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.

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.

Cryosurgical ablation of the prostate.

Cryosurgery for the treatment of prostatic disease, a technique that originated in the mid-1960s and was almost abandoned in the mid-1970s, has re-emerged in the 1990s for the treatment of cancer of the prostate. This renewed interest is due to several factors, including the development of intraoperative ultrasound, the refinement of percutaneous access techniques, and improvements in cryosurgical apparatus. The modern technique features the transperineal percutaneous placement of several (generally five or six) metal probes, each 3 mm in diameter, in the prostate under ultrasound guidance. After insertion, the probes are cooled in a manner that produces complete freezing of the prostate and, if required, extraprostatic extensions of disease. The freezing process is monitored by ultrasound, which provides an image of the boundary of freezing as it advances through the prostate and thereby provides control of the extent of freezing. This review describes the historical background of prostatic cryosurgery and the current status of this new procedure, including the important issues of case selection, technique, and results. The recent nature of this experience precludes judgment of long-term merit, but the favorable short-term results of cryosurgical ablation of the prostate encourage further selective use of this technique in the treatment of prostate cancer. Definition of appropriate patient selection and optimal technique are needed to improve treatment by cryosurgery.

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.

History of cryosurgery.

The use of freezing temperatures for the therapeutic destruction of tissue began in England in 1845-51 when James Arnott described the use of iced salt solutions (about-20 degrees C) to freeze advanced cancers in accessible sites, producing reduction in tumor size and amelioration of pain. Improved freezing techniques were possible early in the 1990s when solidified carbon dioxide came into use and later when liquid nitrogen and nitrous oxide became available. Nevertheless, cryotherapy was a minor technique, used only for the accessible lesions of skin and mucosa. With the development of modern cryosurgical apparatus by Cooper in 1961, a resurgence of interest in cryosurgery was initiated and techniques for diverse clinical conditions, including visceral cancer, evolved, After the initial widespread clinical trials matured in the 1970s, some applications of the technique fell into disuse while others became standard treatment. Late in the 1980s, further improvements in apparatus and imaging techniques have permitted increased clinical use in neoplastic disease, including visceral cancer.

Recurrent basal cell carcinoma treated with cryosurgery.

BACKGROUND: Although there are reports of cure rates achieved by cryosurgery for primary basal cell carcinomas (BCCs), there are few data on the cryosurgical treatment of recurrent BCCs. OBJECTIVE: Our purpose was to discuss case selection, cryosurgical management, and results of therapy. METHODS: Cryosurgery was performed in 54 patients with 56 recurrent BCCs. The treatment consisted of aggressive freezing including an adequate margin of surrounding tissue. RESULTS: Wound healing was favorable and the cosmetic results were excellent. Two recurrences were found and were referred for Mohs micrographic surgery. CONCLUSION: We conclude that cryosurgical treatment of selected recurrent BCCs yields results that compare favorably with other methods of treatment.

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.

Evaluation and treatment of carotid stenosis in open-heart surgery patients.

Indications for identification and treatment of extracranial carotid artery disease in candidates for open-heart surgery (OHS) remain unsettled. We evaluated the efficacy of OPG-GEE screening and our nonrandomized use of carotid endarterectomy in 2312 OHS patients from 1975 to 1989. Data was analyzed using the chi 2 squared and Fisher's exact tests. OPG was performed in 1602/2312 (69%) of the patients. OPG was positive in 122/1602 patients (7.6%) and negative in 1480/1602 (92.4%) patients. Of the patients with positive OPG, 31 patients had insignificant carotid bifurcation disease, 32 patients had total internal carotid artery occlusion, and 59 patients had operable carotid bifurcation lesions. Selective use of angiography identified an additional 8 patients with operable carotid bifurcation lesions (total 67, 33 symptomatic and 34 asymptomatic). Overall stroke rate for 2312 patients was 40/2312 (1.7%) [30 day mortality rate 60/2312 (3.2%)]. Stroke incidence was significantly increased (P < 0.01) in patients with a positive OPG, 8/122 (6.60%) vs those with negative OPG (23/1480, 1.6%). However, it was most marked in patients with operable bifurcation lesions (6/67, 9.0%). Stroke was not increased in patients with carotid occlusion or positive OPG without significant carotid bifurcation disease (2/63, 3.20%). Carotid endarterectomy in patients with operable bifurcation lesions was associated with a decreased (P < 0.05) stroke rate after OHS (1/44, 2.30% vs 5/23, 21.7%). Our data suggests identification of significant carotid disease and carotid endarterectomy will decrease stroke after OHS.

