Circular STAG2 RNA Modulates Bladder Cancer Progression via miR-145-5p/TAGLN2 and Is Considered as a Biomarker for Recurrence.

The current study aimed to elucidate the regulatory mechanisms of the circRNA hsa_circ_0139697 (circSTAG2(16-25)) in BCa and to consider the opportunity of using circSTAG2(16-25) isolated from BCa patient urine as a marker for disease development prediction. The selection of this circRNA was determined by the special role of its parental gene STAG2 in BCa biology. The circRNA hsa_circ_0139697 was chosen from 25 STAG2 circRNAs due to its differential expression in the urine of BCa patients and healthy volunteers. Higher levels of circSTAG2(16-25) were detected in urine samples obtained from patients with recurrent tumors. A higher expression of circSTAG2(16-25) was also detected in more tumorigenic BCa cell lines. The overexpression of circSTAG2(16-25) in BCa cells induced the elevation of proliferation, motility, and invasion. To study the mechanisms of circSTAG2(16-25) activity, we confirmed that circSTAG2(16-25) can bind miR-145-5p in vitro as was predicted by bioinformatic search. miR-145-5p was shown to suppress some genes that promoted BCa progression. One of these genes, TAGLN2, encodes the protein Transgelin 2, which plays a role in BCa cell motility and invasion. Therefore, the possible mechanism of action of circSTAG2(16-25) could be sponging the tumor suppressor miR-145-5p, which results in activation of TAGLN2. In addition, circSTAG2(16-25) might be considered as a potential biomarker for recurrence prediction.

Organoid model of urothelial cancer: establishment and applications for bladder cancer research.

3D cancer cell models are suitable for drug evaluation because they more precisely mimic tissue architecture than 2D cultures. To study cytotoxicity of anticancer agents, the most sensitive CellTiter-Glo 3D assay is used. However, this is an end point assay, so it is not possible to consider the variance of the starting material amount in the final reading. It is difficult to maintain an even plating density of 3D organoids for cytotoxicity analysis. We present a simple, 3D bladder cancer culture that can be maintained, cryopreserved and used for molecular and drug response studies. We applied a simple modification of the drug response assay for 3D cultures by measuring the background signal with the CellTiter Blue assay before drug application.

Revisiting the pineal gland: a review of calcification, masses, precocious puberty, and melatonin functions.

Introduction: The pineal gland, an endocrine organ of the posterior cranial fossa famously involved in sleep and wakefulness, has continually been a topic of scientific advancement and curiosity. Methods: We review present an up-to-date review including the anatomy, embryology, and physiology of the pineal gland and its ability to secrete hormones including melatonin, pathophysiology of pineal gland tumors, cysts, and calcifications, their clinical presentation including their association with parkinsonism and precocious puberty, and various treatment approaches. Results: Exploring the biochemistry of melatonin, various calcification morphologies, and pineal tumors may uncover a wider role and the exhaustive case study consolidation allows clinicians to carefully review the literature and aid their treatment approaches. Conclusion: It is imperative that clinicians and diagnosticians are able to distinguish manifestations of an overlooked gland.

Clostridium perfringens enterotoxin as a potential drug for intravesical treatment of bladder cancer.

The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC). Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model. We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment. CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells. C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and phosphorylation status of EphA2 and Src after C-CPE treatment and found changes in expression and phosphorylated status of these regulatory molecules. These observations show that after additional preclinical studies CPE and C-CPE in combinations with other drugs can be considered as a potential modalities for intravesical treatment of BC because of its ability to effectively destroy BC cells expressing claudin 4 and low toxicity against normal urothelium.

Metachronous Malignant Solitary Fibrous Tumor of Kidney: Case Report and Review of Literature.

Solitary fibrous tumors are well described in the pleura, but rare extra-pleural neoplasms have been reported. We describe a patient with a solitary left renal fibrous tumor who after undergoing a nephrectomy, presented 8 years later with a contralateral metachronous solitary fibrous tumor. Malignant metastatic extra-pleural solitary fibrous tumors are extremely rare, and to our knowledge, this is the first case of contralateral recurrence of solitary renal fibrous tumor. The patient underwent a robotic assisted partial nephrectomy of the right renal mass. Both tumors showed overlapping histopathology.

