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Optimal Freeze Cycle Length for Renal Cryotherapy
Investigative Urology
Optimal Freeze Cycle Length for Renal Cryotherapy Jennifer Lee Young,* Elham Khanifar, Navneet Narula, Cervando Gerardo Ortiz-Vanderdys, Surendra Babu Kolla, Donald Lowell Pick, Petros George Sountoulides, Oskar Grau Kaufmann, Kathryn Elizabeth Osann, Victor Buu Huynh, Adam Geoffrey Kaplan, Lorena Aurora Andrade, Michael Ken Louie, Elspeth Marguerita McDougall† and Ralph Victor Clayman‡ From the Departments of Urology (JLY, CGOV, SBK, DLP, PGS, OGK, VBH, AGK, LAA, MKL, EMM, RVC), Pathology (EK, NN) and Medicine (KEO), University of California-Irvine, Orange, California
Purpose: To our knowledge the optimal freeze cycle length in renal cryotherapy is unknown. Ten-minute time based freeze cycles were compared to temperature based freeze cycles to ⫺20C. Materials and Methods: Laparoscopic renal cryotherapy was performed on 16 swine. Time based trials consisted of a double 10-minute freeze separated by a 5-minute thaw. Temperature based trials were double cycles of 1, 5 or 10-minute freeze initiated after 1 of 4 sensors indicated ⫺20C. A 5-minute active thaw was used between freeze cycles. Control trials consisted of cryoneedle placement for 25 minutes without freeze or thaw. Viability staining and histological analysis were done. Results: There was no difference in cellular necrosis between any of the temperature based freeze cycles (p ⫽ 0.1). Time based freeze cycles showed more nuclear pyknosis, indicative of necrosis, than the 3 experimental freeze cycles for the renal cortex (p ⫽ 0.05) but not for the renal medulla (p ⫽ 0.61). Mean time to ⫺20C for freeze cycle 1 was 19 minutes 10 seconds (range 9 to 46 minutes). In 4 of 21 trials (19%) ⫺20C was never attained despite freezing for 25 to 63 minutes. Conclusions: There was no difference in immediate cellular necrosis among double 1, 5 or 10-minute freeze cycles. Cellular necrosis was evident on histological analysis for trials in which ⫺20C was attained and in freeze cycles based on time alone. With a standard 10-minute cryoablation period most treated parenchyma 1 cm from the probe never attained ⫺20C. Cell death appeared to occur at temperatures warmer than ⫺20C during renal cryotherapy.
Abbreviations and Acronyms TTC ⫽ triphenyl tetrazolium chloride Submitted for publication September 7, 2010. Study received institutional animal care and use committee approval. Supported by Galil Medical. * Correspondence: 333 City Blvd. West, Suite 2100, Orange, California 92868 (telephone: 714456-6717; FAX: 714-456-5062; e-mail: jlyoung@ uci.edu). † Financial interest and/or other relationship with Astellas, Karl Storz, Intuitive Surgical, Ethicon Endo-Surgical, EndoCare and METI. ‡ Financial interest and/or other relationship with Cook Urological, Applied Urology, Intuitive, Omeros, Greenwald, Orthopedic Service, Galil Medical, Boston Scientific, Vascular Technology and Karl Storz.
Key Words: kidney, cryosurgery, necrosis, laparoscopy, swine THE increased use of abdominal imaging has increased the incidence of asymptomatic renal masses with subsequent stage migration.1 The detection rate of incidentally discovered renal masses increased from 7% to 13% in the early 1970s to 48% to 66% in recent years with most being low stage renal cell carcinoma.2– 4 Enhancing renal masses exhibit heterogeneous behavior with 20% being benign and 20% to 25% with potentially
aggressive behavior.5,6 The standard of care for clinically localized renal cell carcinoma is surgical resection via radical or partial nephrectomy.7,8 Radical nephrectomy predisposes the patient to chronic kidney disease, cardiovascular risk and increased mortality.9,10 Recent American Urological Association guidelines11 expanded treatment options to include thermal ablation12,13 and active surveillance.14,15
0022-5347/11/1861-0283/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 283-288, July 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.03.034
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RESEARCH, INC.
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OPTIMAL FREEZE CYCLE LENGTH FOR RENAL CRYOTHERAPY
Since thermal ablation is a relatively new treatment modality, few standards have been developed for its most efficacious use. Currently ice ball size, freeze cycle length or thermocouple based end points are used to judge procedure adequacy. We determined the optimal freeze cycle length for renal cryotherapy.
MATERIALS AND METHODS A total of 16 female Yorkshire farm pigs weighing between 30 and 42 kg were used in this study, which was approved by the institutional animal care and use committee at our institution. The animals were fasted the night before surgery. After premedication with 0.05 mg/kg atropine intramuscularly anesthesia was induced with 6 mg/kg Telazol® and 2 mg/kg xylazine intramuscularly. An ear vein cannula was placed and the animal was intubated. Anesthesia was induced with 4% to 5% isoflurane maintained at 1% to 2%. At the conclusion of the study the animals were sacrificed using 0.1 ml/kg Eutha-6 (Western Medical Supply, Arcadia, California). After anesthesia administration animals were placed in the left lateral decubitus position. Pneumoperitoneum was established with a Veress needle in the right lower quadrant and set to 15 mm Hg. A 12 mm laparoscopic port was placed below the umbilicus. Two additional 12 mm ports were placed lateral to the rectus muscle under direct vision. The right kidney was exposed using blunt and sharp dissection. The ablation site was randomized (random.org) to the upper or lower pole. A paper ruler was placed in the abdomen. Electrocautery was used to demarcate the cryoprobe site, and 1 cm cephalad and caudad to plan placement of 2 multithermal sensors. A 1.47 mm (17 gauge) IceRod™ cryoprobe was placed percutaneously 2 cm deep to the kidney surface. According to manufacturer recommendations at the end of the second 10-minute freeze cycle the IceRod attains ⫺40C in a 16 ⫻ 41 mm area around the probe, ⫺20C in a 27 ⫻ 50 mm area around the probe and 0C in a 40 ⫻ 58 mm area around the probe with a 2 to 3 mm margin of error. A Multi-Point Thermal Sensor™ was placed percutaneously at the marked sites 2 cm deep to the kidney surface. The sensor probes are 1.47 mm in diameter (17 gauge) with thermal sensors at 5, 15, 25 and 35 mm. Thus, the 5 and 15 mm sensors were within the kidney parenchyma. Sensor accuracy is ⫾ 2C. A Presice™ Cryoablation System was used with argon gas at 4,300 psi. In the 3 experimental arms cryotherapy was administered until ⫺20C was shown by 1 of the 4 temperature sensors (fig. 1). After the target temperature was achieved a 1, 5 or 10-minute freeze cycle was initiated. Active thaw with helium gas at 2,200 psi was then given until the same sensor showed 0C. A 5-minute thaw cycle was then administered. Cryotherapy was again administered until the same sensor showed ⫺20C. A second freeze cycle the same duration as the first was initiated. The right kidney hilum was ligated to preserve the acute cryolesion.
