Antigen-specific helper T-cells (CD4+), cytotoxic T-cells (CD8+), and regulatory T-cells (Treg cells) can be activated by recognizing and binding to the presented antigens. They stimulate the immune response by the secretion of cytokines, destroy tumor cells directly or lead to immune suppression, respectively. The MHC class I generates the cytotoxic response. However, exogenous antigens are presented on MHC class II molecules. Dendritic cells are better capable of cross-presentation (the process by which exogenous antigens enter the MHC class I pathways) than the macrophages and, thus, are more effective in promoting cytotoxic T-cell activity. Several factors can influence the modulation of the immune system either negatively or positively by cryoablation. Necrosis, the major component of cell destruction in the heart of the ablation area, leads to increased dendritic cell maturation and 39, 40 macrophage activation . Whereas, apoptosis can be recognized as a physiological process, which can lead to suppression of the immune 41 response . Also, the mixture of cytokines that are released after cryoablation influences the nature of the initiated immune response being stimulatory or suppressive. Dendritic cells can provoke the cellular response and, therefore, play an eminent role in cryoablation. Since immature dendritic cells are bone marrow derived and are transported by the blood flow, the vascular response of the cryoablated area will influence the impact 0 that these cells may have. At a quick-freeze rate (â&#x2C6;&#x2020; 20 to â&#x2C6;&#x2020; 50 C/minute), 42 intracellular ice occurs as deadly to the cell and necrosis results . In a mice 43 model with established breast cancer, Sabel et al have demonstrated the potential role of the immune response in relation to the freezing rate. Cryoablation using a high freeze rate resulted in a significant increase in tumor-specific T-cells, a reduction of pulmonary metastases and improved survival compared to surgical excision of the primary tumor. However, cryoablation using a low freeze rate resulted in an increase in regulatory Tcells, a significant increase in pulmonary metastases and decreased survival compared to surgical excision of the primary tumor. Adjuvant therapy Renal tumor size is a significant predictor for the risk of intraoperative complications including incomplete ablation performing cryosurgery. Therefore, in selected patients with larger renal tumors and with imperative reasons for choosing cryoablation as nephron-sparing treatment method, adjuvant therapy can be considered. Adjuvant therapy, can accentuate tissue destruction by modulating the response to known mechanisms of 35 cryoinjury . Adjuvant therapy can focus at the promotion of cryogenic

139

injury, target volume reduction, and the combination of both. Thermophysical adjuvants can enhance the immediate cryogenic cell injury. Chemotherapeutic adjuvants act at the molecular level and induce apoptosis. Cytokines or vascular-based agents modulate the inflammatory response. Immunomodulating adjuvants stimulate immune cells to enhance tissue destruction. Procedures that theoretically can support renal cancer cryosurgery are: -

Renal arterial embolization (RAE) Neo-angiogenic inhibiting drugs (NAID) Trans renal arterial apoptotic enhancing embolization (TRAAEE)

Common indications for RAE are symptomatic angiomyolipomas, palliation of unresectable renal cancer, hemorrhage, vascular lesions, malignant 44 hypertension, and sequelae of end-stage renal disease . Reasons for RAE as adjuvant therapy to cryoablation can be reducing the rate of postcryosurgical bleeding and increase the treatment efficacy by reducing tumor volume. Furthermore, lowering the percentage of Treg cells or reducing their activity can improve the anti-tumor immune situation and help reduce the chance for tumor recurrence and metastasis. In patients with intermediate and advanced renal cancer the effect of RAE versus RAE + cryoablation on 45 the differentiation of Treg cells was studied by Li et al . Compared to RAE, RAE + cryoablation showed a wider range of tumor necrosis and a more significant drop in the percentage of Treg cells. So far, there are no studies conducted using RAE + cryoablation as a curative option for renal cancer. When NAID-therapy is considered as neo-adjuvant therapy to cryoablation 9 for renal cancer, a histological diagnosis is obligatory . The revised Response Evaluation Criteria in Solid Tumors can be used to assess the 46 response to the adjuvant treatment of renal cancer . Kroon et al reported that, compared to tumors larger than 7 cm, smaller renal tumor size was 47 related to more effective downsizing . They recognized that the potential benefit of neo-adjuvant treatment to downsize the primary tumor for nephron-sparing surgery including cryoablation may exist, particularly in tumors sized 5 to 7 cm. Several in-vitro and in-vivo studies have shown the ability of 48-52 chemotherapeutics to augment cell death at cryoablation . No study using cryo-chemo combination therapy has shown an augmentation up to the ice ball border. Limitations are the limited dose of the agent at the target tissue level and cancer specific sensitivity to the type of drug used. So far, systemic chemotherapy is not commonly used in the treatment of renal cancer. However, targeted adjuvant delivery to enhance the apoptotic sensitivity of only the target tissue can be performed by direct injection of the tissue, systemically using nano-sized carriers, and combined with trans-

140

arterial embolization. Trans-arterial chemoembolization (TACE) is frequently used as a therapy for liver tumours and involves the combination of a 53 chemotherapeutic agent with a drug carrier that is delivered intra-arterially . The purpose of embolization is to reduce arterial inflow, diminish washout of the chemotherapeutic agent, and prolong contact time between cancer cells and the chemotherapeutic agents. So far, no studies have been published addressing trans-arterial embolization in combination with apoptotic agents as therapeutic method for the treatment of renal cancer. TNF-Îą can act as an additional stressor to the microvasculature of the tissue and aid in the maturation of immune cells at the time of local tumor destruction during 50, 54 cryoablation . Jiang et al showed that targeted delivery of TNF-Îą using gold nanoparticles coated with the drug achieved a significant augmentation 50 in tissue injury and greatly reduced systemic side effects . Follow-up imaging of renal tumor cryosurgery

Within 5 years follow-up of all renal cancer patients treated with curative intent, 25% of the patients presents with metachronous metastases and an 1, 2 additional 20% with synchronous metastases . However, the risk for metastatic development in patients with small renal cancers is small. Within 55, 56 10 years follow-up 1-8.4% will be diagnosed with metastases . The biological potential of small renal cell carcinomas (RCC) seems to be mild. This reflects in the relative merit cancer specific survival of stage 1 disease 57 . However, it is reported that lesions of 3-4 cm diameter have a significant higher risk for high grade and advanced-stage disease compared to smaller 58 lesions . Klatte et al. reported that, following cryosurgery, the chance for 59 local progression or recurrence is 8.5% . They also found that the risk for local progression increased with 85% per 1 cm increase of tumor size. The mean follow-up for cryosurgery in this study was 29 months. Although this 60, 61 follow-up time is short, local recurrence is rarely reported after 2 years . The rate of reported distant metastases following renal cancer cryoablation 62 is 1% . In general, risk factors for renal cancer metastatic development are high nuclear grade and vascular invasion. However, determining vascular invasion and the accuracy of grading based on biopsy specimen is under discussion. So far, principal guidelines have failed to provide evidence based follow-up 7, 8 schedule for T1a and T1b renal cancers . The proposed algorithm for the follow-up of cryoablated RCC from the EAU guidelines stratifies for risk factors but fails to clarify what these risk factors are and how they are assessed. For example, they do not distinguish between histological subtypes. However, Leibovich et al. reported that in patients diagnosed with renal cancer, the histological subtype of clear cell carcinoma is an

141

independent predictor of progression of distant metastasis and cancer63 specific survival . The risk profile for the occurrence of cancer related incidents of cryoablated renal tumors needs to be assessed. In the first instance, the risk for distant or local events needs to be assessed. Thereafter, the most cost-effective imaging modality with a significant sensitivity for detecting local and or distant disease should be selected. This can be a combination of different imaging modalities and may include diagnostic markers and histology obtained by image-guided core needle biopsies. Pulmonary metastases are the most common metastasis of renal cancer. However, the risk for the development of distant metastases is approximately 1% and, therefore, a regular check-up including chest X-ray or CT-scan can be questioned. Furthermore, if pulmonary metastases occur, it can be discussed whether it is clinically relevant to detect these lesions before they become symptomatic. A regular CT-scan of the chest is probably clinically irrelevant, not cost-effective and exposes the patient to unnecessary radiation doses. Other common sites of metastatic disease from RCC include lymph nodes, liver and the skeletal system, which all are best evaluated with CT or MR imaging. 64, 65

The risk for multifocal RCC is 5-8% . Tsivian et al. reported that lesion size (2-4 cm), male gender, family history of renal cancer, histologic subtype, 64 and grade were independently associated with occult multifocality . Histologic subtypes other than clear cell had an increased risk for occult multifocality of which the papillary subtype had the strongest association which conferred a seven-fold odds increase. Rare subtypes such as medullary and collecting duct RCC increased the odds by >10-fold. The risk for local progression or recurrence following renal mass 59 cryoablation is 8.5% . Our and other studies clarified that tumor diameter is related to the occurrence of incomplete ablation and local recurrence of cryoablation. Lehman et al reported that tumor size appeared to be a key 66 metric for incomplete ablations following LCA . The majority of local 59 recurrences will be detected in the first two years following cryoablation . Multi-institutional long-term follow-up data can be used for the development of a nomogram that will aid to stratify the risk for treatment failure, new renal sites of RCC, and metastases. Based on the risk grade the follow-up can be categorized concerning how often checks should be carried out and what investigational tests are needed. However, under the influence of all kind of circumstances there always will be exceptions to the rule demanding a tailored approach. In summary, the most clinical important and, thus, relevant events following renal mass cryosurgery are the development of a local recurrence or new

142

renal masses (multifocality). Therefore, it seems most relevant to focus at the detection of these events in the follow-up. Incomplete ablation should be detected as soon as reasonable and first time 18 imaging can be performed with a minimum of 2 weeks following ablation . So far, the best method to assess treatment failure is cross sectional imaging of the upper abdomen using contrast CT or MRI. MRI is equivalent to CT for evaluation of renal mass cryosurgery. However, CT has the advantage of widespread availability, more rapid examination time in comparison with MRI, and lower cost than MRI. In case at first time imaging the ablation is considered successful, than consecutive imaging is used to detect local recurrences and new renal masses. Local recurrences tend to occur in the first years following cryoablation and patients with an increased risk for local recurrence should be examined more frequently than patients with low risk. A local recurrence will meet two imaging characteristics, mass growth and vitality of tissue. Both can be used for the detection of a local recurrence. The detection rate of new renal masses is related to the volume of the suspected mass. There is a temptation to lower the radiation exposure by using ultrasonography (US). However, US is less accurate than CT for revealing small renal masses and, thus, currently contrast CT is the first choice. Still, small solid lesions (<1.5 cm diameter) can be missed at diagnostic imaging. In the knowledge that the majority of small RCC are expected to grow with a mean of 2.8 mm diameter a year, one can possibly 67 sustain with a yearly follow-up . Furthermore, one can discuss whether it is clinically relevant to diagnose new tumors < 2 cm diameter at follow-up of small renal masses treated with cryoablation. The highest risk for a local recurrence is in the first years following ablation and the preferred way to detect a local recurrence is contrast CT or MR imaging. After three years follow-up the risk for local recurrence is low but new tumors can occur. After the time frame of three years following surgery, it can be suggested that imaging other than contrast CT would sustain in a reasonable detection rate of clinically relevant new tumors or local recurrence. Randomized control studies are needed to answer this question. Conclusion to future prospects

Four cryobiological tissue response mechanisms to cryogenic ablation of tissues are mainly significant at the border of the ice-ball where they most likely result in only limited damage because of the insufficiently low temperatures. Adjuvant therapies might be beneficial in achieving complete tissue destruction in this zone. Working mechanisms of adjuvant drug

143

therapies will be related to the basic biological principles of cryosurgical induced cell injury. Currently, these adjuvant strategies remain experimental and need further study focusing at the choice of the adjuvant, the time interval between the adjuvant addition and cryotherapy, the delivery of the adjuvant to the tumor, and the dose of the adjuvant. Risk factors for recurrence and metastases from small renal masses that underwent cryoablation still need further investigation. Long term follow-up and histological diagnosis are necessary in order to identify the risk factors and can help design the best method for the follow-up of a renal mass after cryosurgery.