Metabolic alterations in bladder cancer: applications for cancer imaging.

Treatment planning, outcome and prognosis are strongly related to the adequate tumor staging for bladder cancer (BC). Unfortunately, a large discrepancy exists between the preoperative clinical and final pathologic staging. Therefore, an advanced imaging-based technique is crucial for adequate staging. Although Magnetic Resonance Imaging (MRI) is currently the best in vivo imaging technique for BC staging because of its excellent soft-tissue contrast and absence of ionizing radiation it lacks cancer-specificity. Tumor-specific positron emission tomography (PET), which is based on the Warburg effect (preferential uptake of glucose by cancer cells), exploits the radioactively-labeled glucose analogs, i.e., FDG. Although FDG-PET is highly cancer specific, it lacks resolution and contrast quality comparable with MRI. Chemical Exchange Saturation Transfer (CEST) MRI enables the detection of low concentrations of metabolites containing protons. BC is an attractive target for glucose CEST MRI because, in addition to the typical systemic administration, glucose might also be directly applied into the bladder to reduce toxicity-related complications. As a first stage of the development of a contrast-specific BC imaging technique we have studied glucose uptake by bladder epithelial cells and have observed that glucose is, indeed, consumed by BC cells with higher intensity than by non-transformed urothelial cells. This effect might be partly explained by increased expression of glucose transporters GLUT1 and GLUT3 in transformed cells as compared to normal urothelium. We also detected higher lactate production by BC cells which is another cancer-specific manifestation of the Warburg effect. In addition, we have observed other metabolic alterations in BC cells as compared to non-transformed cells: in particular, increased pyruvate synthesis. When glucose was substituted by glutamine in culture media, preferential uptake of glutamine by BC cells was observed. The preferential uptake of glucose by BC cells gives an opportunity to develop NMR based imaging procedures where glucose or its derivatives can serve as a contrasting agent. In addition, metabolic alterations observed in BC cells could provide the basis for development of new anti-cancer therapeutics.

Aristolochic acid-induced apoptosis and G2 cell cycle arrest depends on ROS generation and MAP kinases activation.

Ingestion of aristolochic acids (AAs) contained in herbal remedies results in a renal disease and, frequently, urothelial malignancy. The genotoxicity of AA in renal cells, including mutagenic DNA adducts formation, is well documented. However, the mechanisms of AA-induced tubular atrophy and renal fibrosis are largely unknown. To better elucidate some aspects of this process, we studied cell cycle distribution and cell survival of renal epithelial cells treated with AAI at low and high doses. A low dose of AA induces cell cycle arrest in G2/M phase via activation of DNA damage checkpoint pathway ATM-Chk2-p53-p21. DNA damage signaling pathway is activated more likely via increased production of reactive oxygen species (ROS) caused by AA treatment then via DNA damage induced directly by AA. Higher AA concentration induced cell death partly via apoptosis. Since mitogen-activated protein kinases play an important role in cell survival, death and cell cycle progression, we assayed their function in AA-treated renal tubular epithelial cells. ERK1/2 and p38 but not JNK were activated in cells treated with AA. In addition, pharmacological inhibition of ERK1/2 and p38 as well as suppression of ROS generation with N-acetyl-L-cysteine resulted in the partial relief of cells from G2/M checkpoint and a decline of apoptosis level. Cell cycle arrest may be a mechanism for DNA repair, cell survival and reprogramming of epithelial cells to the fibroblast type. An apoptosis of renal epithelial cells at higher AA dose might be necessary to provide space for newly reprogrammed fibrotic cells.

A claudin 3 and claudin 4-targeted Clostridium perfringens protoxin is selectively cytotoxic to PSA-producing prostate cancer cells.