Figure 1. Freeze cycle in progress with 1.47 mm (17 gauge) cryoprobe in center, 1 thermal sensor cephalad and 1 caudad. All probes were placed percutaneously 2 cm deep to kidney surface.
The animal was repositioned and the procedure was repeated on the left kidney. Trials were randomized by freeze cycle and side. A temperature of ⫺20C was deemed unobtainable in 4 trials since the temperature reached a plateau despite having the probe activated for 25 to 63 minutes. These trials were terminated and a trial was then performed on the other pole. At the conclusion of the procedure the kidneys were immediately harvested via a midline incision. The time based control arm consisted of 6 trials using a double 10-minute freeze separated by a 5-minute active thaw. The sham control arm consisted of cryoprobe and sensor placement for 25 minutes without any freeze or thaw. A 3 mm axial slice was taken from each kidney through the center of the cryolesion and placed in 10% neutral buffered formalin for histological analysis. A 3 mm axial slice from the cryolesion was placed in TTC to confirm that the cryolesion corresponded to nonviable tissue. This dye forms a red stain in the presence of an intact dehydrogenase enzyme system.16 Accordingly areas of viable tissue stain red and areas of necrotic tissue that lack dehydrogenase activity fail to stain (fig. 2). Histological analysis was done by an experienced genitourinary pathologist (NN) at our institution and a senior resident (EK) working under her supervision. Each pathologist was blinded to the study groups. Gross lesion size was measured as well as ischemic zone dimensions. An adjacent slice from the lesion that included surrounding normal kidney tissue was submitted for histological assessment. After fixation in 10% formalin the tissue was embedded in paraffin. Sections (4 m) were cut, stained with hematoxylin and eosin, and evaluated microscopically. The cortex and the medulla were evaluated for epithelial coagulative necrosis/nuclear pyknosis, nuclear fragmentation, cytoplasmic vacuolization, vascular congestion, vascular ectasia and hemorrhage, including tubular and interstitial. In addition, the glomeruli in the cortex
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system was assessed to assess whether the urothelium was intact or sloughed. Each of these features was graded semiquantitatively as 1—less than 5% of the area involved, 2— 6% to 50% involved and 3— greater than 50% involved (fig. 3). A separate histological analysis of viability was performed of the border of the cryolesion and within the cryolesion 0.5 mm from the border for representative sections. Evidence of nuclear injury (pyknosis and karyorrhexis) was assessed in the cortex. The amount of nuclear injury was graded as 1—less than 5%, 2— 6% to 50% and 3— greater than 50%. Statistical analysis was performed by an independent statistician (KEO) with SYSTAT® 11.0. Time to 0C and time to ⫺20C were compared between freeze cycles using the Kruskal-Wallis nonparametric test. Groups were compared with respect to histological damage using the Kruskal-Wallis test. Temperature data were normally distributed and are shown as the mean, SD and range.
RESULTS
Figure 2. Kidney axial slice. A, gross view shows mottled cryolesion. B, after TTC staining viable adjacent kidney stained red, indicating intact dehydrogenase enzyme system.
were assessed for evidence of injury, including hemorrhage and nuclear fragmentation. The interlobar arteries were evaluated to determine whether the endothelium was preserved or showed denudation. The pelvicaliceal
During the 3 experimental freeze cycles a total of 21 cryolesions were created in 19 kidneys. Mean time to ⫺20C for the first freeze for all 3 trial freeze cycles was 19.2 minutes. Mean time to 0C for the 1, 5 and 10 minutes freeze cycles was 2.2, 3.3 and 3.0 minutes, respectively. Mean time to ⫺20C for the second freeze cycle was 6.0, 4.0 and 6.2 minutes for the 1, 5 and 10-minute freeze cycles, respectively. In 4 of 21 trials (19%) ⫺20C was never attained despite freezing for 25 to 63 minutes. There was no statistically significant difference in time to 0C (p ⫽ 0.37) or ⫺20C (p ⫽ 0.15) for freeze cycle 2 among the 3 experimental arms. During the time based control trials 6 cryolesions were created in 6 kidneys. Mean temperature at the end of freeze cycle 2 for the 15 mm (superficial)
Figure 3. Histological views of cortex. A, control with 25-minute cryoprobe only and no freeze or thaw. The nuclei are preserved and there is no evidence of interstitial hemorrhage or vascular injury. Occasional tubules with hemorrhage are seen. Reduced from ⫻20. B, experimental animal with 2, 1 minute freezes in which ⫺20C was achieved once, separated by 5-minute active thaw. Cellular injury is seen as fragmented (long arrows) and pyknotic (short arrows) nuclei. Arrowhead indicates interstitial hemorrhage. Reduced from ⫻40.
