Re-purposing cryoablation: a combinatorial 'therapy' for the destruction of tissue.

It is now recognized that the tumor microenvironment creates a protective neo-tissue that isolates the tumor from the various defense strategies of the body. Evidence demonstrates that, with successive therapeutic attempts, cancer cells acquire resistance to individual treatment modalities. For example, exposure to cytotoxic drugs results in the survival of approximately 20-30% of the cancer cells as only dividing cells succumb to each toxic exposure. With follow-up treatments, each additional dose results in tumor-associated fibroblasts secreting surface-protective proteins, which enhance cancer cell resistance. Similar outcomes are reported following radiotherapy. These defensive strategies are indicative of evolved capabilities of cancer to assure successful tumor growth through well-established anti-tumor-protective adaptations. As such, successful cancer management requires the activation of multiple cellular 'kill switches' to prevent initiation of diverse protective adaptations. Thermal therapies are unique treatment modalities typically applied as monotherapies (without repetition) thereby denying cancer cells the opportunity to express defensive mutations. Further, the destructive mechanisms of action involved with cryoablation (CA) include both physical and molecular insults resulting in the disruption of multiple defensive strategies that are not cell cycle dependent and adds a damaging structural (physical) element. This review discusses the application and clinical outcomes of CA with an emphasis on the mechanisms of cell death induced by structural, metabolic, vascular and immune processes. The induction of diverse cell death cascades, resulting in the activation of apoptosis and necrosis, allows CA to be characterized as a combinatorial treatment modality. Our understanding of these mechanisms now supports adjunctive therapies that can augment cell death pathways.

Mechanisms of cryoablation: clinical consequences on malignant tumors.

While the destructive actions of a cryoablative freeze cycle are long recognized, more recent evidence has revealed a complex set of molecular responses that provides a path for optimization. The importance of optimization relates to the observation that the cryosurgical treatment of tumors yields success only equivalent to alternative therapies. This is also true of all existing therapies of cancer, which while applied with curative intent; provide only disease suppression for periods ranging from months to years. Recent research has led to an important new understanding of the nature of cancer, which has implications for primary therapies, including cryosurgical treatment. We now recognize that a cancer is a highly organized tissue dependent on other supporting cells for its establishment, growth and invasion. Further, cancer stem cells are now recognized as an origin of disease and prove resistant to many treatment modalities. Growth is dependent on endothelial cells essential to blood vessel formation, fibroblasts production of growth factors, and protective functions of cells of the immune system. This review discusses the biology of cancer, which has profound implications for the diverse therapies of the disease, including cryosurgery. We also describe the cryosurgical treatment of diverse cancers, citing results, types of adjunctive therapy intended to improve clinical outcomes, and comment briefly on other energy-based ablative therapies. With an expanded view of tumor complexity we identify those elements key to effective cryoablation and strategies designed to optimize cancer cell mortality with a consideration of the now recognized hallmarks of cancer.

Temperature-dependent activation of differential apoptotic pathways during cryoablation in a human prostate cancer model.

BACKGROUND: Critical to the continual improvement of cryoablation efficacy is deciphering the biochemical responses of cells to low-temperature exposure. The identification of delayed-onset cell death has allowed for the manipulation of cellular responses through the regulation of apoptosis. We hypothesized that in addition to delayed apoptotic events associated with mild subfreezing temperatures (10 to -25 °C), cells exposed to ultra-low temperatures (<-30 °C) may undergo rapid, early-onset apoptosis. METHODS: Human prostate cancer model and cells (PC-3) were exposed to temperatures of -60, -30 and -15 °C to simulate a cryoablative procedure. Using a combination of flow-cytometry, fluorescent microscopy and western blot analyses, samples were assessed at various times post thaw to identify the presence, levels and the pathways involved in cell death. RESULTS: Exposure to temperatures <-30 °C yielded a significant apoptotic population within 30 min of thawing, peaking at 90 min (~40%), and by 6 h, only necrosis was observed. In samples only reaching temperatures >-30 °C, apoptosis was not noted until 6-24 h post thaw, with the levels of apoptosis reaching ~10% (-15 °C) and ~25% (-30 °C) at 6 h post thaw. Further, it was found that early-onset apoptosis progressed through a membrane-mediated mechanism, whereas delayed apoptosis progressed through a mitochondrial path. CONCLUSIONS: These data demonstrate the impact of apoptotic continuum, whereby the more severe cryogenic stress activated the extrinsic, membrane-regulated pathway, whereas less severe freezing activated the intrinsic, mitochondrial-mediated path. The rapid induction and progression of apoptosis at ultra-low temperatures provides an explanation as to why such results have not previously been identified following freezing. Ultimately, an understanding of the events and signaling pathways involved in triggering apoptosis following freezing may provide a path for selective induction of the rapid-onset and delayed programmed cell death pathways in an effort to improve the overall cryoablation efficacy.