144

References

Sun M, Thuret R, Abdollah F, Lughezzani G, Schmitges J, Tian Z, Shariat SF, Montorsi F, Patard JJ, Perrotte P, Karakiewicz PI. Age-adjusted incidence, mortality, and survival rates of stage-specific renal cell carcinoma in North America: a trend analysis. Eur Urol. 2011; 59:135-141.

Nguyen MM, Gill IS, Ellison LM. The evolving presentation of renal carcinoma in the United States: trends from the Surveillance, Epidemiology, and End Results program. J Urol. 2006; 176:2397-2400; discussion 400.

Russo P. Localized renal cell carcinoma. Curr Treat Options Oncol. 2001; 2(5): 447 - 455.

Frank I, Blute ML, Cheville JC, Lohse CM, Weaver AL, Zincke H. Solid renal tumors: an analysis of pathological features related to tumor size. J Urol. 2003; 170:22172220.

Hollingsworth JM, Miller DC, Daignault S, Hollenbeck BK. Rising incidence of small renal masses: a need to reassess treatment effect. J Natl Cancer Inst. 2006; 98(18): 1331 - 1334.

Dutch Guideline Committee for renal cancer. Niercelcarcinoom. Versie 2.0, evidenced based 2010-12-01. Integraal Kankercentrum Nederland, http://oncoline.nl.

Schmidbauer J, Remzi M, Memarsadeghi M, Haitel A, Klingler HC, Katzenbeisser D, Wiener H, Marberger M. Diagnostic accuracy of computed tomography-guided percutaneous biopsy of renal masses. Eur Urol. 2008; 53(5): 1003 - 1011.

Volpe A, Finelli A, Gill IS, Jewett MA, Martignoni G, Polascik TJ, Remzi M, Uzzo RG. Rationale for percutaneous biopsy and histologic characterization of renal tumors. Eur Urol. 2012; 62(3): 491 â&#x20AC;&#x201C; 504. DOI: 10.1016/J.eururo.2012.05.009.

Herts BR, Baker ME. The current role of percutaneous biopsy in the evaluation of renal masses. Semin Urol Oncol. 1995; 13(4): 254 - 261.

Baust JG, Gage AA. The molecular base of cryosurgery. BJU Int 2005; 95(9): 1187 - 1191.

145

Baust JG, Gage AA, Clarke D, Baust JM, VanBuskirk R. Cryosurgery-a putative approach to molecular-based optimization. Cryobiology. 2004; 48(2): 190 - 204.

Gage AA, Baust J. Mechanisms of tissue injury in cryosurgery. Cryobiology. 1998; 37(3): 171 - 186.

Hoffmann NE, Bischof JC. The cryobiology of cryosurgical injury. Urology. 2002; 60(2 Suppl 1): 40 - 49.

Ames CD, Vanlangendonck R, Venkatesh R, Gonzales FC, Quayle S, Yan Y, Humphrey PA, Landman J. Enhanced renal parenchymal cryoablation with novel 17-gauge cryoprobes. Urology 2004; 64: 173 â&#x20AC;&#x201C; 175.

Lagerveld BW, van Horssen P, Laguna MP, van den Wijngaard JPHM, Siebes M, Wijkstra H, de la Rosette JJMCH, Spaan JAE. The gradient change of arterial vascular anatomy after cryoablation. J Urol. 2011; 186(2): 681 - 686.

Rupp CC, Hoffmann NE, Schmidlin FR, Swanlund DJ, Bischof JC, Coad JE. Cryosurgical changes in the porcine kidney: histologic analysis with thermal history correlation. Cryobiology. 2002; 45(2): 167 - 182.

Lewis T, Love WS. Vascular reactions of the skin to injury. Part III: Some effects of freezing, of cooling and of warming. Heart. 1926; 13: 27 - 60. Mundt ED. Studies on the pathogenesis of cold injury: Microcirculatory changes in tissue injured by freezing. Proc Symp Artic Biol Med. 1964; 4, 51 - 72.

21 22

Quantanilla R, Krusen F, Essex H. Studies on frostbite with special reference to treatment and the effect on minute blood vessels. Am J Physiol. 1947; 149(1): 149 - 161.

Rabb JM, Renaud ML, Brandt A, Witt CW. Effect of freezing and thawing on the microcirculation and capillary endothelium of hamster cheek pouch. Cryobiology. 1974; 11(6): 508 - 518.

Barker JH, Bartlett R, Funk W, et al. The effect of superoxide dismutase on the skin microcirculation after ischaemia and reperfusion. Prog Appl Microcirc. 1987; 12: 276 - 281.

Zook N, Hussmann J, Brown R, Russell R, Kucan J, Roth A, Suchy H. Microcirculatory studies of frostbite injury. Ann Plast Surg. 1998; 40(3): 246 - 255.

Rothenborg H. Cutaneous circulation in rabbits and humans before, during and after cryosurgical procedures, measured by Xenon-133 clearance. Cryobiology. 1970; 6(6): 507 - 511.

Zacarian SA, Stone D, Clater M. Effects cryogenic temperatures on microcirculation in the Golden hamster cheek pouch. Cryobiology. 1970; 7(1): 27 - 39.

Bourne MH, Piepkorn MW, Clayton F, Leonard LG. Analysis of microvascular changes in frostbite injury. J Surg Res 1986; 40(1): 26 - 35.

Bellman S, Adams-Ray J. Vascular reactions after experimental cold injury; a microangiographic study of rabbit ears. Angiology. 1956; 7(4): 339 - 367.

Giampapa VC, Oh C, Aufses AH Jr. The vascular effect of cold injury. Cryobiology. 1981; 18(1): 49 - 54.

146

Daum PS, Bowers WD Jr, Tejada J, Hamlet MP. Vascular casts demonstrate microcirculatory insufficiency in acute frostbite. Cryobiology. 1987; 24(1): 65 - 73.

Kimura M, Rabbani Z, Mouraviev V, Tsivian M, Vujaskovic Z, Satoh T, Baba S, Baust JM, Baust JG, Polascik TJ. Morphology of hypoxia following cryoablation in a prostate murine model: its relationship to necrosis, apoptosis, and microvessel density. Cryobiology. 2010; 61: 148 – 154.

Dolcet X, Llobet D, Pallares J, Matias-Guiu X. NF-kB in development and progression of human cancer. Virchows Arch. 2005; 446(5): 475 - 482.

Clarke DM, Robilotto AT, Rhee E, VanBuskirk RG, Baust JG, Gage AA, Baust JM. Cryoablation of renal cancer: variables involved in freezing-induced cell death. Technol Cancer Res Treat. 2007; 6(2): 66 - 79.

Goel R, Anderson K, Slaton J, Schmidlin F, Vercellotti G, Belcher J, Bischof JC. Adjuvant approaches to enhance cryosurgery. J Biomech Eng. 2009; 131(7): 074003.

Soanes WA, Ablin RJ, Gonder MJ. Remission of metastatic lesions following cryosurgery in prostatic cancer: immunologic considerations. J Urol. 1970; 104: 154 – 159.

Tanaka S. Cryosurgical treatment of advanced breast cancer. Skin Cancer 1995; 10: 9 – 18.

Sabel MS. Cryo-immunology: a review of the literature and proposed mechanisms for stimulatory versus suppressive immune responses. Cryobiology. 2009; 58(1): 1 - 11.

Sauter B, Albert ML, Fransisco L, Larsson M, Somersan S, Bhardwaj N. Consequences of cell death. Exposure to necrotic tumor cells, but not primary tissue cells or apoptotic cells, induces the maturation of immunostimulatory dendritic cells. J Exp Med. 2000; 191: 423 – 434.

Gallucci S, Lolkema M, Matzinger P. Natural adjuvants: endogenous activators of dendritic cells. Nat Med. 1999; 5: 1249 – 1255.

Peng Y, Martin DA, Kenkel J, Zhang K, Ogden CA, Elkon KB. Innate and adaptive immune response to apoptotic cells. J Autoimmun. 2007; 29: 303 – 309.

Bischof JC, Cristof K, Rubinsky B. A morphological study of the cooling rate response of normal and neoplastic human liver tissue: Cryosurgical implications. Cryobiology 1993; 30: 482 – 492.

Sabel MS, Su G, Griffith KA, Chang AE. Rate of freeze alters the immunologic response after cryoablation of breast cancer. Ann Surg Oncol 2010; 17: 1187 – 1193.

Schwartz MJ, Smith EB, Trost DW, Vaughan ED Jr. Renal artery embolization: clinical indications and experience from over 100 cases. BJU Int. 2007; 99: 881 886.

147

Li Y, Guo Z, Liu CF, Xing WG, Si TG, Liu F, Guo JZ, Xing JZ. Effect of transcatheter renal arterial embolization combined with cryoablation on regulatory + + CD4 CD25 T lymphocytes in the peripheral blood of patients with advanced renal carcinoma. Cryobiology. 2012; 65: 56 - 59.

Eisenhauer EA, Therasse P, Bogaerts J, Schwartz LH, Sargent D, Ford R, Dancey J, Arbuck S, Gwyther S, Mooney M, Rubinstein L, Shankar L, Dodd L, Kaplan R, Lacombe D, Verweij J. New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1). Eur J Cancer. 2009; 45(2): 228 – 247.