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sensor was ⫺3.8C ⫾ 15.2C (range ⫺23 to 13) for the cephalad sensor and 8.8C ⫾ 19.8C (range ⫺14 to 31) for the caudad sensor. Mean temperature at end of freeze cycle 2 for the 5 mm (deep) sensor was 6.5C ⫾ 20.5C (range ⫺22 to 22) for the cephalad sensor and 11.3C ⫾ 11.8C (range 2 to 22) for the caudad sensor. During the sham control trials zero cryolesions were made in 6 kidneys. Mean temperature at the end of 25 minutes for the 15 mm (superficial) sensor was 40.0C ⫾ 0.9C (range 39 to 41) for the cephalad sensor and 40.8C ⫾ 1.0C (range 40 to 42) for the caudad sensor. Mean temperature at the end of 25 minutes for the 5 mm (deep) sensor was 40.2C ⫾ 1.2C (range 39 to 42) for the cephalad sensor and 40.2C ⫾ 1.2C (range 39 to 42) for the caudad sensor. On gross examination of the kidney after TTC staining the cryolesion corresponded to nonviable tissue and the surrounding kidney corresponded to viable tissue. In the experimental cycles cryolesion length range was 26.0 to 38.9 mm, width was 24.0 to 39.0 mm and depth was between 13.2 and 25.5 mm. On histological examination there was no difference in cellular necrosis among any of the experimental freeze cycles for the cortex (p ⫽ 0.1) or the medulla (p ⫽ 0.81). The time based control arm showed no difference in cellular necrosis from that in the experimental arms in the medulla (p ⫽ 0.61) but showed more necrosis in the cortex (p ⫽ 0.05). There was significantly more necrosis in the experimental arms (p ⫽ 0.001) and in the time based control arm (p ⫽ 0.004) than in the sham control arm. Analysis of the cryolesion border revealed sharp demarcation with intratubular and interstitial hemorrhage. In the cryolesion 0.5 mm from the border the 1-minute experimental freeze cycle trials showed 1 grade 1 and 1 grade 2 nuclear injuries. The 5-minute freeze cycle showed 2 grade 1 and 1 grade 2 nuclear injuries. The 10-minute freeze cycle showed 3 grade 1 and 1 grade 2 nuclear injuries. Time based control trials showed 1 grade 1 and 5 grade 2 nuclear injuries.
DISCUSSION Although literature varies on the temperature necessary for cell death from ⫺16C17 to ⫺50C18 for neoplastic tissue, currently 20C is thought to be the target temperature to kill malignant renal cells.19,20 Several freeze cycles have been evaluated to date, during which temperature was recorded for some but not recorded for most. Stephenson et al studied renal cryotherapy with 4, 3.5 mm cryoprobes in a canine model via open and laparoscopic access.21 A single freeze-thaw cycle was used and lesions were monitored by ultrasound. Histology revealed a central region of complete necrosis bounded by a 2 mm region of marginal injury that
showed regenerative activity at 8 days. Long and Faller studied the feasibility of renal cryotherapy in a porcine model with 3 protocols, including a 3 to 4-minute freeze with a single cryoprobe, a 6 to 7-minute freeze with a single cryoprobe and a double 10-minute freeze with 2 cryoprobes spaced 2 mm apart.22 The single cryoprobes resulted in discrete lesions without perirenal damage. The 2 probes and double freeze produced large areas of infarction with significant perirenal reaction in 5 of the 6 animals and hydronephrosis/renal loss in 2. Based on histological analysis they recommended treating 3 to 4 cm exophytic lesions amenable to surface ultrasound monitoring. When investigating the most appropriate freezing duration and the number of probes needed to cause necrosis without morbidity, Auge et al studied 5, 10 and 15-minute freeze cycles with a single or double 3.4 mm probe and active thaw in swine via open surgery.23 Temperature was measured 5, 10, 15 and 20 mm from the cryoprobe and ranged from ⫺10C to ⫺85C at the 5 mm sensor. At 4 to 7-day survival the single 5-minute freeze was inadequate to cause tissue necrosis, the 10-minute freeze resulted in necrosis without complications and the 15-minute freeze caused necrosis and renal fracture. A 10-minute freeze with a single or double probe was recommended. To evaluate the ideal number of freeze cycles and the type of thaw process Woolley et al evaluated single and double 15-minute freeze cycles with active or passive thaw using a 3 mm probe (Endocare, Irvine, California) in dogs via laparoscopic access after 4-week survival.24 Mean temperature recorded in the cryoprobe attained ⫺142C ⫾ 1.0C. The double freeze cycle produced a significantly larger area of liquefactive necrosis than the single freeze. A double 15-minute freeze with an active thaw was recommended to effectively ablate tissue and decrease operative time. To evaluate laparoscopic cryotherapy Bishoff et al treated the 2 poles of each kidney in 6 farm pigs with single and double 5 and 15-minute freeze cycles using a 4.8 mm probe.25 Temperature recorded within the probe attained ⫺180C. Kidneys were harvested for histopathology immediately, or at 1 or 13 weeks. They concluded that longer freeze cycles and double freeze cycles did not create further destruction compared to shorter and single freeze cycles. They recommended a single 15-minute freeze cycle with ice ball extension at least 1 cm beyond the lesion. Weld et al used intraoperative ultrasound and histological analysis to evaluate the ablation area created by an IceRod after a double 10-minute freeze and a 3-minute active thaw in the porcine kidney via a laparoscopic approach.26 They found a 19 mm
OPTIMAL FREEZE CYCLE LENGTH FOR RENAL CRYOTHERAPY