Use of 1,25α dihydroxyvitamin D3 as a cryosensitizing agent in a murine prostate cancer model.

Cryotherapy has emerged as a primary treatment option for prostate cancer (CaP); however, incomplete ablation in the periphery of the cryogenic lesion can lead to recurrence. Accordingly, we investigated the use of a non-toxic adjunctive agent, vitamin D3 (VD3), with cryotherapy to sensitize CaP to low temperature-induced, non-ice rupture-related cell death. VD3 (calcitriol) has been identified as a possible adjunct in the treatment of cancer because of its antiproliferative and antitumorigenic properties. This study aimed to identify the cellular responses and molecular pathways activated when VD3 (calcitriol) is combined with cryotherapy in a murine CaP model. Single freeze-thaw events above -15 °C had little effect on cancer cell viability; however, pretreatment with calcitriol in conjunction with cryo significantly increased cell death. The -15 °C calcitriol combination increased cell death to 55% following a single freeze compared with negligible cell loss by freezing or calcitriol alone. Repeated cryo combination yielded 90% cell death compared with 65% in dual freeze-only cycles. Western blot analysis following calcitriol cryosensitization regimes confirmed the activation of apoptosis. Specifically, proapoptotic Bid and procaspase-3 were found to decrease at 1 h following combination treatment, indicating cleavage to the active forms. A parallel in vivo study confirmed the increased cell death when combining cryotherapy with calcitriol pretreatment. The development of an adjunctive therapy combining calcitriol and cryotherapy represents a potentially highly effective, less toxic, minimally invasive treatment option. These results suggest a role for calcitriol and cryo as a combinatorial treatment for CaP, with the potential for clinical translation.

Integrin involvement in freeze resistance of androgen-insensitive prostate cancer.

Cryoablation has emerged as a primary therapy to treat prostate cancer. Although effective, the assumption that freezing serves as a ubiquitous lethal stress is challenged by clinical experience and experimental evidence demonstrating time-temperature-related cell-death dependence. The age-related transformation from an androgen-sensitive (AS) to an androgen-insensitive (AI) phenotype is a major challenge in the management of prostate cancer. AI cells exhibit morphological changes and treatment resistance to many therapies. As this resistance has been linked with alpha6beta4 integrin overexpression as a result of androgen receptor (AR) loss, we investigated whether alpha6beta4 integrin expression, as a result AR loss, contributes to the reported increased freeze tolerance of AI prostate cancer. A series of studies using AS (LNCaP LP and PC-3 AR) and AI (LNCaP HP and PC-3) cell lines were designed to investigate the cellular mechanisms contributing to variations in freezing response. Investigation into alpha6beta4 integrin expression revealed that AI cell lines overexpressed this protein, thereby altering morphological characteristics and increasing adhesion characteristics. Molecular investigations revealed a significant decrease in caspases-8, -9, and -3 levels in AI cells after freezing. Inhibition of alpha6beta4 integrin resulted in increased caspase activity after freezing (similar to AS cells) and enhanced cell death. These data show that AI cells show an increase in post-freeze susceptibility after inhibition of alpha6beta4 integrin function. Further understanding the role of androgen receptor-related alpha6beta4 integrin expression in prostate cancer cells responses to freezing might lead to novel options for neo-adjunctive treatments targeting the AR signaling pathway.