Kroon BK, de Bruijn R, Prevoo W, Horenblas S, Powles T, Bex A. Probability of downsizing primary tumors of renal cell carcinoma by targeted therapies is related to size at presentation. Urology. 2013; 81:111-115.

Clarke DM, Baust JM, VanBuskirk RG, Baust JG. Addition of anticancer agents enhances freezing-induced prostate cancer cell death: implications of mitochondrial involvement. Cryobiology. 2004; 49: 45 – 61.

Le Pivert P, Haddad RS, Aller A, Titus K, Doulat J, Renard M, Morrison DR. Ultrasound guided combined cryoablation and microencapsulated 5-Fluoracil inhibits growth of human prostate tumors in xenogenic mouse model assessed by luminescence imaging. Technol Cancer Res Treat. 2004; 3: 135 – 142.

Jiang J, Goel R, Iftekar MA, Visaria R, Belcher JD, Vercelotti GM, Bischof JC. Tumor necrosis factor-alpha-induced accentuation in cryoinjury: mechanisms in vitro and in vivo. Mol Cancer Ther. 2008; 7: 2547 – 2555. Forest V, Peoc’h M, Ardiet C, Campos L, Guyotat D, Vergnon JM. Benefit of a combined treatment of cryotherapy and chemotherapy on tumour growth and late cryo-induced angiogenesis in a small-cell lung cancer model. Lung Cancer. 2006; 54: 79 – 86.

Clarke DM, Baust JM, VanBuskirk RG, Baust JG. Chemo-cryo combination therapy: an adjunctive model for the treatment of prostate cancer. Cryobiology. 2001; 42:274 -285.

Gadaleta CD, Ranieri G. Trans-arterial chemoembolization as a therapy for liver tumours: New clinical developments and suggestions for combination with angiogenesis inhibitors. Crit Rev Oncol Hem. 2011; 80: 40 – 53.

Udagawa M, Kudo-Saito C, Hasegawa G, Yano K, Yamamoto A, Yaguchi M, Toda M, Azuma I, Iwai T, Kawakami Y. Enhancement of immunologic tumor regression by intratumoral administration of dendritic cells in combination with cryoablative tumor pretreatment and Bacillus Calmette-Guerin cell wall skeleton stimulation. Clin Cancer Res. 2006; 12: 7465 – 7475.

Chawla SN, Crispen PL, Hanlon, Greenberg RE, Chen DY, Uzzo RG. The natural history of observed enhancing renal masses: meta-analysis and review of the world literature. J Urol. 2006; 175:425-431.

Klatte T, Patard JJ, de Martino, et al. Tumor size does not predict risk of metastatic disease or prognosis of small renal cell carcinomas. J Urol. 2008; 179:1719-1726.

Sun M, Abdollah F, Bianchi M, Trinh QD, Jeldres C, Tian Z, Shariat SF, Widmer H, Zorn K, Menon M, Montorsi F, Perrotte P, Karakiewicz PI. A stage-for-stage and grade-for-grade analysis of cancer-specific mortality rates in renal cell carcinoma according to age: a competing-risks regression analysis. Eur Urol. 2011; 60(6): 1152 - 1159.

148

Remzi M, Ozsoy M, Klingler HC, Susani M, Waldert M, Seitz C, Schmidbauer J, Marberger M. Are small renal tumors harmless? Analysis of histopathological features according to tumors 4 cm or less in diameter. J Urol. 2006; 176(3): 896 899.

Klatte T, Grubmüller B, Waldert M, Weibl P, Remzi M. Laparoscopic cryoablation versus partial nephrectomy for the treatment of small renal masses: systematic review and cumulative analysis of observational studies. Eur Urol. 2011; 60: 435 - 443.

Aron M, Kamoi K, Remer E, Berger A, Desai M, Gill I. Laparoscopic renal cryoablation: 8-year, single surgeon outcomes. J Urol. 2010; 183: 889 - 895.

Kunkle DA, Uzzo RG. Cryoablation or radiofrequency ablation of the small renal mass: a meta-analysis. Cancer. 2008; 113(10): 2671 - 2680. Doi: 10.1002/cncr.23896.

Tsivian M, Moreira DM, Caso JR, Mouraviev V, Madden JF, Brataslavsky G, Robertson CN, Albala DM, Polascik TJ. Predicting occult multifocality of renal cell carcinoma. Eur Urol. 2010; 58: 118 - 126.

Siracusano S, Novara G, Antonelli A, Artibani W, Bertini R, Carini M, Carmignani G, CicillatoS, Cunico SC, Lampropoulou N, Longo N, Martorana G, Minervini A, Mirone V, Simeone C, Simonato A, Valotto C, Zattoni F, Ficarra V; members of the SATURN project – LUNA foundation. Prognostic role of tumour multifocality in renal cell carcinoma. BJU Int. 2012; 110: E443 – 448. Doi: 10.1111/j.1464-410X.2012.1112.x.

Lehman DS, Hruby GW, Phillips CK, Mckiernan JM, Benson MC, Landman J. Laparoscopic renal cryoablation: efficacy and complications for larger renal masses. J Endourol. 2008; 22(6): 1123 - 1128.

Chawla SN, Pierre JS, Hanlon AL, Greenberg RE, Chen DY, Uzzo RG. The natural history of observed enhancing renal masses: meta-analysis and review of the world literature. J Urol. 2006; 175: 425 – 431.

149

150

summary

The cellular biophysical response to freezing and thawing is directly related to the behavior of water molecules in the intracellular and extracellular environment. Whether tissue is damaged as a result of freezing and thawing is dependent on cell injury, tissue structure injury and the functional impairment of the tissue compounds. The associated different cryobiological responses leading to cell death can be caused by four main factors, of which 1, 2 the first is immediate cell injury . The second is that of freezing induced 3 apoptosis . The third cause that can lead to tissue injury is the 4, 5 immunological response to cryoablation . And the fourth cause that is considered to play a pivotal role in contribution to the freezing injury is the initiated vascular response. Multiple, mutually dependent, mechanisms are responsible for initiating vascular stasis and subsequent tissue necrosis after 6 freeze . In previous reports, damage to the endothelium, ischemiareperfusion injury, inflammation and the resulting loss of microcirculatory support are essential for defining the limits of the cryolesion and the 6-11 triggering of the induction of apoptosis and cellular necrosis . Chapter 2 describes a novel method for studying the renal arterial vascular anatomy and distribution of vessel diameter. In this pilot study, we assessed the combined use of a fluorescent casting technique, cryomicrotome imaging, and three-dimensional (3-D) computer analysis as a new static method for visualizing and reconstructing the vascular anatomy in a porcine 12 renal model . Using this technique, we were able to visualize and reconstruct the renal arterial blood supply in 3-D. The arterial geometry of the complete porcine kidney could be visualized at a resolution of up to 50Âľm of the whole. Following cryosurgery, cessation of blood flow within the ablated area will start with the immediate destruction of microvessels and will be exacerbated by the vascular response and this will ultimately lead to the cessation of 6, 13 blood flow in the larger vessels . In Chapter 3 we examined if the method as described in chapter 2 could be used to visualize this process and thereby establish a microvascular 14 diameter threshold for acute vascular damage . Here again we used the porcine kidney for investigational purposes. The key finding was that we were able to show that renal parenchymal cryoablation injury immediately destroys arteries smaller than 180Âľm. However, the branching structures of larger arteries remained anatomically intact and connected to vascular structures in the surrounding tissue. Studies of contrast-enhanced imaging report that on the day following clinical renal mass cryoablation showed that, 15, 16 in some cases, blood flow could be found in the cryoablation zone . As described in chapter 6, this phenomenon was also noticed using contrastenhanced ultrasound. However, one should beware that residual vascular structures that conduct contrast medium through the ablated area may go unnoticed in images by different type of contrast-imaging methods because

152

of resolution issues. According to the distribution pattern of the fluorescent cast, it was postulated that contrast medium passes rapidly through the residual larger vessels in the ablated region but is not distributed to the smaller vessels in the ablated area, although it is distributed in bordering non-ablated tissue. Because of that in the acute phase following cryoablation still patent vessels could be noticed within the ablation zone we conducted the study as described in Chapter 4. In this in vivo study we attempted to quantify temporary changes in the vascular anatomical structure and blood 17 flow after cryosurgery in one of the poles of porcine kidney . Experiments were divided into five groups of 4 kidneys each with different survival times, including group 1 - 20 minutes, group 2 - 4 hours, group 3 - 2 days, group 4 1 week and group 5 - 2 weeks. In each pig simultaneous open bilateral renal cryoablation was performed on the anterior side of the lower or upper pole. Total freezing time was 20 minutes using a double freeze-thaw cycle. One of the key findings was that some anatomically arterial vessels were still present after 2 days but after one week no detectable vascular structures remained within the ablation zone. In the same pig study cohort, we investigated whether these anatomically intact vessels after cryoablation also transport blood. For this, fluorescently labeled microspheres were injected in the descending aorta before kidney removal. We found that in the first group the average relative microsphere density in the ablated zone decreased to 2% compared with that of the control value in the other groups the microsphere density was practically zero. In the first group, larger vessels in the ablation zone that remained patent in the ablation zone were found to transport hardly any blood to the ablated tissue. In this early phase, microspheres were transported to the outer perimeter of the non-ablated renal parenchyma, but were practically absent in the ablated core. However, by 1 week after ablation, the vascular structures in the ablation crater had disappeared completely and microvessels in the border zone were only connected to vascular structures approaching the crater wall from outside. The key finding in this study is that in a clinical setting after renal mass cryoablation, it is expected that blood vessel transport has ceased and vessel destruction should be fully complete after 2 weeks. It has therefore been postulated that since vessel destruction is complete after 2 weeks intravenous contrast measurement can be considered a true indicator of ablation success. Contrast-enhanced computed tomography- (CT)/ magnetic resonance- (MR)/ ultrasonography (US) imaging within 2 weeks following renal mass cryoablation may signal a false positive because of slight focal or rim enhancement of the cryoablation zone. A key point in the process of choosing the best-fit treatment for a patient is to estimate the intraoperative and postoperative risk for complications. Since 18, 19 the introduction of nephrometry indices in 2009 , several study groups reported that highly renal tumor anatomical complexity is related to a higher risk of complications compared to those with low complexity following 18, 20-22 extirpative surgery . Recently, this association was also found in