diameter zone of complete ablation 2 cm from the tip of the IceRod. This equates to necrosis 9.5 mm from the cryoprobe, which correlates with the nonlethal temperatures that we found 1 cm from the cryoprobe. Thus, there is good data correspondence between these 2 peer reviewed studies. In humans Edmunds et al noted that a single 15-minute freeze with an active thaw using a 3 mm probe was sufficient for complete destruction of 1.5 to 1.8 cm renal masses on histology after immediate partial nephrectomy in 2 patients.27 Cellular necrosis was evident on histological analysis in trials in which ⫺20C was attained and in freeze cycles based on time alone. With a 10-minute cryoablation period most treated parenchyma 1 cm from the probe never attained ⫺20C. This suggests that temperatures above ⫺20C, even above 0C, cause cellular necrosis. Although histology was performed in the acute phase, there would likely be additional damage with time due to endothelial damage and microvascular thrombosis. The extended time at cold temperature may have also contributed to cell death. This would explain our results clinically since we now know that the temperature in most patients was never close to ⫺20C with time based treatments but there has been a paucity of recurrences. The relative resistance to necrosis in the medulla in time based trials is difficult to explain since a similar finding was not seen in temperature based trials. Cells in the renal medulla are known to exist in a hostile milieu characterized by hypoxia due to relatively low blood flow, oxygen diffusion and the high metabolic demands of medullary cells.28 Also, these cells are exposed to wide variations in extracellular solute concentrations and abundant reactive oxygen species. Renal medullary cells survive in this environment by the high glycolytic capacity to decrease dependence on oxygen, high quantities of heat shock proteins and a transcriptional response to hypoxia. Other adaptations include enhanced expression of aldose reductase to combat osmotic stress, abundant superoxidase to endure oxidative stress and the ability to rapidly release organic osmolytes to reduce cell volume in the face of cellular edema. This multitude of adaptive responses of the renal medulla may explain why the time based control arm showed more necrosis in the cortex but no difference in medullary necrosis in the experimental arms. A study limitation is that histology was done immediately. Thus, direct cell injury from ice crystal formation was evaluated but not subsequent
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vascular injury and thrombosis. The study could be repeated with animal survival to evaluate each mechanism of necrosis. Another limitation is that this study was performed in healthy renal tissue. Malignant tissue may require longer or colder freeze cycles due to its vascularity. Also, penetration of each cryoneedle may have varied depending on kidney size among the different animals. In future studies this could be formally measured by intraoperative ultrasound or by measuring summary statistics of kidney size to determine tight CIs around the mean volume. Lastly, this was a pilot study with a small sample size due to the expense of the animals involved. Power to detect differences was consequently limited. Only differences greater than 80% could be reliably detected, given the small number of test kidneys. Currently our treatment regimen is to place sufficient cryoprobes to achieve a ⫺20C isotherm, as measured by multiple temperature sensing needles at the upper and lower tumor borders. We use 2, 10-minute freeze cycles and a 5-minute thaw cycle, and are satisfied if at any time during the freeze cycle the temperature decreases to ⫺20C or lower since our study showed that the interval at ⫺20C has no bearing on the amount of cell necrosis. Our regimen may be overly cautious, given the reported excellent results with unmonitored double 10-minute freeze cycles, during which we know in many cases ⫺20C was not attained. However, until we have additional data on the minimal temperature needed to incur cell death during a sustained 10minute period, we continue to use the ⫺20C goal.
CONCLUSIONS Three experimental cycles and 2 control arms were evaluated to delineate the optimal freeze cycle for renal cryotherapy. There was no difference in immediate cellular necrosis between double 1, 5 or 10minute freeze cycles after ⫺20C was attained. Time based cycles, ie 2, 10-minute freezes separated by a 5-minute thaw, showed more cellular necrosis than the experimental ⫺20C freeze cycles for the cortex but not for the medulla. With a standard 10-minute cryoablation period most treated parenchyma 1 cm from the probe never attains ⫺20C and commonly does not even attain 0C. However, cellular necrosis was evident on histological analysis for the timed trials and appeared to be no different (medulla) or better (cortex) than in animals in which ⫺20C was attained and maintained for a specific period.
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9. McKiernan J, Simmons R, Katz J et al: Natural history of chronic renal insufficiency after partial and radical nephrectomy. Urology 2002; 59: 816.
2. Volpe A, Panzarella T, Rendon RA et al: The natural history of incidentally detected small renal masses. Cancer 2004; 100: 738.
10. Thompson RH, Boorjian SA, Lohse CM et al: Radical nephrectomy for pT1a renal masses may be associated with decreased overall survival compared to partial nephrectomy. J Urol 2008; 179: 468.
3. Wehle MJ, Thiel DD, Petrou SP et al: Conservative management of incidental contrast-enhancing renal masses as a safe alternative to invasive therapy. Urology 2004; 64: 49.
11. Campbell SC, Novick AC, Belldegrun A et al: Guideline for management of the clinical T1 renal mass. J Urol 182: 1271.
4. Kato M, Suzuki T, Suzuki Y et al: Natural history of small renal cell carcinoma: evaluation of growth rate, histological grade, cell proliferation and apoptosis. J Urol 2004; 172: 863.
12. Kunkle DA, Egleston BL and Uzzo RG: Excise, ablate or observe: the small renal mass dilemma—a meta-analysis and review. J Urol 2008; 179: 1227.
5. Kutikov A, Fossett LK, Ramchandani P et al: Incidence of benign pathologic findings at partial nephrectomy for solitary renal mass presumed to be renal cell carcinoma on preoperative imaging. BJU Int 2008; 68: 737.
13. Gill IS, Remer EM, Hasan WA et al: Renal cryoablation: outcome at 3 years. J Urol 2005; 173: 1903.
6. Snyder ME, Bach A, Kattan MW et al: Incidence of benign lesions for clinically localized renal masses smaller than 7 cm in radiological diameter: influence of sex. J Urol 2006; 176: 2391. 7. Frank I, Blute ML, Leibovich BC et al: Independent validation of the 2002 American Joint Committee on cancer primary tumor classification for renal cell carcinoma using a large, single institution cohort. J Urol 2005; 173: 1889. 8. Hafez KS, Fergany AF and Novick AC: Nephron sparing surgery for localized renal cell carcinoma: impact of tumor size on patient survival, tumor recurrence and TNM staging. J Urol 1999; 162: 1930.