Experimental cryosurgery investigations in vivo.

Cryosurgery is the use of freezing temperatures to elicit an ablative response in a targeted tissue. This review provides a global overview of experimentation in vivo which has been the basis of advancement of this widely applied therapeutic option. The cellular and tissue-related events that underlie the mechanisms of destruction, including direct cell injury (cryolysis), vascular stasis, apoptosis and necrosis, are described and are related to the optimal methods of technique of freezing to achieve efficacious therapy. In vivo experiments with major organs, including wound healing, the putative immunological response following thawing, and the use of cryoadjunctive strategies to enhance cancer cell sensitivity to freezing, are described.

Cryoablation induces necrosis and apoptosis in lung adenocarcinoma in mice.

This study evaluated cryoablation on subcutaneously transplanted tumors of lung adenocarcinoma LA795 in T739 mice in vivo, in an effort to assess the feasibility of cryoablation in treatment of NSCLC. Subcutaneously transplanted lung adenocarcinoma LA795 was implanted into T739 mice yielding tumors of approximately 2.5 cm in diameter. Following cryoablation, the various modes of cell death were studied: necrosis in the central frozen zone by light microscopy and apoptosis in periphery of the frozen zone by in situ end labeling (TUNEL). Bc1-2 and bax expression were detected by immunohistochemical SABC procedures, and the cleavage and activation of Caspase 3 and PARP in peripheral zone by Western blot. We find that in central cryoablated zone, necrosis was the dominant mode of cell death occurring at three hours and four days post-thaw. The first three-hour necrosis peak involved approximately 47% of the tumor while the four-day peak increased in volume to 68% of the tumor. In peripheral cryoablation zone, definite cell apoptosis could be observed by morphological examination under light microscope and TUNEL staining, peaking at 8-16 h after cryoablation. Immunohistochemical results yielded little change in bcl-2 protein expression before and after cryoablation. However, bax protein expression was up-regulated significantly after cryoablation. In addition, cleavage and activation of Caspase-3 and PARP occurred in the peripheral freeze zone after the treatment. It indicated that Cryoablation efficiently induces cell death both by necrosis and apoptosis. Cryoablation appears to induce apoptosis in the peripheral freeze zone through the intrinsic mitochondrial caspase pathway based on bax upregulation. This observation allows us to suggest that cryoablation may be combined with chemotherapy to increase cancer destruction.

Issues critical to the successful application of cryosurgical ablation of the prostate.

The techniques of present-day cryosurgery performed with multiprobe freezing apparatus and advanced imaging techniques yield predictable and encouraging results in the treatment of prostatic and renal cancers. Nevertheless, and not unique to cryosurgical treatment, the rates of persistent disease demonstrate the need for improvement in technique and emphasize the need for proper management of the therapeutic margin. The causes of persistent disease often relate to a range of factors including selection of patients, understanding of the extent of the tumor, limitations of the imaging techniques, and failure to freeze the tumor periphery in an efficacious manner. Of these diverse factors, the one most readily managed, but subject to therapeutic error, is the technique of freezing the tumor and appropriate margin to a lethal temperature [Baust, J. G., Gage, A. A. The Molecular Basis of Cryosurgery. BJU Int 95, 1187-1191 (2005)]. This article describes the recent experiments that examine the molecular basis of cryosurgery, clarifies the actions of the components of the freeze-thaw cycle, and defines the resultant effect on the cryogenic lesion from a clinical perspective. Further, this review addresses the important issue of management of the margin of the tumor through adjunctive therapy. Accordingly, a goal of this review is to identify the technical and future adjunctive therapeutic practices that should improve the efficacy of cryoablative techniques for the treatment of malignant lesions.

Targeted induction of apoptosis via TRAIL and cryoablation: a novel strategy for the treatment of prostate cancer.