Prostate cancer is the second leading cause of non-cutaneous cancer-related death in males, and effective strategies for treatment of metastatic disease are currently limited. The tight junction proteins, claudin 3 and claudin 4, serve as cell-surface receptors for the pore-forming Clostridium perfringens enterotoxin [CPE]. Most prostate cancer cells overexpress claudin 3 and claudin 4, and claudins are aberrantly distributed over the plasma membrane, making these cells particularly sensitive to cytolysis by CPE. Prostate cancer cells secrete PSA locally that is proteolytically active; however, circulating PSA is inactivated via binding to protease inhibitors. To overcome systemic toxicity of CPE, a modified protoxin was constructed with a tethered ligand attached to the C-terminus connected by a flexible linker containing a PSA-specific protease cleavage site. This engineered protoxin selectively and efficiently lyses PSA-producing prostate cancer cells whereas CLDN3 and CLDN4 positive cells that do not express PSA are resistant to cytolysis.

Early detection of carcinoma in situ of the bladder: a comparative study of white light cystoscopy, narrow band imaging, 5-ALA fluorescence cystoscopy and 3-dimensional optical coherence tomography.

PURPOSE: We compared the efficacy and potential limitations of white light cystoscopy, narrow band imaging, 5-ALA fluorescence cystoscopy and 3-dimensional optical coherence tomography for early diagnosis of bladder carcinoma in situ. MATERIALS AND METHODS: By expressing simian virus 40T antigen in the urothelium carcinoma in situ typically develops in SV40T transgenic mice in about 8 to 20 weeks and then frank high grade papillary urothelial carcinoma starts to emerge. A total of 18 control and 29 SV40T mice were examined during weeks 8 to 22 by white light cystoscopy, fluorescence cystoscopy, narrow band imaging and 3-dimensional optical coherence tomography. Results were validated by histology. Newly improved algorithms for computer aided detection were applied to acquired 3-dimensional optical coherence tomography images to enhance the quantitative diagnosis of carcinoma in situ in near real time. RESULTS: Of 29 carcinoma in situ samples 27 were detected by 3-dimensional optical coherence tomography, 1 by white light cystoscopy, 26 by narrow band imaging and 13 by fluorescence cystoscopy. Of the 18 histologically confirmed benign cases 17 were detected by 3-dimensional optical coherence tomography, 14 by white light cystoscopy, 5 by narrow band imaging and 18 by fluorescence cystoscopy. The diagnostic sensitivity of white light cystoscopy (3.4%) and fluorescence cystoscopy (44.8%), and the specificity of narrow band imaging (27.8%) were significantly enhanced by 3-dimensional optical coherence tomography to 93.1% and 94.4%, respectively (p <0.01). CONCLUSIONS: Three-dimensional optical coherence tomography with quantitative computer aided detection can significantly enhance the sensitivity of white light cystoscopy and fluorescence cystoscopy, and the specificity of narrow band imaging for early diagnosis of carcinoma in situ. This suggests the potential of narrow band imaging guided 3-dimensional optical coherence tomography for future clinical detection of carcinoma in situ when effective image guidance is desirable.

Diagnosis of bladder cancer with microelectromechanical systems-based cystoscopic optical coherence tomography.

OBJECTIVES: To examine the utility and potential limitations of microelectromechanical systems-based spectral-domain cystoscopic optical coherence tomography (COCT) so as to improve the diagnosis of early bladder cancer. METHODS: An optical coherence tomography catheter was integrated into the single instrument channel of a 22F cystoscope to permit white-light-guided COCT over a large field of view (4.6 mm wide and 2.1 mm deep per scan at 8 frames/s) and 10-microm resolution. Intraoperative COCT diagnosis was performed in 56 patients, with a total of 110 lesions examined and compared with biopsied histology. RESULTS: The overall sensitivity of COCT (94%) was significantly higher than cystoscopy (75%, P = .02) and voided cytology (59%, P = .005); the major enhancement over cystoscopy was for low-grade pTa-1 cancer and carcinoma in situ (P < .018). The overall specificity of COCT (81%) was comparable to voided cytology (88.9%, P = .49), but significantly higher than cystoscopy (62.5%, P = .02). CONCLUSIONS: The microelectromechanical systems-based COCT, owing to its high resolution and detection sensitivity and large field of view, offers great potential for "optical biopsy" to enhance the diagnosis of nonpapillary bladder tumors and their recurrences and to guide bladder tumor resection.

In vivo bladder imaging with microelectromechanical-systems-based endoscopic spectral domain optical coherence tomography.