14. Oda T, Miyao N, Takahashi A et al: Growth rates of primary and metastatic lesions of renal cell carcinoma. Int J Urol 2001; 8: 473. 15. Chawla SN, Crispen PL, Hanlon AL et al: The natural history of observed enhancing renal masses: meta-analysis and review of the world. J Urol 2006; 175: 425. 16. Fishbein MC, Meerbaum S, Rit J et al: Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J 1981; 101: 593. 17. Schmidlin FR, Rupp CC, Hoffmann NE et al: Measurement and prediction of thermal behavior and acute assessment of injury in a pig model of renal cryosurgery. J Endourol 2001; 15: 193.
18. Gage AA and Baust J: Mechanisms of tissue injury in cryosurgery. Cryobiology 1998; 37: 171. 19. Uchida M, Imaide Y and Sugimoto K: Percutaneous cryosurgery for renal tumours. Br J Urol 1995; 75: 132. 20. Chosy SG, Nakada SY, Lee FT et al: Monitoring renal cryosurgery: predictors of tissue necrosis in swine. J Urol 1998; 159: 1370. 21. Stephenson RA, King DK and Rohr LR: Renal cryoablation in a canine model. Urology 1996; 47: 772. 22. Long JP and Faller GT: Percutaneous cryoablation of the kidney in a porcine model. Cryobiology 1999; 38: 89. 23. Auge BK, Santa-Cruz RW and Polascik TJ: Effect of freeze time during renal cryoablation: a swine model. J Endourol 2006; 20: 1101. 24. Woolley ML, Schulsinger DA, Durand DB et al: Effect of freezing parameters (freeze cycle and thaw process) on tissue destruction following renal cryoablation. J Endourol 2002; 16: 519. 25. Bishoff JT, Chen RB, Lee BR et al: Laparoscopic renal cryoablation: acute and long-term clinical, radiographic, and pathologic effects in an animal model and application in a clinical trial. J Endourol 1999; 13: 233. 26. Weld KJ, Hruby G, Humphrey PA et al: Precise characterization of renal parenchymal response to single and multiple cryoablation probes. J Urol 2006; 176: 784. 27. Edmunds TB Jr, Schulsinger DA, Durand DB et al: Acute histologic changes in human renal tumors after cryoablation. J Endourol 2000; 14: 139. 28. Neuhofer W and Beck FX: Survival in hostile environments: strategies of renal medullary cells. Physiology 2006; 21: 171.
Optimal Freeze Cycle Length for Renal Cryotherapy Jennifer Lee Young,* Elham Khanifar, Navneet Narula, Cervando Gerardo Ortiz-Vanderdys, Surendra Babu Kolla, Donald Lowell Pick, Petros George Sountoulides, Oskar Grau Kaufmann, Kathryn Elizabeth Osann, Victor Buu Huynh, Adam Geoffrey Kaplan, Lorena Aurora Andrade, Michael Ken Louie, Elspeth Marguerita McDougall† and Ralph Victor Clayman‡ From the Departments of Urology (JLY, CGOV, SBK, DLP, PGS, OGK, VBH, AGK, LAA, MKL, EMM, RVC), Pathology (EK, NN) and Medicine (KEO), University of California-Irvine, Orange, California
Purpose: To our knowledge the optimal freeze cycle length in renal cryotherapy is unknown. Ten-minute time based freeze cycles were compared to temperature based freeze cycles to ⫺20C. Materials and Methods: Laparoscopic renal cryotherapy was performed on 16 swine. Time based trials consisted of a double 10-minute freeze separated by a 5-minute thaw. Temperature based trials were double cycles of 1, 5 or 10-minute freeze initiated after 1 of 4 sensors indicated ⫺20C. A 5-minute active thaw was used between freeze cycles. Control trials consisted of cryoneedle placement for 25 minutes without freeze or thaw. Viability staining and histological analysis were done. Results: There was no difference in cellular necrosis between any of the temperature based freeze cycles (p ⫽ 0.1). Time based freeze cycles showed more nuclear pyknosis, indicative of necrosis, than the 3 experimental freeze cycles for the renal cortex (p ⫽ 0.05) but not for the renal medulla (p ⫽ 0.61). Mean time to ⫺20C for freeze cycle 1 was 19 minutes 10 seconds (range 9 to 46 minutes). In 4 of 21 trials (19%) ⫺20C was never attained despite freezing for 25 to 63 minutes. Conclusions: There was no difference in immediate cellular necrosis among double 1, 5 or 10-minute freeze cycles. Cellular necrosis was evident on histological analysis for trials in which ⫺20C was attained and in freeze cycles based on time alone. With a standard 10-minute cryoablation period most treated parenchyma 1 cm from the probe never attained ⫺20C. Cell death appeared to occur at temperatures warmer than ⫺20C during renal cryotherapy.
Abbreviations and Acronyms TTC ⫽ triphenyl tetrazolium chloride Submitted for publication September 7, 2010. Study received institutional animal care and use committee approval. Supported by Galil Medical. * Correspondence: 333 City Blvd. West, Suite 2100, Orange, California 92868 (telephone: 714456-6717; FAX: 714-456-5062; e-mail: jlyoung@ uci.edu). † Financial interest and/or other relationship with Astellas, Karl Storz, Intuitive Surgical, Ethicon Endo-Surgical, EndoCare and METI. ‡ Financial interest and/or other relationship with Cook Urological, Applied Urology, Intuitive, Omeros, Greenwald, Orthopedic Service, Galil Medical, Boston Scientific, Vascular Technology and Karl Storz.