Adjuvant therapies contribute to the successful treatment of cancer. Our previous reports have shown that combining cryoablation with cytotoxic agents enhances cell death. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a cytotoxic agent that preferentially induces apoptosis in a variety of human cancer cells. Human prostate cancer cells (PC-3) are resistant to many cytodestructive agents, including cryoablation and TRAIL. Here, we evaluated the effects of TRAIL combined with cryoablation on PC-3 and normal prostate (RWPE-1) cell death. Exposure of PC-3 cells to freezing (-10 degrees C) or TRAIL (500 ng/ml) results in minimal cell death, whereas a complete loss of viability is observed with the simultaneous combination. The synergistic effect was found to be due to a marked increase in apoptosis. Western blot analysis revealed a significant level of caspase-8 and -3 cleavage between 12 and 24 h post-exposure. Caspase activation assays provided similar results and also indicated a role for caspase-9. Inhibitors to caspase-8 and -9 along with a pan-caspase inhibitor were incorporated to determine which pathway was necessary for the combined efficacy. Inhibition of caspase-8 significantly blocked the combination-induced cell death compared to cells that did not receive the inhibitor (63% compared to 10% viable). The addition of the caspase-9 inhibitor resulted in only a minimal protection. Importantly, the combination was not effective when applied to normal prostate cells. The results describe a novel therapeutic model for the treatment of prostate cancer and provide support for future in vivo studies.

Progress toward optimization of cryosurgery.

Cryosurgery for diverse neoplastic and non-neoplastic diseases has expanded in applicability in recent years, especially since intraoperative ultrasound became available as a method of monitoring the process of tissue freezing. However, persistence of disease after presumably adequate cryosurgical treatment has disclosed deficiencies in the technique, perhaps due to faulty application of the freeze-thaw cycles or due to shortcomings in the imaging method. Clearly cryosurgical technique is less than optimal. The optimal dosimetry for tissue freezing, the recent improvements in imaging techniques, and the need for adjunctive therapy are defined in this review, which assesses the progress toward improving the efficacy of cryosurgery.

Chemo-cryo combination therapy: an adjunctive model for the treatment of prostate cancer.

Despite continuing research and the development of alternate therapeutic options, prostate cancer remains problematic. Chemotherapy has played a minor role as a treatment option due to its lack of efficacy. Whereas cryotherapy has received renewed attention as a treatment modality, it too fails to offer an absolute curative option. Previously, we reported on the utilization of a therapeutic model, which, in combination, increases cell death in a canine renal cell model. Based upon that study, we investigated a combination therapy model as an alternative for the treatment modality for prostate cancer. We hypothesized that the combination of chemotherapy and cryosurgery would result in enhanced cell death, thereby presenting a more effective treatment of prostate cancer. A human prostate cancer cell (PC-3) model was exposed to 5-fluorouracil (5-FU) for 2 and 4 days (prefreeze), freezing (-5 to -100 degrees C), or a combination of the two treatments, and each was assessed for effectiveness over a 2-week posttreatment period. Additionally, investigation into the mechanisms of cell death initiated by the respective therapies was performed through DNA cleavage analysis. For chemotherapy, cultures exposed to 5-FU (2-4 days) yielded a 15-25% loss in cell survival. For cryotherapy, cultures exposed to a temperature window of -5 to -20 degrees C yielded an initial 5-70% loss of viability but cells propagated over time. Cultures exposed to temperatures of -25 to -80 degrees C yielded a 90-99% (+/-4.5%) initial loss in viability with repopulation observed by 12 days postthaw. Cells frozen to -100 degrees C yielded 100% (+/-0.3%) loss of viability and exhibited no signs of propagation. For chemo-cryo therapy, combination treatment at milder temperatures (-5 to -25 degrees C) resulted in an enhanced loss of cell viability compared to that for either treatment alone. Combination treatment at lower temperatures (-40 to -80 degrees C) resulted in a complete loss of cell viability. DNA fragmentation analysis at 48 h posttreatment revealed that dead (detached) cells treated with 5-FU died primarily through apoptosis, whereas dead cells from freezing (-15 degrees C) alone died primarily through freeze-rupture and necrosis. Detached cell analysis from combination treatment at -15 degrees C revealed the presence of apoptotic, necrotic, and freeze-rupture cell death. Scanning electron micrographs of cells exposed to freezing contributing to cell death. These data demonstrate that the combination of 5-FU at sublethal doses and freezing temperatures improves human prostate cancer cell death efficacy. Further, we suggest that chemo-cryo therapy offers a potential alternative treatment for the control and eradication of prostate cancer.