We report the recent technical improvements in our microelectromechanical systems (MEMS)-based spectral-domain endoscopic OCT (SDEOCT) and applications for in vivo bladder imaging diagnosis. With the technical advances in MEMS mirror fabrication and endoscopic light coupling methods, the new SDEOCT system is able to visualize morphological details of the urinary bladder with high image fidelity close to bench-top OCT (e.g., 10 mum12 mum axial/lateral resolutions, >108 dB dynamic range) at a fourfold to eightfold improved frame rate. An in vivo animal study based on a porcine acute inflammation model following protamine sulfate instillation is performed to further evaluate the utility of SDEOCT system to delineate bladder morphology and inflammatory lesions as well as to detect subsurface blood flow. In addition, a preliminary clinical study is performed to identify the morphological features pertinent to bladder cancer diagnosis, including loss of boundary or image contrast between urothelium and the underlying layers, heterogeneous patterns in the cancerous urothelium, and margin between normal and bladder cancers. The results of a human study (91% sensitivity, 80% specificity) suggest that SDEOCT enables a high-resolution cross-sectional image of human bladder structures to detect transitional cell carcinomas (TCC); however, due to reduced imaging depth of SDEOCT in cancerous lesions, staging of bladder cancers may be limited to T1 to T2a (prior to muscle invasion).

High body mass index in muscular patients and flank position are risk factors for rhabdomyolysis: case report after laparoscopic live-donor nephrectomy.

BACKGROUND AND PURPOSE: Rhabdomyolysis is well known after traumatic crush injuries or ischemia involving muscles. Postoperatively, it most likely is secondary to surgical positioning and patient muscle mass. We report a case after laparoscopic live-donor nephrectomy. CASE REPORT: A muscular 35-year-old man underwent elective left laparoscopic live-donor nephrectomy in a 70 degrees flank position with four ports. He was in the right-side lying position with hip flexion (flank position) for approximately 4 hours. A kidney bridge had been placed between the iliac crest and the rib cage. Postoperatively, the patient had light-pinkish urine and low urine output. There was marked induration of the buttocks and significant pedal and scrotal edema. With judicious use of alkalinization and diuretics, the patient did not require dialysis, and renal function returned to base level by postoperative day 20. The recipient of the kidney had a normal postoperative course. CONCLUSION: Rhabdomyolysis is a syndrome of muscle necrosis and release of intracellular components into the circulation. Acute renal failure secondary to myoglobinuria is a common complication. We currently use little flexion of the table during donor nephrectomy and bring the table to a neutral position immediately after kidney retrieval. Postoperatively, one needs a high index of suspicion for rhabdomyolysis to avoid or at least promptly recognize this rare but potentially serious condition after any operation lasting >or=4 hours.

Extraperitoneal laparoscopic prostatectomy (adenomectomy) for obstructing benign prostatic hyperplasia: transvesical and transcapsular (Millin) techniques.

PURPOSE: We describe extraperitoneal laparoscopic resection of large prostatic adenomas (<100 g) as an alternative to open simple prostatectomy by both the transcapsular or Millin and the transvesical approaches. PATIENTS AND METHODS: We have performed more than 20 laparoscopic prostatectomies (adenomectomies) for benign prostatic hyperplasia (BPH) for glands >100 g. The initial two cases, with follow-up longer than 1 year, are included in this report. Using an extraperitoneal approach, enucleation of the obstructing prostatic lobes was performed with the aid of a Harmonic Scalpel and laparoscopic claw forceps. Hemostatic sutures were placed at 5 and 7 o'clock. The urethrovesical junction (transvesical) or capsulotomy (Millin) were closed in an interrupted fashion using intracorporeal sutures. RESULTS: Both procedures were successful. The total operative time was 180 minutes for first the case and 120 minutes for the second. The adenoma removed was approximately 138 g in the first case and 102 g in the second case. The estimated blood loss was <50 mL and <200 mL, respectively. The postoperative courses were unremarkable. Analgesic requirements were minimal, and the patient was discharged on postoperative day 2 and 3, respectively. A follow-up examination at 1, 3, 6, and 12 months showed that the flow rate is >20 mL and the postvoiding residual volume 0, with normal continence and sexual potency in both men. CONCLUSIONS: Extraperitoneal laparoscopic simple prostatectomy is a simple straightforward technique. Minimal bleeding, a reduced transfusion rate, shorter hospitalization, and faster recovery are additional advantages. This minimally invasive technique is a reasonable alternative to open simple prostatectomy for large glands with reduced morbidity.