Key Words: kidney, cryosurgery, necrosis, laparoscopy, swine THE increased use of abdominal imaging has increased the incidence of asymptomatic renal masses with subsequent stage migration.1 The detection rate of incidentally discovered renal masses increased from 7% to 13% in the early 1970s to 48% to 66% in recent years with most being low stage renal cell carcinoma.2– 4 Enhancing renal masses exhibit heterogeneous behavior with 20% being benign and 20% to 25% with potentially
aggressive behavior.5,6 The standard of care for clinically localized renal cell carcinoma is surgical resection via radical or partial nephrectomy.7,8 Radical nephrectomy predisposes the patient to chronic kidney disease, cardiovascular risk and increased mortality.9,10 Recent American Urological Association guidelines11 expanded treatment options to include thermal ablation12,13 and active surveillance.14,15
0022-5347/11/1861-0283/0 THE JOURNAL OF UROLOGY® © 2011 by AMERICAN UROLOGICAL ASSOCIATION EDUCATION
Vol. 186, 283-288, July 2011 Printed in U.S.A. DOI:10.1016/j.juro.2011.03.034
AND
RESEARCH, INC.
www.jurology.com
283
284
OPTIMAL FREEZE CYCLE LENGTH FOR RENAL CRYOTHERAPY
Since thermal ablation is a relatively new treatment modality, few standards have been developed for its most efficacious use. Currently ice ball size, freeze cycle length or thermocouple based end points are used to judge procedure adequacy. We determined the optimal freeze cycle length for renal cryotherapy.
MATERIALS AND METHODS A total of 16 female Yorkshire farm pigs weighing between 30 and 42 kg were used in this study, which was approved by the institutional animal care and use committee at our institution. The animals were fasted the night before surgery. After premedication with 0.05 mg/kg atropine intramuscularly anesthesia was induced with 6 mg/kg Telazol® and 2 mg/kg xylazine intramuscularly. An ear vein cannula was placed and the animal was intubated. Anesthesia was induced with 4% to 5% isoflurane maintained at 1% to 2%. At the conclusion of the study the animals were sacrificed using 0.1 ml/kg Eutha-6 (Western Medical Supply, Arcadia, California). After anesthesia administration animals were placed in the left lateral decubitus position. Pneumoperitoneum was established with a Veress needle in the right lower quadrant and set to 15 mm Hg. A 12 mm laparoscopic port was placed below the umbilicus. Two additional 12 mm ports were placed lateral to the rectus muscle under direct vision. The right kidney was exposed using blunt and sharp dissection. The ablation site was randomized (random.org) to the upper or lower pole. A paper ruler was placed in the abdomen. Electrocautery was used to demarcate the cryoprobe site, and 1 cm cephalad and caudad to plan placement of 2 multithermal sensors. A 1.47 mm (17 gauge) IceRod™ cryoprobe was placed percutaneously 2 cm deep to the kidney surface. According to manufacturer recommendations at the end of the second 10-minute freeze cycle the IceRod attains ⫺40C in a 16 ⫻ 41 mm area around the probe, ⫺20C in a 27 ⫻ 50 mm area around the probe and 0C in a 40 ⫻ 58 mm area around the probe with a 2 to 3 mm margin of error. A Multi-Point Thermal Sensor™ was placed percutaneously at the marked sites 2 cm deep to the kidney surface. The sensor probes are 1.47 mm in diameter (17 gauge) with thermal sensors at 5, 15, 25 and 35 mm. Thus, the 5 and 15 mm sensors were within the kidney parenchyma. Sensor accuracy is ⫾ 2C. A Presice™ Cryoablation System was used with argon gas at 4,300 psi. In the 3 experimental arms cryotherapy was administered until ⫺20C was shown by 1 of the 4 temperature sensors (fig. 1). After the target temperature was achieved a 1, 5 or 10-minute freeze cycle was initiated. Active thaw with helium gas at 2,200 psi was then given until the same sensor showed 0C. A 5-minute thaw cycle was then administered. Cryotherapy was again administered until the same sensor showed ⫺20C. A second freeze cycle the same duration as the first was initiated. The right kidney hilum was ligated to preserve the acute cryolesion.
Figure 1. Freeze cycle in progress with 1.47 mm (17 gauge) cryoprobe in center, 1 thermal sensor cephalad and 1 caudad. All probes were placed percutaneously 2 cm deep to kidney surface.
The animal was repositioned and the procedure was repeated on the left kidney. Trials were randomized by freeze cycle and side. A temperature of ⫺20C was deemed unobtainable in 4 trials since the temperature reached a plateau despite having the probe activated for 25 to 63 minutes. These trials were terminated and a trial was then performed on the other pole. At the conclusion of the procedure the kidneys were immediately harvested via a midline incision. The time based control arm consisted of 6 trials using a double 10-minute freeze separated by a 5-minute active thaw. The sham control arm consisted of cryoprobe and sensor placement for 25 minutes without any freeze or thaw. A 3 mm axial slice was taken from each kidney through the center of the cryolesion and placed in 10% neutral buffered formalin for histological analysis. A 3 mm axial slice from the cryolesion was placed in TTC to confirm that the cryolesion corresponded to nonviable tissue. This dye forms a red stain in the presence of an intact dehydrogenase enzyme system.16 Accordingly areas of viable tissue stain red and areas of necrotic tissue that lack dehydrogenase activity fail to stain (fig. 2). Histological analysis was done by an experienced genitourinary pathologist (NN) at our institution and a senior resident (EK) working under her supervision. Each pathologist was blinded to the study groups. Gross lesion size was measured as well as ischemic zone dimensions. An adjacent slice from the lesion that included surrounding normal kidney tissue was submitted for histological assessment. After fixation in 10% formalin the tissue was embedded in paraffin. Sections (4 m) were cut, stained with hematoxylin and eosin, and evaluated microscopically. The cortex and the medulla were evaluated for epithelial coagulative necrosis/nuclear pyknosis, nuclear fragmentation, cytoplasmic vacuolization, vascular congestion, vascular ectasia and hemorrhage, including tubular and interstitial. In addition, the glomeruli in the cortex
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system was assessed to assess whether the urothelium was intact or sloughed. Each of these features was graded semiquantitatively as 1—less than 5% of the area involved, 2— 6% to 50% involved and 3— greater than 50% involved (fig. 3). A separate histological analysis of viability was performed of the border of the cryolesion and within the cryolesion 0.5 mm from the border for representative sections. Evidence of nuclear injury (pyknosis and karyorrhexis) was assessed in the cortex. The amount of nuclear injury was graded as 1—less than 5%, 2— 6% to 50% and 3— greater than 50%. Statistical analysis was performed by an independent statistician (KEO) with SYSTAT® 11.0. Time to 0C and time to ⫺20C were compared between freeze cycles using the Kruskal-Wallis nonparametric test. Groups were compared with respect to histological damage using the Kruskal-Wallis test. Temperature data were normally distributed and are shown as the mean, SD and range.