The roles of iNOS in liver ischemia-reperfusion injury.

To determine the contribution of the inducible nitric oxide synthase (iNOS) to hepatic injury following warm ischemia-reperfusion, we developed a model of partial hepatic ischemia-reperfusion in mice and studied the injury response in iNOS knockout (KO) mice. Compared with wild types, iNOS KO animals exhibited lower plasma transaminase levels after 1 and 6 h of reperfusion following 1 h of ischemia. At the 3-h time point, enzyme levels were not different between the two groups. iNOS mRNA was not detectable in the ischemic hepatic lobes of wild-type mice until 3 h of reperfusion; however, perfusion studies identified a significant delay in reperfusion of the ischemic lobe in the iNOS KO mice at the 1-h time point with similar perfusion rates at 3 and 6 h compared with wild type. By way of comparison, mice deficient in the endothelial NOS (eNOS) were also assessed for the degree of hepatic damage 3 h post-reperfusion. Plasma transaminase levels were significantly increased in eNOS KO animals compared with wild-type controls. These data suggest that systemic as well as local sources of iNOS regulate reperfusion, and local iNOS contributes to hepatic injury, while eNOS is protective in warm hepatic ischemia-reperfusion.

A molecular basis of cryopreservation failure and its modulation to improve cell survival.

The requirement for more effective cryopreservation (CP) methodologies in support of the emerging fields of cell bioprocessing and cell therapy is now critical. Current CP strategies appropriately focus on minimizing the damaging actions of physicochemical stressors and membrane disruption associated with extra- and intracellular ice formation that occurs during the freeze-thaw process. CP protocols derived from this conceptual paradigm, however, yield suboptimal survival rates. We now provide the first report on the identification of delayed-onset cell death following CP and the significance of modulating molecular biological aspects of the cellular responses (apoptosis) to low temperature as an essential component to improve postthaw outcome. In this study we quantitatively examined the molecular basis of cell death associated with CP failure in a canine renal cell model. In addition, we report on the significant improvement in CP outcome through the modulation of these molecular mechanisms by the utilization of an organ preservation solution. HypoThermosol. Further, the utilization of HypoThermosol as the preservation medium and the modulation of molecular-based cell death have led to a paradigm shift in biologic preservation methodologies. The recognition of molecular mechanisms associated with CP-induced cell death offers the promise of improved CP of more complex and/or fragile biological systems such as stem cells, engineered tissues, and human organs.

Characterization and cloning of a Tenebrio molitor hemolymph protein with sequence similarity to insect odorant-binding proteins.

The yellow mealworm beetle, Tenebrio molitor, produces a number of moderately abundant low molecular weight hemolymph proteins ( approximately 12 kDa) which behave in a similar manner during purification and share antigenic epitopes. The cDNA sequence of the major component (THP12) was determined and the deduced protein sequence was found to be similar to those of insect odorant-binding proteins. Southern blot analysis suggests that at least some of the diversity in this family of proteins is encoded at the gene level. Both northern and western blot analysis indicate that THP12 is present in a variety of developmental stages and both sexes. THP12 was originally classified as an antifreeze protein, but the lack of antifreeze activity in the recombinant protein, as well as the clear separation of the antifreeze activity from THP12 following HPLC purification, has ruled out this function. The abundance of THP12, the similarity of THP12 to insect odorant-binding proteins, and the presence of hydrophobic cavities inside the protein (Rothemund et al., A new class of hexahelical insect proteins revealed as putative carriers of small hydrophobic ligands. Structure, 7 (1999) 1325-1332.) suggest that THP12 may function to carry non-water soluble compounds in the hemolymph. THP12 is also similar, particularly in structurally important regions, to other insect proteins from non-sensory tissues, suggesting the existence of a large family of carrier proteins which may perform diverse functions throughout the insect.