Instrumentation for laparoscopic renal surgery--Padron Endoscopic Exposing Retractor (PEER) and Endoholder: point of technique.

During laparoscopic surgery, as in open surgery, exposure is critical. However, this can be difficult during laparoscopy due to limited haptic feedback and the loss of 3-dimensional visualization. Excessive force may be inadvertently applied by assistants when anatomic structures are retracted; similarly, the retractors may be unknowingly moved because the limited field of view with the laparoscope precludes constant visualization of the retracting instrument. To overcome these problems, we have been using a 5- or 10-mm PEER retractor in combination with an articulating arm instrument holder (Endoholder) to aid laparoscopic renal surgery. The adjustable spring-loaded articulating instrument holder (Endoholder) consists of 4 components, including table attachment, a base rod, flexible extension arm, and precision clamp. The clamp accommodates variously sized instruments, and the flexible extension arm rotates 360 degrees to aid in positioning. The instrument holder is clamped to the table via the base rod over a sterile drape. A PEER Retractor, Roto-lok ratchet (5- or 10-mm diameter and 32-cm length) is placed intracorporeally to retract and position the kidney for hilar, upper, and lower pole dissection. The PEER retractor's handle is secured in place using the precision clamp of the instrument holder. The articulating instrument holder and PEER retractor are used for our renal, adrenal, and ureteral laparoscopic procedures. Placement of the retractor through a 5- or 10-mm port and deployment can be done quickly. Adequate and stable positioning of the retractor provides excellent and secure visualization of the operative field. These instruments have been used in more than 200 cases without any complication except 1 minor liver laceration. The articulating instrument holder with the PEER retractor is a very useful aid during laparoscopic renal surgery. This instrument reduces the chances of inadvertent injury to viscera by the assistant while maintaining an excellent anatomic view throughout the procedure. This will have a significant impact on the advancement of laparoscopy and its acceptance by every urologist.

Prostate cancer cell adhesion to bone marrow endothelium: the role of prostate-specific antigen.

Bone metastasis is the most frequent complication of prostate cancer (PC). Elucidation of the biological basis of this specificity is required for the development of approaches for metastatic inhibition. We investigated the possibility that the preferential attachment of PC cells to bone marrow endothelium (as opposed to endothelium from other organs) affects this specificity. We selected, from peptide phage-displayed libraries, peptide ligands to surfaces of PC cells (C4-2B) attenuated (30-40%) binding of C4-2B cells to bone marrow endothelial cells (BMECs). We then determined the molecules on the surface of C4-2B cells interacted with the selected peptides using column affinity chromatography and a cDNA expression phage-displayed library generated from C4-2B cells in T7 phage. We identified a phage from the cDNA library that specifically bound to one of the selected peptides-L11. This phage displayed the amino acid sequence homologous for the COOH-terminal portion of prostate-specific antigen (PSA). To examine the possible direct involvement of PSA in the interactions between PC and BMECs, we performed a cell-cell adhesion assay. Antibodies to PSA attenuated PC cells adhesion to BMECs. In addition, exogenous proteolytically active PSA modulated this adhesion. Finally, inactivation of mRNA coding PSA by a small interfering RNA (siRNA) diminished C4-2B cell adhesion to BMECs. These results indicate that PSA expressed as secreted and surface-associated molecules in C4-2B cells is involved in cell-cell interactions and/or digests components of bone marrow endothelium for preferential adhesion and penetration of PC cells. The suggested experimental approach is a promising strategy for identification of cell surface molecules involved in intercellular interactions.

[Inhibition of lectin induced peripheral blood lymphocyte proliferation by colorectal cancer extract: a preliminary report of three cases].