153

23-27

patients following ablative treatment . In Chapter 5 we investigated the impact of the RENAL and PADUA nephrometry indices on the assessment of intraoperative and postoperative complications in a cohort of 99 patients with primary renal tumors treated with LCA. A total of 23 intraoperative complications occurred in 19 of the 99 procedures. In the postoperative period up to 30 days, a total of 23 complications occurred in 20 patients. For the risk of intraoperative complications, there was a significant correlation (p<0.05) with tumor diameter in millimeters (p=0.008), tumor surface (p=0.008), tumor volume (p=0.005), the RENAL domains “R-size” (p<0.027), “N-nearness to the collecting system” (p<0.034), “RENAL score” (p<0.047), and the domain “diameter” of the PADUA-index (p<0.027). In terms of multivariate analysis, the tumor diameter, measured in millimeters, was a determining factor for predicting of intraoperative complications of LCA. The domains “diameter” of the RENAL and PADUA indices were both significantly correlated with the risk for intraoperative complications but were not determining factors. Lehman et al reported that tumor size appeared to be a key metric for incomplete ablations and the risk for complications 28 following LCA . This study confirms that patients with larger tumors have an increased risk for treatment failure. The threshold tumor diameter of 35mm (odds ratio 5.32; 95% confidence interval) was found to be predictive for an increased risk for intraoperative complications performing LCA. The study showed no significant correlation between postoperative complications and the total score or anatomical complexity grade of both nephrometry indices. Since only the RENAL domain “N-nearness to the collecting system” was the only significant factor predicting for postoperative complications, no multivariate analysis was performed. Other factors as surgical approach (retro- or transperitoneal), upper pole located tumors and hilar located tumors did not significantly correlate with an increased risk for intraoperative complications performing LCA. Currently, using complication as a variable, the RENAL and PADUA indices cannot be used as a discriminator between partial nephrectomy and cryoablation. A clear limitation of these nephrometry indices is that they are conducted by categorically scoring anatomical parameters reflecting the event of complicated surgery. However, the reference, or cut-off, for the categorical division per parameter is arbitrary. In future, adjustments to the categorical indexation of the nephrometry indices may improve the decision-making process regarding which type of treatment for a small renal mass is desired. The observation that the standard follow-up, using contrast-enhanced computed tomography (CT), in patients treated with cryoablation for a small renal mass and diagnosed with declined renal function runs the risk of jeopardizing the remaining renal function led to the study of alternative imaging methods. The development of contrast-induced nephropathy is a significant complication of intravascular contrast medium use that is related 29, 30 to excess morbidity and mortality . The most important risk factor is preexisting renal impairment, which increases the risk for contrast induced

154

nephropathy by more than 20 times . Therefore, alternative imaging not requiring radiographic contrast medium should be considered if the alternate imaging could adequately address the diagnostic questions. In Chapter 6, using Contrast Enhanced UltraSonography (CEUS) as an alternative method of imaging in patients with renal tumors treated with 16, 32 cryoablation is described . In this study, an Acuson Sequoia ultrasound (Siemens Medical, Mountain View, CA, USA) and a 4C1 transducer, equipped with contrast pulse sequence (CPS) was used. The major benefits of CEUS are its easy applicability, low cost, and safe contrast medium. Furthermore, the high sensitivity to micro-vascular perfusion might enable CEUS to image the short-term dynamic process of vascular injury after cryoablation. Perfusion defects after renal mass cryoablation were identified and scored in 6 patients (perfusion score 0-4). In most patients (n=5), no CPS signals were detected inside the lesion (perfusion score 0), and thus according to CPS imaging, no perfusion was present. In all five patients, these findings corresponded with the contrast CT/MRI results. In one patient, no perfusion was detected using either CPS or CT imaging one month after cryoablation. However, 8 months after the ablation using CPS imaging, perfusion was observed inside the lesion (perfusion score 3 and 2) whereas no enhancement was found using contrast CT. The implication of this finding could not be that CPS imaging is a more sensitive technique for detecting reperfusion than CT. However, the other possibility is a false-positive CPS result. Barwari et al have studied, following LCA, the difference in 33 enhancement that contrast CT/MR and CEUS were able to detect . At three months follow up assessed in 32 patients, they found a specificity and negative predictive value of 92% and 77% respectively. At one year follow up assessed in 21 patients, the specificity found was 90% with a negative predictive value of 100%. However, the absence of focal recurrences in this series makes it difficult to draw conclusions on the sensitivity and positive predictive value of CEUS as a method used for follow-up. It can be questioned how the perfusion patterns of focal recurrences of ablated tumors will appear. However, the results of the present study show that CPS imaging can be used to describe the perfusion characteristics of the cryoablation lesions. Therefore, this justifies the implementation of larger prospective studies with long term follow-up in order to determine the exact value of this technique for detecting and discriminating vital tumor remnant, recurrent tumor or reperfusion of the scar tissue. Currently, CEUS is not likely to replace standard CT or MR imaging, however, it can be a valuable alternative in selected cases. For patients with a small renal mass treated with CA, and with relative or imperative contra-indications for the use of contrast-agents at CT or MR imaging, CEUS may be a reasonable alternative technique for imaging at follow-up. In the future, however, this method can possibly be used to lower the total number of contrast CT/MR needed at long-term follow-up.

155

Another alternative imaging method for the follow up of cryoablated solid 18 renal tumors is described in Chapter 7. The F-FDG PET-CT findings 18 before and after laparoscopic renal cryoablation were reported. F-FDG PET-CT can possibly be a reasonable alternative to the routine mode of imaging in those patients with relative or imperative contra-indications for the use of intravenous administrated contrast-agents used in CT or MR imaging. 18 The hypothesis of this study was that if F-FDG metabolisms could be detected in renal mass before cryoablation, it should not be detectable in the target zone after cryoablation. In this series we performed a PET-CT before ablative surgery in 7 cases. It was clearly noticed that metabolic uptake of 18 F-FDG can be detected in solid renal lesions and, therefore, this could be used as a reference to discriminate for metabolic activity after renal mass cryoablation. In all those cases we found a significant decrease of metabolic FDG-uptake in the centre of the cryoablated tumors. However, in four patients, metabolic activity was noticed at the rim of the ablation assessed 18 by F-FDG PET-CT after LCA. In one patient this was found in the peritumoral fat at the third consecutive PET-CT after almost three years post-LCA, whereas previous scans showed no peripheral metabolic activity. Therefore, this must be considered as possible metastases. In the three other patients at the first PET-CT following cryoablation, the metabolic activity of peripheral rim was encircling the metabolic inactive ablation zone as noticed. This seems comparable to earlier reported findings at CT and MRI where rim enhancement around the ablation zone was noticed in 34-36 several cases 1 month after treatment . This peri-ablational enhancement is considered benign. During the first months after ablation this finding is of no consequence; however, if it were to occur later at followup, that could be interpreted as a sign of recurrence and will typically appear as irregular peripheral enhancement. In one patient, FDG-accumulation was found in the rim of the ablation zone at the dorsal-central border. Initially this was not recognized as suspect for focal recurrence. However, contrastenhanced CT showed focal enhancement, and therefore the patient underwent consecutive biopsy and cryosurgery. Histology confirmed the presence of viable RCC tissue. The major limitation of this study was that only a few patients, at different times after CA, were studied in order to 18 examine the possibility of using F-FDG PET-CT imaging for characterization of the ablated renal lesion. No longitudinal data were collected and no direct comparison with CT/MRI was intended. Furthermore, the majority of PET-CT findings are not confirmed by the lack of histology. Therefore, the accuracy of PET-CT could not be assessed.

156

Sam envatting De cryo-biofysische respons van een cel is afhankelijk van het gedrag van de watermoleculen in het intra- en extracellulair milieu. Of weefseldestructie als gevolg van bevriezing en ontdooiing plaatsvindt hangt af van cellulaire beschadiging, afbraak van weefsel verbindende structuren en functionele beschadiging van de algehele samenstelling van weefsel. Er zijn vier verschillende cryobiologische reacties die leiden tot celdood. De eerste is de 1, 2 directe cellulaire beschadiging leidend tot acute celdood . Een andere is 3 cryo-geïnduceerde apoptose leidend tot indirecte celdood . Een door bevriezing geïnitieerde immunologische reactie is de derde aanleiding tot 4, 5 indirecte celdood . De vierde reactie, de vasculaire respons, speelt een centrale rol in de bijdrage tot weefselschade als gevolg van bevriezing. Verschillende, onderling afhankelijke, processen zijn verantwoordelijk voor het door bevriezing ontstaan van blokkade van de doorbloeding en de 6 weefselnecrose als gevolg hiervan . Volgens eerdere rapportages, zijn beschadiging van het endotheel, schade tengevolge van ischemie gevolgd door hernieuwde perfusie, lokale ontstekingreactie en het verlies van de microcirculatie, essentieel ter bepaling van de uitbreiding van de cryoablatie zone. Tevens bevorderen deze de aanzet van apoptose en cellulaire 6-11 necrose . Hoofdstuk 2 beschrijft een nieuwe methode om de arteriële vasculaire anatomie en de verhouding van verschillende vaatdiameters in een nier te bestuderen. In deze pilotstudie staat het gecombineerde gebruik van een fluorescerend afgietsel techniek, de beeldvorming met behulp van cryomicrotoom en een driedimensionale (3-D) computer analyse centraal. Deze combinatie wordt beschreven als een statisch model voor het zichtbaar maken en reconstrueren van de anatomische vaatboom in een 12 model van een varkensnier . Het bleek met deze techniek mogelijk om de renale arteriële bloedvoorziening in 3-D zichtbaar te maken en te reconstrueren. Zo werd de arteriële geometrie van een complete varkensnier zichtbaar tot een resolutie van 50 µm. Het beëindigen van de bloedcirculatie in de ablatie zone na cryochirurgie begint met de onmiddellijke destructie van de microvasculatuur en is afgerond wanneer de vasculaire respons uiteindelijk ook de bloedcirculatie 6, 13 in de grotere vaattakken tot stilstand brengt . In Hoofdstuk 3 is bestudeerd of de methode zoals die in hoofdstuk 2 beschreven is gebruikt kon worden om het hierboven genoemde fenomeen zichtbaar te maken en een bepaalde vaatdiameter vast te stellen waarbij de 14 acute microvasculaire schade optreed . Hiervoor werd wederom de varkensnier als studiemodel gebruikt. Een fundamentele bevinding was dat het op deze wijze mogelijk was om zichtbaar te maken dat cryoablatie van