Cell viability improves following inhibition of cryopreservation-induced apoptosis.

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. HypoThermosol' (HTS), a hypothermic (4 degrees C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME + 5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (approximately 30%) and 5-d recovery (approximately 90%). Cells cryopreserved in CryoStor CS 5, a HTS derivative containing 5% DMSO, yielded approximately 75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME + 5% DMSO, HTS, or CryoStor CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use of HTS (a dextran-based, intracellular-type solution) without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.

Cryosurgical effects on growing vessels.

Cryosurgical treatment of unresectable hepatic malignancies has proven beneficial in adults. Concerns regarding its use in children include the effect on growth and the risk of injury to adjacent structures. To test the effect of cryoablation on adjacent vascular structures in a growing animal, liquid nitrogen cryoablation was performed on a juvenile murine model. Sprague Dawley rats underwent double freeze-thaw cryoablation of the abdominal aorta with interposed liver tissue. Serial sacrifices were performed over 120 days. Comparisons were made with sham-operated controls. Overall, animal growth paralleled that of sham controls through all time points. Gross examination of aortic diameter also showed similar growth in vessel size between the groups. Histologic analysis demonstrated injury after cryoablation with smooth muscle cell vacuolization, followed by cell death. Aortic media layer collapse resulted from cellular loss, however, elastin fiber composition was maintained. Aortic patency was preserved despite evidence of cellular injury and aortic wall remodeling. An associated thermal sink effect on the opposing wall was identified. After cryoablation adjacent to the abdominal aorta in adolescent rats, vascular patency is maintained and animal growth and structural function is preserved, despite cellular injury and wall compression. These observations suggest that cryoablation may be a useful treatment adjunct in young subjects.

Endoscopic cryotherapy: experimental results and first clinical use.

BACKGROUND: Cryotherapy or the application of extreme cold has many potential applications in gastroenterology including tissue destruction and hemostasis but until now its development has been prevented by the lack of a delivery device suitable for use through the endoscope. We report here our experience with prototype devices using both liquid nitrogen driven by a cryosurgical system and cryogenic refrigerants (nitrous oxide and carbon dioxide) at or near ambient temperature. METHODS: Cryotherapy was applied to the distal esophageal mucosa of dogs via a flexible catheter passed through an upper endoscope. In other dogs, cryotherapy was used for hemostasis in a bleeding ulcer model. The procedure was also used for palliation in a 58-year-old man with unresectable adenocarcinoma of the stomach with pyloric channel obstruction. RESULTS: Freezing of the superficial mucosa was nearly instantaneous. All dogs survived the procedure and appeared to thrive. Histologic evaluation revealed significant necrosis of the superficial epithelial layer accompanied by a fibrinocellular infiltrate on the surface. These markers of acute injury subside by the fourth to sixth day and are replaced by regenerating epithelium, a process that is virtually complete by day 10. In the hemostasis experiments, bleeding ceased immediately after cryospraying of the lesions but resumed on thawing in most cases. Application of cryotherapy in the patient resulted in reduction of the pyloric mass with no immediately apparent adverse effects. CONCLUSIONS: These data, although preliminary, demonstrate the feasibility of endoscopic cryotherapy using a simple hand-held device. This device has broad potential for use in gastroenterology including ablation of superficial epithelium, debulking of large tumors and hemostasis.

Mechanisms of tissue injury in cryosurgery.