BACKGROUND: Tumor infiltrating lymphocytes (TIL) in colorectal cancer are a manifestation of local, cell mediated immune response to the malignant tumor. Tumor progression is due to impairment of the host ability to control tumor growth. Several studies suggested possible causes for such impairment, however, the precise factor(s) underlying such malfunction is uncertain. AIM: To compare the possible effects of colorectal cancer (CRC) and normal colonic mucosa extracts on lectin induced blastogenesis of the same patients' peripheral blood lymphocytes (PBL) proliferation. METHODS: CRC and normal mucosa extracts were obtained from 3 patients undergoing curative surgery for colon adenocarcinoma. Proliferation assays used PBL from the CRC patients, incubated with Concanavalin A (ConA) and Phytohemoglobin (PHA) in the presence of CRC or normal mucosa extract and in medium alone. Proliferation was measured by H3 Thymidine incorporation following 48 hours on incubation. RESULTS: Exposure of ConA induced PBL proliferation assay to CRC extract yielded a 98.7% inhibition measured by counts/minute (cpm) of incorporated H3 Thymidine compared to normal colonic mucosa extract (1,214 +/- 594 cpm vs. 95,335 +/- 6,997 cpm respectively, p = 0.0018). PHA stimulated proliferation exposed to CRC extract showed a 99.7% decrease in blastogenic activity compared to normal mucosa extract (362 +/- 175 cpm vs. 62,375 +/- 16,591 cpm respectively, p = 0.0234). CONCLUSIONS: These preliminary results suggest that CRC extract contain factor(s) capable of profoundly inhibiting lectin induced proliferation of PBL. Shedding of suppressor substances may be one of the possible mechanisms by which the tumor evades the effector arm of the cell-mediated immune response. Characterization of such factors may aid in intratumor, local and systemic cellular immune response reconstitution.

Effect of freezing parameters (freeze cycle and thaw process) on tissue destruction following renal cryoablation.

BACKGROUND AND PURPOSE: Renal cryoablation is a successful nephron-sparing treatment alternative for selected patients with small renal tumors. The purpose of this study was to compare the effects of the number of freeze cycles (one v two) and the thaw process (active v passive) on renal tissue following cryodestruction. MATERIALS AND METHODS: Sixteen female mongrel dogs (19.9 +/- 2.1 kg) were randomly divided into four groups and underwent transabdominal laparoscopic access by standard techniques. Tissue freezing was performed using argon gas following interstitial cryoprobe (3 mm) placement into the upper and lower poles of the left kidney. Single active (SA), single passive (SP) double active (DA) or double passive (DP) 15-minute treatment cycle(s) were carried out via the CRYOcare Cryosurgical Unit (Endocare, Irving, CA) on eight kidneys each. An active thaw process with helium gas or a passive thaw process was initiated after each freeze period. The cryoprobe was removed when the temperature reached 0 degrees C. Four weeks following cryosurgery, animals were sacrificed, and the renal tissue was evaluated grossly and histologically. RESULTS: Interstitial cryoprobe temperatures decreased from 31.3 degrees C +/- 1.4 degrees C to -142 degrees C +/- 1.0 degrees C following the 15-minute freeze cycle. The temperature reached 0 degrees C significantly faster following active thaw than with the passive process (2.13 +/- 0.24 min/freeze cycle and 15.18 +/- 2.97 min/freeze cycle, respectively; P < 0.0001). Grossly, each lesion consisted of a central area of necrosis surrounded by a rim of white tissue. On microscopic examination, each lesion consisted of a central area of liquefaction necrosis (LN) surrounded by various degrees of fibrosis and granulation tissue admixed with residual parenchyma. The size of the LN was significantly different in tissues subjected to double and single freeze cycles when compared across both thaw processes (active and passive). There was no significant difference in the overall lesion volume following DA, DP, SA, or SP. CONCLUSIONS: Renal cryodestruction via laparoscopic access achieves complete tissue ablation without complications. The double freeze cycle produced significantly larger areas of LN than the single freeze regardless of the thaw process. The type of thaw process did not affect the amount of tissue damage. Utilizing a double 15-minute freeze cycle with the faster active thaw process will effectively cryoablate renal tissue as well as significantly reduce overall operative time.