157

het nierparenchym onmiddellijk de arteriën kleiner dan 180 µm vernietigd. Terwijl in deze acute fase na bevriezing grotere vaten anatomisch intact blijven en in verbinding staan met vasculaire structuren buiten het ablatie gebied. Rapportages van contrast beeldvorming op de dag na cryoablatie vermelden dat in sommige gevallen bloedcirculatie aanwezig is in de ablatie 15, 16 zone . In hoofdstuk 6 is beschreven dat dit fenomeen ook is vastgesteld wanneer echografie met intravasculair contrastmedium word gebruikt. Door de beperking van de resolutie van verschillende methoden van contrastbeeldvorming kan het echter mogelijk zijn dat resterende contrast transporterende vasculaire structuren onopgemerkt blijven. Volgens het distributiepatroon van het fluorescerend afgietsel wordt verondersteld dat het contrastmedium binnen de ablatie zone snel door de grotere vaten passeert maar dat er geen distributie naar kleinere vaten binnen de ablatie zone is, waar dat wel daarbuiten plaatsvindt. Het feit dat in de acute fase na cryoablatie nog steeds patente vasculatuur in de ablatie zone aangetroffen werd, was aanleiding voor het verdere onderzoek zoals is beschreven in Hoofdstuk 4. In deze in vivo studie werd gepoogd de tijdelijke veranderingen in de anatomische vaatstructuur en bloedcirculatie in één van 17 de polen van de varkensnier, als gevolg van cryochirurgie, te bestuderen . De experimenten werden verdeeld over vijf groepen, met elk vier nieren. Iedere groep heeft een verschillende overlevingstijd na cryoablatie: groep 1 – twintig minuten, groep 2 – vier uren, groep 3 – twee dagen, groep 4 – een week en groep 5 – twee weken. In ieder varken werd simultaan de anterieure zijde van de onder- dan wel de bovenpool van de linker en rechter nier met cryochirurgie behandeld. De totale bevriezingstijd was twintig minuten gedurende een zogenaamde dubbele vries-dooi cyclus. Een belangrijke bevinding van deze studie was dat enkele arteriële bloedvaten na twee dagen nog anatomisch intact waren maar dat na een week er geen vasculaire structuren meer gevonden werden binnen de ablatiezone. In deze zelfde studiegroep onderzochten wij of de anatomisch intacte vasculatuur na cryoablatie ook nog bloed transporteert. Hiervoor werden fluorescent gekleurde microsferen in de aorta descendens geïnjecteerd voordat de nier werd uitgenomen. In vergelijk met de controle groep nam de gemiddelde relatieve densiteit van microsferen in de ablatie zone af met 2%. In alle andere groepen was de microsfeer densiteit nagenoeg 0%. De grotere vaten in de eerste groep die anatomisch intact waren bleken nauwelijks bloed te transporteren naar het weefsel in de ablatie zone. In deze vroege fase, werden de microsferen wel getransporteerd naar het buitenste grensgebied van de ablatie zone maar niet meer naar het centrale deel van de ablatie zone. Eén week na cryoablatie zijn er in de ablatie regio geen vasculaire structuren meer zichtbaar, maar zijn de microsferen die aangetroffen worden in de periferie van de ablatie zone afkomstig uit het stroomgebied van bloedvaten direct buiten het ablatie gebied. Gezien deze constatering verwachten wij dat, in een klinische situatie, twee weken na complete cryoablatie van een niertumor in het ablatie gebied, een totale destructie van alle vasculaire structuren heeft plaatsgevonden en zodoende

158

het bloedtransport is gestopt. Wij veronderstellen dat na twee weken, wanneer de vasculaire destructie in het ablatie gebied is afgerond, beeldvorming met intraveneus contrast gebruikt kan worden als een indicator voor het ter bepaling van het succes van de behandeling. In de eerste twee weken na cryoablatie kan de beoordeling van de ablatie zone door middel van CT, magnetic resonance (MR) of echografische (US) beeldvorming met intraveneus contrast ten onrechte worden beschouwd als een incomplete ablatie op basis van de aanwezigheid van nog aanwezige focale dan wel rand aankleuring. Bij de beslissing welke behandeling voor een patiënt het meest in aanmerking komt speelt de inschatting van het intra-operatieve en postoperatieve complicatie risico een belangrijke rol. Sinds de introductie van de 18, 19 nefrometrie indices in 2009 hebben verschillende studiegroepen laten zien dat in het geval van extirpatieve chirurgie een hoge anatomische complexiteit van niertumoren is gerelateerd aan een hoger risico voor complicaties, vergeleken met patiënten met een lage anatomische 18, 20-22 complexiteit van de niertumor . Recent onderzoek liet deze associatie ook zien bij patiënten met niertumoren die behandeld zijn met ablatie 23-27 technieken . In Hoofdstuk 5 bestudeerden wij de correlatie tussen de RENAL en PADUA nefrometrie indices en de intra-operatieve en post-operatieve complicaties in een cohort van 99 patiënten met een primaire niertumor die zijn behandeld met laparoscopische cryoablatie. In totaal 23 intra-operatieve complicaties deden zich voor in 19 van de 99 procedures. Gedurende de post-operatieve periode van 30 dagen, deden zich 23 complicaties voor bij 20 patiënten. Het risico voor chirurgische complicaties had een significante correlatie (p<0.05) met de diameter van de tumor in millimeters (p=0.008), de tumor oppervlakte (p=0.008), het tumor volume (p=0.005), de RENAL domeinen “R-size” (p<0.027) en “N-nearness to the collecting system” (p<0.034), “RENAL score” (p<0.047), en het domein “diameter” van de PADUA-index (p<0.027). Bij multivariate analyse bleek de diameter van de tumor, gemeten in millimeters, een determinant voorspellende factor voor intra-operatieve complicaties bij LCA te zijn. Het domein “diameter” van de RENAL en de PADUA indices waren beiden significant gecorreleerd met het risico voor intra-operatieve complicaties, maar bleken niet determinant bij multivariate analyse. Lehman et al. rapporteerden dat de diameter van de tumor een bepalende factor is voor het voorspellen van incomplete ablaties en het 28 risico van complicaties bij LCA . Onze studie bevestigd dat patiënten met grotere tumoren meer kans hebben op falen van de behandeling. Een grenswaarde van 35 mm diameter van de tumor (odds ratio 5.32; 95% betrouwbaarheidsinterval) bleek voorspellend voor een verhoogd risico op het voorkomen van intra-operatieve complicaties bij LCA. In de studie werd geen significante correlatie aangetoond tussen post-operatieve complicaties en de totaal score van de anatomische complexiteitsgraad van beide

159

nefrometrie indices. Omdat alleen het RENAL domein “N-nearness to the collecting system” een significante voorspeller bleek voor het voorkomen van post-operatieve complicaties, werd geen multivariate analyse verricht. Andere factoren zoals de chirurgische benadering (retro- of transperitoneaal), tumoren gelokaliseerd in de bovenpool van de nier en tumoren gelegen in de hilus van de nier bleken geen significante correlatie te hebben met het voorkomen van intra-operatieve complicaties bij LCA. De RENAL en PADUA indices kunnen momenteel niet behulpzaam zijn bij het maken van een keuze tussen een partiële nefrectomie of een laparoscopische cryoablatie, als het doel is om de kans op een complicatie als gevolg van de behandeling te minimaliseren. Een duidelijke beperking van de nefrometrische indices, gebruikt ter beschouwing van de mogelijkheid op gecompliceerde chirurgie, is dat zij tot stand zijn gebracht door de anatomische parameters categorisch te scoren. Echter de referentie of grenswaarden van iedere categoriale verdeling per parameter zijn arbitrair gekozen. Aanpassingen van de categoriale verdelingen van de nefrometrische indices zal dan ook noodzakelijk zijn om deze indices te kunnen gebruiken voor de advisering en de besluitvorming rond de meest gewenste operatieve methode voor een kleine niertumor. De observatie dat de standaard follow-up door middel van intraveneuscontrast CT bij patiënten met een beperkte nierfunctie en behandeld door middel van cryoablatie. voor een kleine niertumor niet zonder risico is voor de bestaande nierfunctie heeft geleid tot het onderzoeken van alternatieve follow-up methoden van beeldvorming. Een door contrast veroorzaakte nefropathie is een significante complicatie van het gebruik van intravasculair contrastmedium en is gerelateerd aan een overdaad aan morbiditeit en 29, 30 mortaliteit . De belangrijkste bijdragende factor in deze is de reeds bestaande beperking van de nierfunctie welke het risico op progressie van de nefropathie als gevolg van contrast gebruik, verhoogd met een factor 20 31 . In geselecteerde gevallen is het wenselijk wanneer alternatieve beeldvorming, zonder het gebruik maken van radiografisch intraveneus contrastmedium, adequaat de diagnostische vraagstelling kan beantwoorden. In Hoofdstuk 6, is het gebruik van Contrast Versterkte Echografie (CEUS) als alternatieve beeldvormingstechniek bij patiënten met 16, 32 een, met cryoablatie behandelde, niertumor beschreven . In deze studie werd gebruik gemaakt van een Acuson Sequoia echo-apparaat (Siemens Medical, Mountain View, CA, USA) en een 4C1 transducer toegerust met Contrast Pulse Sequence (CPS). De voordelen van CEUS zijn de eenvoudige toepasbaarheid, de lage kosten en het gebruik van een veilig contrastmedium. De hoge sensitiviteit van CEUS voor het detecteren van microvasculaire perfusie maakt dat de methode, kort na cryoablatie, in theorie geschikt is voor de beeldvorming van het dynamische proces van de vasculaire veranderingen. De perfusie defecten in de ablatie zone na cryoablatie van een niertumor werden geïdentificeerd en gescoord bij zes patiënten (perfusie score 0-4). In de meeste gevallen (n=5) werden geen