As the modern era of cryosurgery began in the mid 1960s, the basic features of cryosurgical technique were established as rapid freezing, slow thawing, and repetition of the freeze-thaw cycle. Since then, new applications of cryosurgery have caused numerous investigations on the mechanism of injury in cryosurgery with the intent to better define appropriate or optimal temperature-time dosimetry of the freeze-thaw cycles. A diversity of opinion has become evident on some aspects of technique, but the basic tenets of cryosurgery remain unchanged. All the parts of the freeze-thaw cycle can cause tissue injury. The cooling rate should be as fast as possible, but it is not as critical as other factors. The coldest tissue temperature is the prime factor in cell death and this should be -50 degreesC in neoplastic tissue. The optimal duration of freezing is not known, but prolonged freezing increases tissue destruction. The thawing rate is a prime destructive factor and it should be as slow as possible. Repetition of the freeze-thaw cycle is well known to be an important factor in effective therapy. A prime need in cryosurgical research is related to the periphery of the cryosurgical lesion where some cells die and others live. Adjunctive therapy should influence the fate of cells in this region and increase the efficacy of cryosurgical techniques.

Minimally invasive cryosurgery--technological advances.

The technological advances which have caused renewed interest in cryosurgery are the development of intraoperative ultrasound to monitor the therapeutic process and the development of new cryosurgical equipment designed to use supercooled liquid nitrogen. The thin, highly efficient probes, available in several sizes, can be placed in diseased sites via endoscopy or percutaneously in minimally invasive procedures. The manner of use is to place the probe in the desired location in the diseased tissue with ultrasound guidance. If required by the size or location of the tumor, as many as five probes can be inserted and cooled to -195 degrees C simultaneously. The process of freezing is monitored by ultrasound which displays a hypoechoic (dark) image when the tissue if frozen. Rapid freezing, slow thawing, and repetition of the freeze/thaw cycle are standard features of technique. Clinical applications which have become common in the past 4 years include the treatment of prostatic cancer and liver tumors. The cases selected for cryosurgery are generally those for which no conventional treatment is possible. However, especially in prostatic cancer, the operative morbidity is so low and the results of therapy are sufficiently good in the short term to merit consideration of use in earlier stages of the disease. Diverse tumors in other sites, such as the brain, bronchus, bone, pancreas, kidney, and uterus, have also been treated in small numbers by cryosurgery. Judging from this experience, further expansion in the use of cryosurgical techniques seems certain.

A comparative assessment of cryosurgical devices: application to prostatic disease.

OBJECTIVES: To determine the comparative freezing ability of the Cryotech (CT) and AccuProbe (CMS) cryosurgical systems. METHODS: Four conditions designed to model clinical situations were produced: (1) Single-probe performance in water at 17 degrees C; (2) five-probe performance in water at 17 degrees C; (3) single-probe performance in gel at 22 degrees C; and (4) single-probe performance in bovine liver. Parameters evaluated included temperatures at various time points (rates to and final low temperature), configuration of a freeze zone, and shaft freezing characteristics. In addition, isotherms were measured at predetermined distances from the center of the freeze zone. RESULTS: Both systems provided freezing of various media under operational conditions. In water, the CMS 3-mm probe delivered more rapid freezing temperature rates than the 3-mm CT probe, with a 110 degrees C difference in probe surface temperature. In gel, the CMS probe increased freeze volume fourfold versus a twofold increase for the CT probe. In bovine liver, there was nearly equivalent performance with respect to geometry of the freeze ball. Extrapolation of the CT cooling curve indicated temperature equivalence at 30 minutes. A larger shaft diameter 4.9-mm CT probe produced results similar to the CMS probe in all the tested media. In addition, the freeze configuration of the CMS probe was spherical; the CT configuration was more cylindrical. CMS probe (equivalent diameter) tip temperatures were on average 100 degrees C lower. CONCLUSIONS: Our tests demonstrated differences between the CMS and CT probe. The major differences are in the configuration of the freeze zone and shaft freezing. In equivalent conditions, the CMS 3-mm probe delivered more rapid cooling rates, a more spherical freeze ball, and lower absolute temperatures than the CT 3-mm probe. The larger CT probe produces equivalent freezing temperatures to the CMS probe, albeit with a more spherical shape. However, these in vitro systems may not adequately reflect varied prostate morphology. Further research is under way to determine if these differences affect relative efficacy of cryotherapy of the prostate.