RESULTS
Figure 2. Kidney axial slice. A, gross view shows mottled cryolesion. B, after TTC staining viable adjacent kidney stained red, indicating intact dehydrogenase enzyme system.
were assessed for evidence of injury, including hemorrhage and nuclear fragmentation. The interlobar arteries were evaluated to determine whether the endothelium was preserved or showed denudation. The pelvicaliceal
During the 3 experimental freeze cycles a total of 21 cryolesions were created in 19 kidneys. Mean time to ⫺20C for the first freeze for all 3 trial freeze cycles was 19.2 minutes. Mean time to 0C for the 1, 5 and 10 minutes freeze cycles was 2.2, 3.3 and 3.0 minutes, respectively. Mean time to ⫺20C for the second freeze cycle was 6.0, 4.0 and 6.2 minutes for the 1, 5 and 10-minute freeze cycles, respectively. In 4 of 21 trials (19%) ⫺20C was never attained despite freezing for 25 to 63 minutes. There was no statistically significant difference in time to 0C (p ⫽ 0.37) or ⫺20C (p ⫽ 0.15) for freeze cycle 2 among the 3 experimental arms. During the time based control trials 6 cryolesions were created in 6 kidneys. Mean temperature at the end of freeze cycle 2 for the 15 mm (superficial)
Figure 3. Histological views of cortex. A, control with 25-minute cryoprobe only and no freeze or thaw. The nuclei are preserved and there is no evidence of interstitial hemorrhage or vascular injury. Occasional tubules with hemorrhage are seen. Reduced from ⫻20. B, experimental animal with 2, 1 minute freezes in which ⫺20C was achieved once, separated by 5-minute active thaw. Cellular injury is seen as fragmented (long arrows) and pyknotic (short arrows) nuclei. Arrowhead indicates interstitial hemorrhage. Reduced from ⫻40.
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OPTIMAL FREEZE CYCLE LENGTH FOR RENAL CRYOTHERAPY
sensor was ⫺3.8C ⫾ 15.2C (range ⫺23 to 13) for the cephalad sensor and 8.8C ⫾ 19.8C (range ⫺14 to 31) for the caudad sensor. Mean temperature at end of freeze cycle 2 for the 5 mm (deep) sensor was 6.5C ⫾ 20.5C (range ⫺22 to 22) for the cephalad sensor and 11.3C ⫾ 11.8C (range 2 to 22) for the caudad sensor. During the sham control trials zero cryolesions were made in 6 kidneys. Mean temperature at the end of 25 minutes for the 15 mm (superficial) sensor was 40.0C ⫾ 0.9C (range 39 to 41) for the cephalad sensor and 40.8C ⫾ 1.0C (range 40 to 42) for the caudad sensor. Mean temperature at the end of 25 minutes for the 5 mm (deep) sensor was 40.2C ⫾ 1.2C (range 39 to 42) for the cephalad sensor and 40.2C ⫾ 1.2C (range 39 to 42) for the caudad sensor. On gross examination of the kidney after TTC staining the cryolesion corresponded to nonviable tissue and the surrounding kidney corresponded to viable tissue. In the experimental cycles cryolesion length range was 26.0 to 38.9 mm, width was 24.0 to 39.0 mm and depth was between 13.2 and 25.5 mm. On histological examination there was no difference in cellular necrosis among any of the experimental freeze cycles for the cortex (p ⫽ 0.1) or the medulla (p ⫽ 0.81). The time based control arm showed no difference in cellular necrosis from that in the experimental arms in the medulla (p ⫽ 0.61) but showed more necrosis in the cortex (p ⫽ 0.05). There was significantly more necrosis in the experimental arms (p ⫽ 0.001) and in the time based control arm (p ⫽ 0.004) than in the sham control arm. Analysis of the cryolesion border revealed sharp demarcation with intratubular and interstitial hemorrhage. In the cryolesion 0.5 mm from the border the 1-minute experimental freeze cycle trials showed 1 grade 1 and 1 grade 2 nuclear injuries. The 5-minute freeze cycle showed 2 grade 1 and 1 grade 2 nuclear injuries. The 10-minute freeze cycle showed 3 grade 1 and 1 grade 2 nuclear injuries. Time based control trials showed 1 grade 1 and 5 grade 2 nuclear injuries.