160

CPS-signalen gedetecteerd in de ablatie zone en werd dit beschouwd als afwezigheid van perfusie (perfusie score 0). In al deze gevallen kwam deze bevinding overeen met de resultaten zoals geconstateerd bij contrastCT/MR. Bij één patiënt werd 1 maand na cryoablatie, zowel bij CPS als bij CT-beeldvorming, geen perfusie in de ablatie zone geconstateerd. Acht maanden na de ablatie werd met CEUS perfusie geconstateerd in de ablatie zone (perfusie score 2-3). Echter de contrast-CT liet geen aanwijzing voor perfusie zien. Dit impliceert niet perse dat de CPS beeldvorming meer sensitief is voor de detectie van perfusie dan CT. Er bestaat ook de mogelijkheid van een vals-positieve CPS bevinding. Barwari et al. bestudeerden de overeenstemming tussen de detectie van contrast, 33 aangetoond door middel van contrast CT/MR, en die van CEUS na LCA . Bij 32 patiënten met drie maanden follow-up vonden zij respectievelijk een specificiteit en een negatief voorspellende waarde van 92% en 77%. Bij 21 patiënten met een follow-up van 1 jaar was de specificiteit 90% met een negatief voorspellende waarde van 100%. In deze studie zijn er geen patiënten met een bewezen locaal recidief niertumor na cryoablatie. Er kan dus geen goede uitspraak worden gedaan over de sensitiviteit en de positief voorspellende waarde van CEUS. Zo kunnen we op basis hiervan niet concluderen dat CEUS een geschikt alternatieve methode is voor de followup. Het is onduidelijk hoe precies de perfusie patronen van een locaal recidief na cryoablatie zullen zijn. De huidige studie laat echter wel zien dat CPS beeldvorming gebruikt kan worden om de perfusie karakteristieken van cryoablatie gebieden te beschrijven. Prospectieve studies met een lange termijn follow-up zullen nodig zijn om te kunnen bepalen of deze methode geschikt is voor de detectie van- en het onderscheid te kunnen maken tussen incomplete ablatie, recidief tumor of benigne reperfusie van littekenweefsel. Op dit moment is er geen reden standaard contrast CT of MR beeldvorming te vervangen door CEUS. Echter CEUS kan als alternatieve methode gebruikt worden voor de follow-up van patiënten die een cryoablatie voor een kleine niertumor hebben ondergaan en waarbij een relatieve dan wel imperatieve contra-indicatie bestaat voor het gebruik van intraveneuze contrastmiddelen bij CT en/of MR. In de toekomst kan de methode mogelijk wel ingezet worden om het totaal aantal benodigde contrast CT/MR studies, gedurende een lange termijn follow-up, te verminderen. Een volgende alternatieve beeldvormende techniek als methode voor de follow-up van kleine niertumoren, behandeld met cryoablatie, is beschreven 18 in Hoofdstuk 7. Hierin worden de bevindingen van F-FDG PET-CT beeldvorming voor en na laparoscopische cryoablatie besproken. Voor patiënten met een relatieve of imperatieve contra-indicatie voor het gebruik 18 van intraveneus contrast, ten tijde van CT- of MR beeldvorming, kan de FFDG PET-CT mogelijk een alternatieve vorm van beeldvorming zijn. De 18 hypothese van deze studie was dat wanneer F-FDG metabolisme gevonden werd in een niertumor voorafgaand aan cryoablatie, dit in principe na een complete ablatie van de tumor niet meer aangetroffen werd. Bij

161

zeven patiënten werd een F-FDG PET-CT voorafgaand aan de cryoablatie 18 verricht. In alle gevallen werd duidelijk metabole opname van F-FDG in de tumor geconstateerd en kon deze bevinding worden gebruikt als referentie ter beoordeling van metabole activiteit in de laesie na cryoablatie. In het centrum van de ablatie zone werd in alle gevallen een duidelijke afname van 18 metabole F-FDG activiteit gevonden. Bij vier patiënten werd echter wel metabole activiteit in de rand van de ablatie zone gevonden. Bij één patiënt 18 werd na drie jaar nieuw ontstane F-FDG activiteit gevonden in het peritumorale vet. De PET-CT scans die eerder in de follow-up waren verricht hadden deze activiteit niet laten zien. Deze nieuwe activiteit moet dan ook worden beschouwt als mogelijke metastase. Bij de drie overige patiënten, werd ten tijde van de eerste PET-CT na cryoablatie, metabole randactiviteit gevonden rondom de metabole inactieve ablatie zone. In eerdere studies, een maand na cryoablatie, is een vergelijkbare activiteit van randaankleuring 34-36 rond de ablatie zone gerapporteerd bij CT/MR . Deze aankleurende zone rondom de ablatie word echter als benigne beschouwd. Tot enkele maanden na de ablatie heeft deze bevinding geen consequenties. Echter wanneer dit beeld zich als nieuw fenomeen voordoet later gedurende de follow-up dan moet het worden geïnterpreteerd als een recidief. De aankleuring is dan niet rondom, maar focaal en heeft een irregulair aspect. Bij één patiënt werd een FDG accumulatie gevonden in de dorso-centrale rand van de ablatie zone. Initieel werd dit niet duidelijk herkend en dus niet geïnterpreteerd als tumorrecidief, echter contrast-CT-scan liet focale aankleuring zien verdacht voor tumorrecidief. Het focaal recidief werd gebiopteerd en vervolgens behandeld door middel van CT-geleide percutane cryoablatie. Histologisch onderzoek bevestigde de aanwezigheid van vitale tumorcellen passend bij niercelcarcinoom. Het geringe aantal patiënten en de PET-CT studies verricht op verschillende tijden na cryoablatie zijn de belangrijkste beperkingen van deze studie. Er werden geen longitudinale data vergaard en in de opzet van de studie is er geen vergelijk met CT/MRI gepland. De accuratie van de PET-CT bevindingen zijn in de meeste gevallen niet bevestigd door middel van histologie.

162

Referenties

Baust JG, Gage AA. The molecular base of cryosurgery. BJU Int. 2005; 1187 - 1191.

Baust JG, Gage AA, Clarke D, Baust JM, VanBuskirk R. Cryosurgery-a putative approach to molecular-based optimization. Cryobiology. 2004; 48(2): 190 - 204.

Clarke DM, Robilotto AT, Rhee E, VanBuskirk RG, Baust JG, Gage AA, Baust Cryoablation of renal cancer: variables involved in freezing-induced death. Technol Cancer Res Treat. 2007; 6(2): 66 - 79.

Sabel MS. Cryo-immunology: a review of the literature and proposed mechanisms for stimulatory versus suppressive immune responses. Cryobiology 2009; 58(1): 1 - 11.

Hoffmann NE, Bischof JC. The cryobiology of cryosurgical injury. Urology 2002; 60(2 Suppl 1): 40 – 49.

Lewis T, Love WS. Vascular reactions of the skin to injury. Part III: Some effects of freezing, of cooling and of warming. Heart 1926; 13: 27 - 60.

Mundt ED. Studies on the pathogenesis of cold injury: Microcirculatory changes in tissue injured by freezing. Proc Symp Artic Biol Med 1964; 4, 51 - 72.

Quantanilla R, Krusen F, Essex H. Studies on frostbite with special reference to treatment and the effect on minute blood vessels. Am J Physiol 1947; 149(1): 149 - 161.

Rabb JM, Renaud ML, Brandt A, Witt CW. Effect of freezing and thawing on the microcirculation and capillary endothelium of hamster cheek pouch. Cryobiology 1974; 11(6): 508 - 518.

Lagerveld BW, ter Wee RD, de la Rosette JJ, Spaan JA, Wijkstra H. Vascular fluorescence casting and imaging cryomicrotomy for computerized three-dimensional renal arterial reconstruction. BJU Int. 2007; 100(2): 387 – 391.

Daum PS, Bowers WD Jr, Tejada J, Hamlet MP. Vascular casts demonstrate microcirculatory insufficiency in acute frostbite. Cryobiology 1987; 24(1): 65 - 73.

163

95(9):

JM. cell

Anderson JK, Shingleton WB, Cadeddu JA. Imaging associated with percutaneous and intraoperative management of renal tumors. Urol Clin N Am. 2006; 33: 339 - 352.

Kutikov A, Uzzo RG. The R.E.N.A.L. nephrometry score: a comprehensive standardized system for quantitating renal tumor size, location and depth. J Urol. 2009; 182:844-853.

Simhan J, Smaldone MC, Tsai KJ, Canter DJ, Li T, Kutikov A, Viterbo R, Chen DY, Greenberg RE, Uzzo RG. Objective measures of renal mass anatomic complexity predict rates of major complications following partial nephrectomy. Eur Urol. 2011; 60:724-730.

Hew MN, Baseskioglu B, Barwari K, Axwijk PH, Can C, Horenblas S, Bex A, Rosette JJ, Pes MP. Critical appraisal of the PADUA classification and assessment of the R.E.N.A.L. nephrometry score in patients undergoing partial nephrectomy. J Urol. 2011; 186:42-46.

164

Lehman DS, Hruby GW, Phillips CK, McKiernan JM, Benson MC, Landman J. Laparoscopic renal cryoablation: efficacy and complications for larger renal masses. J Endourol. 2008; 22(6): 1123 - 1128.

Levy, EM, Viscoli CM, Horwitz RI. The effect of acute renal failure on mortality. A cohort analysis. JAMA. 1996; 275(19): 1489 - 1494.

Rudnick MR, Goldfarb S, Wexler L, Ludbrook PA, Murphy MJ, Halpern EF, Hill JA, Winniford M, Cohen MB, VanFossen DB. Nephrotoxicity of ionic and nonionic contrast media in 1196 patients: a randomized trial. The Iohexol Cooperative Study. Kidney Int. 1995; 47(1): 254 - 261.

Wink MH, Lagerveld BW, Laguna MP, de la Rosette JJ, Wijkstra H. Cryotherapy for renal-cell cancer: diagnosis, treatment, and contrast-enhanced ultrasonography for follow-up. J Endourol. 2006; 20(7): 456 - 458: discussion 458 459.

Remer EM, Weinberg EJ, Oto A, Oâ&#x20AC;&#x2122;Malley CM, Gill IS. MR imaging of the kidneys after laparoscopic cryoablation. AJR Am J Roentgenol. 2000; 174(3): 635 - 640.

Bolte SL, Ankem MK, Moon TD, Hedican SP, Lee FT, Sadowksi EA, Nakada SY. Magnetic resonance imaging findings after laparoscopic renal cryoablation. Urology. 2006; 67: 485 â&#x20AC;&#x201C; 489.

Rutherford EE, Cast JE, Breen DJ. Immediate and long-term CT appearances following radiofrequency ablation of renal tumours. Clin Radiol. 2008; 63(2): 220 230.

165

166

abbreviations

In alphabetical order ASA AUA ACS a-CCI BMI CA CAIX CCI CCS CEUS CPS CT EAU FDG GFR-MDRD HU IOC IVU LCA MIP MR MRI NSS PADUA PET PN POC RAE RCC RENAL

RFA RN SD SEM SPSS SUV TACE TNM

American Society of Anesthesiologists American Urology Association anatomical classification systems age-adjusted Charlson comorbidity index body mass index cryoablation carbonic anhydrase IX Charlson comorbidity index Clavien-Dindo complication classification contrast enhanced ultrasound contrast pulse sequence computed tomography European Association of Urology fluorodeoxyglucose glomerular filtration rate â&#x20AC;&#x201C; modification of diet in renal diseases Hounsfield units intra-operative complications intravenous urography laparoscopic cryoablation maximum intensity projection magnetic resonance magnetic resonance imaging nephron-sparing surgery preoperative aspects and dimensions used for anatomical classification positron emission tomography partial nephrectomy post-operative complications renal arterial embolization renal cell cancer (R)adius, (E)xophytic/endophytic properties, (N)earness to the collecting system, (A)nterior/posterior location, (L)ocation relative to the polar line. radio frequency ablation radical nephrectomy standard deviation scanning electron microscopy statistical package for social sciences software standard uptake value transarterial chemo-embolization tumour, lymph nodes, metastasis

168

US XR 2-D 3-D

ultrasonography X-ray two-dimensional three-dimensional

169

170

PUBLICATIONS AND ABSTRACTS

SUMMARY PUBLICATIONS Cryotherapy for renal-cell cancer: diagnosis, treatment, and contrast-enhanced ultrasonography for follow-up. Wink MH, Lagerveld BW, Laguna MP, de la Rosette JJ, Wijkstra H. J Endourol. 2006; 20(7): 456 â&#x20AC;&#x201C; 458: discussion 458 - 459. ____________________________________________________________ Contrast-enhanced ultrasonography in the follow-up of cryoablation or renal tumours: a feasibility study. Wink MH, Laguna MP, Lagerveld BW, de la Rosette JJMCH, Wijkstra H. BJU Int. 2007; 99(6): 1371 - 1375. ___________________________________________________________ Vascular fluorescence casting and imaging cryomicrotomy for computerized three dimensional renal arterial reconstruction. Lagerveld BW, ter Wee RD, de la Rosette JJ, Spaan JA, Wijkstra H. BJU Int. 2007; 100(2): 387 - 391. ___________________________________________________________ The immediate effect of kidney cryoablation on renal arterial structure in a porcine model studied by imaging cryomicrotome. Lagerveld BW, van Horssen P, Laguna Pes MP, van den Wijngaard JPHM, Streekstra GJ, de la Rosette JJMCH, Wijkstra H, Spaan JAE. J Urol. 2010; 183(3): 1221 - 1226. ___________________________________________________________ Gradient changes of porcine renal arterial vascular anatomy and blood flow after cryoablation. Lagerveld BW, van Horssen P, Laguna Pes MP, van den Wijngaard JPHM, Siebes M, Wijkstra H, de la Rosette JJMCH, Spaan JAE. J Urol. 2011; 186(2): 681 - 686.

SUBMITTED MANUSCRIPTS under review Accepted: Can RENAL and PADUA nephrometry indices predict for complications of laparoscopic cryoablation for clinical stage T1 renal tumors? Lagerveld BW, Brenninkmeijer M, van der Zee JA, van Haarst EP. Journal of Endourology 2013.

172

Submitted: 18 F-FDG PET-CT findings before and after laparoscopic renal cryoablation; an initial report. Lagerveld BW, Sandkuyl R, Sivro F, van der Zee JA, Baars P. British Journal of Urology International 2014.

RELATED ABSTRACTS Acute vascular changes in renal arterial anatomy after cryosurgery. Spaan J, Lagerveld BW, van Horssen P, van den Wijngaard J, de la Rosette J, Wijkstra H. Experimental Biology. New Orleans, United States of America, April 2009. Acute changes in renal arterial anatomy after cryosurgery in a porcine model. Lagerveld BW, van Horssen P, de la Rosette JJMVH, van den Wijngaard J, Wijkstra H, Spaan JAE, Laguna Pes MP. Urology, Volume 74, Issue 4, Supplement 1, October 2009, Page S253. Gradient changes of porcine renal arterial vascular anatomy and blood flow after cryoablation. Spaan J, Lagerveld BW, van Horssen P, van den Wijngaard J, de la Rosette J, Laguna P, Siebes M, Wijkstra H. Experimental Biology. Anaheim, United States of America, April 2010. Cryoablation induced alterations of porcine renal anatomy and blood flow. Lagerveld BW, van Horssen P, Laguna Pes MP, van den Wijngaard JPHM, Wijkstra H, Spaan JAE, de la Rosette JJMCH. J Urol, Volume 183, Issue 4, Supplement, 2010, Page e34. Laparoscopic renal mass cryoablation: relation between comorbidity and post-operative complications. Lagerveld BW, van Dijk MM, Noë AP, van der Zee JA. th European Association of Urology 27 annual congress. Paris, France, 24-28 February 2012. The value of 18F-FDG PET-CT for the evaluation of treatment success of cryosurgery for small renal mass; a pilot study. Lagerveld BW, Sandkuyl, R, Sivro F, van der Zee J, Baars P. nd Société International d’Urology 32 congress. Fukuoka, Japan, 1-4 October 2012. Comparing the distribution of anatomical complexity of small renal masses treated with cryoablation: PADUA versus R.E.N.A.L. nephrometry score. Lagerveld BW, van Dijk M. van der Zee JA. nd Société International d’Urology 32 congress. Fukuoka, Japan, 1-4 October 2012. De waarde van RENAL- en PADUA-indices bij het voorspellen van complicaties als gevolg van laparoscopische cryoablatie van niertumoren. Lagerveld BW, Brenninkmeijer M, van der Zee JA, van Haarst EP. Najaarsvergadering Nederlandse Vereniging voor Urologie, Nieuwegein, November 2012. Tijdschrift voor Urologie 2012; 2(7):180-181.

173

NAMES AND INSTITUTES OF THE AUTHORS INVOLVED IN THE MANUSCRIPTS AND ABSTRACTS (alphabetical order) Dr. P.C. Baars

Department of Nuclear Medicine, St. Lucas Andreas Hospital, Amsterdam, the Netherlands.

Drs. M. Brenninkmeijer

Department of Urology, Waterland Hospital, Purmerend, the Netherlands.

Dr. H. van Dekken

Department of Pathology, St. Lucas Andreas Hospital, Amsterdam, the Netherlands.

Drs. E.P. van Haarst

Department of Urology, St. Lucas Andreas Hospital, Amsterdam, the Netherlands.

Dr. P. van Horssen

Department of Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, the Netherlands.

Dr. M.P. Laguna Pes

Department of Urology, Academic Medical Center, Amsterdam, the Netherlands.

Drs. A.P. NoĂŤ

the Netherlands.

Prof. dr. J.J.M.C.H. de la Rosette

Department of Urology, Academic Medical Center, Amsterdam, the Netherlands.

Drs. R. Sandkuyl

Department of Urology, Onze Lieve Vrouwe Gasthuis, the Netherlands.

Dr. M. Siebes

Department of Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, the Netherlands.

Dr. F. Sivro

Department of Nuclear Medicine, St. Lucas Andreas Hospital, Amsterdam, the Netherlands.

Prof. dr. ir. J.A.E. Spaan

Department of Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, the Netherlands.

Dr. ir. G.J. Streekstra

Department of Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, the Netherlands.

174

Msc. R.D. ter Wee

Department of Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, the Netherlands.

Prof. dr. Ir. H. Wijkstra

Department of Urology, Academic Medical Center, Amsterdam, the Netherlands.

Drs. M.H. Wink

Department of Radiotherapy, Vlietland Hospital, Schiedam, the Netherlands.

Dr. J.P.H.M. van den Wijngaard

Department of Biomedical Engineering and Physics, Academic Medical Center, Amsterdam, the Netherlands.

Drs. J.A. van der Zee

Department of Urology, St. Lucas Andreas Hospital, Amsterdam, the Netherlands.

175

176

DANKWOORD

Zoals zo velen die mij voorgingen begon ook ik ooit onbevangen aan een promotie traject. Twee eerdere pogingen waren al in de kiem gesmoord. Niet deze keer. Het is nu klaar. Wetenschap bedrijven boeit, daagt uit en is leuk. Iets uit je zelf naar boven halen en delen met anderen geeft veel voldoening. Gelukkig doe je wetenschap niet altijd alleen. Ook bij dit project zijn tallozen op diverse wijzen betrokken geweest. Professioneel, uit wetenschappelijke interesse en uit liefde. Ik dank alle betrokkenen daarvoor. Het brengen tot een afgeronde promotie is een project waarin zich een heel leven kan afspiegelen. Al duizend maal ben ik in gedachten iedereen afgegaan die ik dank verschuldigd ben. Echter, allereerst gaan daarbij mijn gedachten uit naar mijn ouders. Ik dank hen voor de vele levenswijsheden die zij aan mij doorgaven en de liefde waarmee zij mij tot op vandaag hebben omarmt. Prof. Dr. J.J.M.C.H. de la Rosette, beste Jean, Veel hebben we elkaar niet meer gezien de laatste tijd. Dat hoeft ook niet altijd. Dit boekje is een aardige strijd geweest. De wetenschap dat jij altijd doorzettingsvermogen apprecieert gaf mij de steun om vooral door te gaan met deze promotie. Dank voor je vertrouwen. Ik heb bewondering voor je nietsontziende gedrevenheid om grote dingen tot stand te brengen en wens je veel succes met de talloze wetenschappelijke activiteiten waar je bij betrokken bent. Je originaliteit hierin is altijd herkenbaar voor mij. Prof. Dr. Ir J.A.E. Spaan, beste Jos, Tijdens een onderbreking van mijn promotieproject werd jij er bij betrokken en hielp je mij een doorstart te maken. En hoe! Je gaf leiding aan een geweldig team en nam mijn sores er probleemloos bij. Nooit te beroerd om mij te helpen. Dank voor je hulp bij het schrijven en het aanmoedigen om het vooral nu eens af te maken. Ook sinds je met emeritaat bent ben je me blijven pushen. Ik heb veel geleerd van je en ben je daar zeer dankbaar voor. Het was me een waar genoegen om met jou te mogen werken. Mijn copromotoren Dr. Pilar Laguna en Prof. Dr. Ir. Hessel Wijkstra, Beste Pilar, ik denk nog vaak terug aan de periode waarin wij samen de eerste laparoscopisch geassisteerde cryochirurgie voor niertumoren in Nederland uitvoerden. Ik heb van onze laparoscopische sessies veel mogen opsteken. De laparoscopie, waarvoor ik tenslotte naar het AMC was gekomen, stond daar nog in zijn kinderschoenen. Wat ik nu als laparoscopisch chirurg kan heeft in die tijd zijn wortels gekregen. De uitdagingen niet uit de weg gaand en vooral te leren van mijn mislukkingen die mogen leiden tot verbeteringen. Beste Hessel, je hebt mij de eerste kneepjes van “hoe doe je wetenschap” laten zien. Zoveel punten en komma’s als jij bij een tekst kon zetten is ongekend. Maar met het tot stand komen van deze promotie heb ik vaak je