DISCUSSION Although literature varies on the temperature necessary for cell death from ⫺16C17 to ⫺50C18 for neoplastic tissue, currently 20C is thought to be the target temperature to kill malignant renal cells.19,20 Several freeze cycles have been evaluated to date, during which temperature was recorded for some but not recorded for most. Stephenson et al studied renal cryotherapy with 4, 3.5 mm cryoprobes in a canine model via open and laparoscopic access.21 A single freeze-thaw cycle was used and lesions were monitored by ultrasound. Histology revealed a central region of complete necrosis bounded by a 2 mm region of marginal injury that
showed regenerative activity at 8 days. Long and Faller studied the feasibility of renal cryotherapy in a porcine model with 3 protocols, including a 3 to 4-minute freeze with a single cryoprobe, a 6 to 7-minute freeze with a single cryoprobe and a double 10-minute freeze with 2 cryoprobes spaced 2 mm apart.22 The single cryoprobes resulted in discrete lesions without perirenal damage. The 2 probes and double freeze produced large areas of infarction with significant perirenal reaction in 5 of the 6 animals and hydronephrosis/renal loss in 2. Based on histological analysis they recommended treating 3 to 4 cm exophytic lesions amenable to surface ultrasound monitoring. When investigating the most appropriate freezing duration and the number of probes needed to cause necrosis without morbidity, Auge et al studied 5, 10 and 15-minute freeze cycles with a single or double 3.4 mm probe and active thaw in swine via open surgery.23 Temperature was measured 5, 10, 15 and 20 mm from the cryoprobe and ranged from ⫺10C to ⫺85C at the 5 mm sensor. At 4 to 7-day survival the single 5-minute freeze was inadequate to cause tissue necrosis, the 10-minute freeze resulted in necrosis without complications and the 15-minute freeze caused necrosis and renal fracture. A 10-minute freeze with a single or double probe was recommended. To evaluate the ideal number of freeze cycles and the type of thaw process Woolley et al evaluated single and double 15-minute freeze cycles with active or passive thaw using a 3 mm probe (Endocare, Irvine, California) in dogs via laparoscopic access after 4-week survival.24 Mean temperature recorded in the cryoprobe attained ⫺142C ⫾ 1.0C. The double freeze cycle produced a significantly larger area of liquefactive necrosis than the single freeze. A double 15-minute freeze with an active thaw was recommended to effectively ablate tissue and decrease operative time. To evaluate laparoscopic cryotherapy Bishoff et al treated the 2 poles of each kidney in 6 farm pigs with single and double 5 and 15-minute freeze cycles using a 4.8 mm probe.25 Temperature recorded within the probe attained ⫺180C. Kidneys were harvested for histopathology immediately, or at 1 or 13 weeks. They concluded that longer freeze cycles and double freeze cycles did not create further destruction compared to shorter and single freeze cycles. They recommended a single 15-minute freeze cycle with ice ball extension at least 1 cm beyond the lesion. Weld et al used intraoperative ultrasound and histological analysis to evaluate the ablation area created by an IceRod after a double 10-minute freeze and a 3-minute active thaw in the porcine kidney via a laparoscopic approach.26 They found a 19 mm
OPTIMAL FREEZE CYCLE LENGTH FOR RENAL CRYOTHERAPY
diameter zone of complete ablation 2 cm from the tip of the IceRod. This equates to necrosis 9.5 mm from the cryoprobe, which correlates with the nonlethal temperatures that we found 1 cm from the cryoprobe. Thus, there is good data correspondence between these 2 peer reviewed studies. In humans Edmunds et al noted that a single 15-minute freeze with an active thaw using a 3 mm probe was sufficient for complete destruction of 1.5 to 1.8 cm renal masses on histology after immediate partial nephrectomy in 2 patients.27 Cellular necrosis was evident on histological analysis in trials in which ⫺20C was attained and in freeze cycles based on time alone. With a 10-minute cryoablation period most treated parenchyma 1 cm from the probe never attained ⫺20C. This suggests that temperatures above ⫺20C, even above 0C, cause cellular necrosis. Although histology was performed in the acute phase, there would likely be additional damage with time due to endothelial damage and microvascular thrombosis. The extended time at cold temperature may have also contributed to cell death. This would explain our results clinically since we now know that the temperature in most patients was never close to ⫺20C with time based treatments but there has been a paucity of recurrences. The relative resistance to necrosis in the medulla in time based trials is difficult to explain since a similar finding was not seen in temperature based trials. Cells in the renal medulla are known to exist in a hostile milieu characterized by hypoxia due to relatively low blood flow, oxygen diffusion and the high metabolic demands of medullary cells.28 Also, these cells are exposed to wide variations in extracellular solute concentrations and abundant reactive oxygen species. Renal medullary cells survive in this environment by the high glycolytic capacity to decrease dependence on oxygen, high quantities of heat shock proteins and a transcriptional response to hypoxia. Other adaptations include enhanced expression of aldose reductase to combat osmotic stress, abundant superoxidase to endure oxidative stress and the ability to rapidly release organic osmolytes to reduce cell volume in the face of cellular edema. This multitude of adaptive responses of the renal medulla may explain why the time based control arm showed more necrosis in the cortex but no difference in medullary necrosis in the experimental arms. A study limitation is that histology was done immediately. Thus, direct cell injury from ice crystal formation was evaluated but not subsequent
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vascular injury and thrombosis. The study could be repeated with animal survival to evaluate each mechanism of necrosis. Another limitation is that this study was performed in healthy renal tissue. Malignant tissue may require longer or colder freeze cycles due to its vascularity. Also, penetration of each cryoneedle may have varied depending on kidney size among the different animals. In future studies this could be formally measured by intraoperative ultrasound or by measuring summary statistics of kidney size to determine tight CIs around the mean volume. Lastly, this was a pilot study with a small sample size due to the expense of the animals involved. Power to detect differences was consequently limited. Only differences greater than 80% could be reliably detected, given the small number of test kidneys. Currently our treatment regimen is to place sufficient cryoprobes to achieve a ⫺20C isotherm, as measured by multiple temperature sensing needles at the upper and lower tumor borders. We use 2, 10-minute freeze cycles and a 5-minute thaw cycle, and are satisfied if at any time during the freeze cycle the temperature decreases to ⫺20C or lower since our study showed that the interval at ⫺20C has no bearing on the amount of cell necrosis. Our regimen may be overly cautious, given the reported excellent results with unmonitored double 10-minute freeze cycles, during which we know in many cases ⫺20C was not attained. However, until we have additional data on the minimal temperature needed to incur cell death during a sustained 10minute period, we continue to use the ⫺20C goal.
CONCLUSIONS Three experimental cycles and 2 control arms were evaluated to delineate the optimal freeze cycle for renal cryotherapy. There was no difference in immediate cellular necrosis between double 1, 5 or 10minute freeze cycles after ⫺20C was attained. Time based cycles, ie 2, 10-minute freezes separated by a 5-minute thaw, showed more cellular necrosis than the experimental ⫺20C freeze cycles for the cortex but not for the medulla. With a standard 10-minute cryoablation period most treated parenchyma 1 cm from the probe never attains ⫺20C and commonly does not even attain 0C. However, cellular necrosis was evident on histological analysis for the timed trials and appeared to be no different (medulla) or better (cortex) than in animals in which ⫺20C was attained and maintained for a specific period.
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OPTIMAL FREEZE CYCLE LENGTH FOR RENAL CRYOTHERAPY
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