178

kritische tips gebruikt. Het moest en kon gewoon beter. Mijn dank voor het opbouwen van deze kritische noot. Mijn maten, de urologen van het Onze Lieve Vrouwe Gasthuis en het Sint Lucas Andreas Ziekenhuis: Eddi Heldeweg, George van Andel, Han van der Zee, Ernst van Haarst, Marina Hovius, Paul Kauer en Tom Schlattman. Beste heren en dame, dank voor jullie vrijgevigheid. Het is uniek te mogen ervaren dat ondersteuning niet alleen bestaat uit sportieve aanmoedigingen maar ook uit het creëren van tijd en ruimte om met een project als dit bezig te kunnen zijn. Ik hoop dat het jullie niet teveel heeft mogen kosten. Alle co-auteurs, Heren en dames, het was een genoegen om met jullie wetenschap te mogen bedrijven en abstracts of artikelen te schrijven. Dank voor al het gedane werk, de suggesties en de correcties. De paranymfen, Frank, mijn broer, je hebt altijd op de juiste momenten achter mijn grillen gestaan en mij van vaderlijk advies voorzien. Mijn dank voor al dit broederlijk voor mij klaar staan. Reinier, mijn maatje, gespecialiseerd in het mij voorhouden van een spiegel. Maar bovenal een supervriend waarmee het altijd goed vertoeven is. Dat ik trots op jou ben leg ik nog wel eens uit aan de bar. Beste Frank en Reinier, bij het schrijven van dit dankwoord heb ik nog geen idee hoe of jullie het er van af zullen brengen als paranimf. Echter zoals hierboven is beschreven is mijn vertrouwen in jullie groot. Aan Germaine, Jouw bereidwilligheid om mij de ruimte te geven en tijd te laten besteden aan dit project is fenomenaal. Zonder jouw liefde, steun en vertrouwen was dit nooit tot stand was gekomen. Dank voor alles en vergeef mijn egoïsme. Ik zie uit naar al die momenten van: “Zeg luister eens even….”, en ik ben één en al oor. Met dank aan de urologen, promovendi, medewerkers, vrienden en fellows van weleer van de afdeling urologie, AMC. Theo de Reijke, Hairo Garibyan, Herman Bakker, Mathias Oelke en Sigrun Langbein, Chaidir Mochtar, Margot Wink, Patricia Beemster, Marleen van Dijk, Intan Kümmerlin, Thomas Skrekas, Tam Kwaider, Peter Tsakiris, Christa Schaap, Sonja van Rees Vellinga. En ook dank aan Bart Witte. Succes met jouw promotie. Ik denk nog steeds met veel plezier terug aan onze filosoferende sessies en prachtige experimenten. Ik hoop later weer eens met jou een “maf” project te mogen verzinnen en uitwerken. De AMC-artiest Rob Kreuger.

179

Beste Rob, wat heb ik altijd heerlijk met je zitten kletsen. Een man met zoveel passie voor zijn vak als medisch illustrator. Eentje van de oude stempel. Dank voor je mooie tekeningen. Ferida Sivro en Philippe Baars, Dank voor jullie expertise en hulp. De prachtigste plaatjes toveren jullie uit de nucleaire hoed. Het dierexperimenteel laboratorium, beste Goos Huijzer en Adrie Maas. Jullie waren altijd bereid om nog even iets extra’s te doen en een uurtje langer te blijven. Dank voor de vele tips. Mijn beeld van het varken is definitief veranderd. Ik herinner mij levendig de fellows die bij ons op het dierenlaboratorium stonden te zwoegen op varkens. Mijn analyse van hun geploeter is geworden tot mijn huidige stelregel: “Als je iets probeert en het 2x achter elkaar niet lukt dan moet je iets veranderen. Al is dit het kleinste detail.” De afdeling Biomedical Physics and Engineering van het AMC: Natuurlijk gaat mijn dank uit naar allen die verbonden zijn geweest aan de ontwikkeling, het onderhoud en het verrichten van experimenten met de cryomicrotoom. Een fantastische machine. Pepijn van Horssen, Jeroen van den Wijngaard en Renee ter Wee dank voor de samenwerking. Jullie zijn een onuitputtelijke bron van goede ideeën geweest. Dear Matthew Wagner, Dank voor het reviseren van het Engelstalige gedeelte van het manuscript. Het waren de laatste puntjes op de i. Prof. Liba, dear Moshé, Dank voor de mooie gedichten. De sponsoren, Dank voor de gulle giften. De Muppets: Wieger, Skip, Dunk, Tijmen, Tess en Suus Vooral jullie ben ik veel verschuldigd. Weg met dat boekje, het is eindelijk af!! We gaan weer lekker gezellige, leuke en spannende dingen doen.

180

Curriculum vitae

English Brunolf Lagerveld is born at 30th July 1965 in Den Helder, The Netherlands. In 1983, he graduated from high school at the Rijksscholengemeenschap in Zwolle. From 1983-1991, he studied medicine at the State University of Groningen and obtained the degree Master of Science. The Free University th of Amsterdam awarded his Medical Degree at 5 November 1993. Thereafter, he worked as a non-training resident at the department of Surgery, Bronovo Hospital in The Hague. From 1995-1996 he was employed as non-training resident at the department of Plastic Surgery, Leyenburg Hospital in The Hague. In 1997, he worked as non-training resident Urology at the Canisius Wilhemina Hospital in Nijmegen. In January 1998, after admittance to the urological resident training program, he started at the department of General Surgery at the Canisius Wilhelmina Hospital in Nijmegen under the supervision of Dr. E. Bruggink. In 2000 and from July 2002 – September 2003, the academic training in urology took place at the department of Urology at the University Medical Center, St. Radboud, Nijmegen, under the supervision of Prof. dr. F.M.J. Debruyne. In between, from 2001 to July 2002, he was trained under the supervision of Drs. Rodrigues Peirera at the department of Urology at the Rijnstate Hospital in Arnhem. The last 3 months of his urology training were at the University of Amsterdam under the supervision of Prof. dr. J.J.M.C.H. de la Rosette. After been registrated as urologist he joined the next 3 years the department of urology at the same hospital as stafmember. This position was combined with a one-year fellowship in urological laparoscopy. The Dutch Association of Urology (NVU) and the Dutch working-group of Endourology (SWEN) st financially supported him. Since 1 September 2006, he works in Amsterdam as consultant urologist at the department of Urology at the St. Lucas Andreas Hospital as well as the Onze Lieve Vrouwe Gasthuis. Nederlands Brunolf Lagerveld werd geboren op 30 juli 1965 in Den Helder. In 1983 behaalde hij het diploma Atheneum B aan de Rijksscholengemeenschap te Zwolle. Van 1983 tot 1991 studeerde hij geneeskunde aan de Rijksuniversiteit van Groningen alwaar hij zijn doctoraal examen behaalde. Het artsexamen werd verkregen aan de Vrije Universiteit van Amsterdam in 1993. Hierna werkte hij als arts assistent algemene chirurgie in het Bronovo Ziekenhuis in ’s Gravenhage. Van 1995 tot 1997 werkte hij als arts assistent plastische chirurgie in het Leyenburg Ziekenhuis te ’s Gravenhage. In 1997 werkte hij als arts assistent urologie in het Canisius Wilhemina Ziekenhuis in Nijmegen. Na toegelaten te zijn tot de opleiding urologie doorliep hij de vooropleiding algemene chirurgie in het Canisius Wilhelmina Ziekenhuis onder leiding van dr. E. Bruggink. De urologische opleiding vond plaats vanaf 2000 in het Universitair Medisch Centrum St. Radboud te Nijmegen onder leiding van

182

Prof. dr. F.M.J. Debruyne. Het gedeelte van de perifere opleiding urologie in het Rijnstate ziekenhuis in Arnhem was onder leiding van R. Rodrigues Peirera. Met de laatste drie maanden van de urologische opleiding in het Academisch Medisch Centrum aan de Universiteit van Amsterdam onder leiding van Prof. dr. J.J.M.C.H. de la Rosette, werd de opleiding tot uroloog uiteindelijk afgerond. Hierna startte hij in het laatstgenoemde ziekenhuis als staflid van de afdeling urologie. Dit werd gecombineerd met een fellowship in de urologische laparoscopie, namens en met financiĂŤle support van de Nederlandse Vereniging voor Urologie en de Stichting Werkgroep Endourologie Nederland. Vanaf 1 september 2006 is hij in Amsterdam werkzaam als uroloog in het St. Lucas Andreas Ziekenhuis en het Onze Lieve Vrouwe Gasthuis.

183

Recurrence of the enemy inside

Who is more stubborn: the tumour on my kidney frozen, four years ago, or myself, with the frozen cancer, deep inside my body? No question, the facts are there: after exactly four years the tumour wakes up frozen, or not, recurrence: he starts expanding again. Yes, it is again: â&#x20AC;&#x153;To be or not to beâ&#x20AC;? he or me? Stubborn as I am I kept checking, tests, C.T. scans, M.R.I., radiology, medical consultations regular visits to the surgeon every three, six months, every year. But no: he makes resurrection. And yes: I go for a new operation Cryo therapy or not, I am going to have it frozen, again inside my abdomen a new operation-but the same. It seems routine: same town, same hospital, same Doctor, same Cryo therapy. Here we go! Hospitalization, pyjama, shirt and mini slips, on the bed, and to the operation-table. The surgeon is there, also the anaesthetist, the nurses-on the operating-table! They talk to me,

184

ask the crucial question: If it goes wrong with your heart should we wake you up? Yes, by all means, shock, cardio-version, till 120, as the years of Moses, am I not also Moshé, Moses? You go for it! We still talk and they introduce the needle, pump in the infusion, I fall asleep. What is this? I hear: Hallo, you, Moshé! but I can’t answer. I try again, slowly open my eyes. A big space, lights, spirits in the air, not flying, I can’t see wings, they are floating angels in white, blue, green, are they men or women? I hear some noises, they float from side to side, they speak to each other, to the figures lying-on what? They take slowly shape, pale figures in the darkness, lying in the beds-like me! Yes, beds are aligned on one side of the big space it is the recovery-hall an antechamber of the return, back to life, after being “nearly there”, on the threshold of non-return the figures came a long way backand so did I? Were am I, in this world, or the next one, is it Paradise, as it looks or its atrium? Who is here the leader, where is God?

185

Everything is taking form, I can hear: Hallo, Mr. Liba! It is the angel next to me, a nurse in green and she insists: Are you here, are you with us? I am, although I feel like floating no gravitation rules here, am I not on Earth? Welcome back! greets me the nurse and I know: the operation is over. Once again, I won the fight with the enemy inside, they froze him, he is dead, it is my victory, and I will be! Until next time? MoshĂŠ Liba Amsterdam, 18-10-2012

186

Imaging and vascular changes