Cryosurgery for lentigo maligna.

BACKGROUND: The need for treatment of lentigo maligna is related to the cosmetic benefits and to the potential for malignant change. The usual treatment is excision, but often lesional size or location preclude this. OBJECTIVE: Our purpose was to show that cryosurgery is an effective alternative treatment modality for lentigo maligna. METHODS: Thirty white patients were treated with cryosurgery. The lesions ranged from 1.3 to 4.5 cm in diameter. Treatment consisted of freezing with liquid nitrogen delivered by open spray. RESULTS: The lesion recurred in two patients, yielding a recurrence rate of 6.6% during the average follow-up period of 3 years. The two recurrent lesions were successfully re-treated with cryosurgery. Eleven patients observed for more than 5 years showed no recurrence. CONCLUSION: Cryosurgery provides excellent cosmetic and curative results. These results are favorably comparable to excisional surgery.

Progress in cryosurgery.

The Workshop on Cryosurgery at the 28th Annual Meeting of the Society for Cryobiology contained a diversity of papers which fairly represented the present state of cryosurgery in medical practice and which identified directions for future research. Emphasis was clearly on the development of visceral cryosurgery, which appears likely to become of increased clinical importance as a result of combination with ultrasound imaging techniques. This report reviews in brief the important new developments in cryosurgery and focuses on those presentations which were of special interest from the viewpoint of research in cryosurgery.

Cryosurgery in the treatment of cancer.

The range of application of cryosurgical techniques to the treatment of cancer is widely diversified and slowly increasing in scope. From these, one may reach the general conclusion that cryosurgical techniques are a standard method of treatment, competitive with other methods of therapy, in cancer located in some sites. For cancers located in other sites, cryosurgery is only useful as an end resort in selected patients. In some areas, especially in the viscera, cryosurgical techniques are only in developmental stages. Cryosurgery is most useful in easily accessible areas of the body. The results of the treatment of most carcinomas of the skin with cryosurgical techniques are as good as any other method of therapy. In carcinoma involving skin, cryosurgery has a special advantage in those situations when malignant tissue overlies bone. Cryosurgery is also useful in the management of dysplastic disease or carcinoma in situ, principally in the oral cavity and the uterine cervix. These applications are sufficiently valuable to be included in the textbooks concerned with those areas. Invasive cancer in other accessible sites, such as the oral cavity or the rectum, can be cured by cryosurgery, but the reports in the medical literature have not led to general use of the technique or descriptions of the technique in textbooks, except for occasional brief mention. Nevertheless, patients who are at high risk for surgical treatment because of coagulopathy or severe cardiopulmonary disease are appropriate candidates for the use of cryosurgical techniques. In the oral cavity, the possibility of preserving the bony structure is an attractive feature that maintains interest in cryosurgery. Unfortunately, there are no control studies to assist in the judgment of merit and in many cited reports, it is not easy to determine the survival rate or compare results with conventional therapy. In these sites, freezing techniques are more often used to achieve palliation of distressing symptoms by tumor bulk reduction, especially when little else can be done, and under these conditions, chemotherapy and radiotherapy are also commonly used. In less accessible sites, which generally require endoscopic or surgical exposure, cryosurgery is not often used. The treatment of carcinoma of the prostate gland by cryosurgery remains viable because of continued interest in the potentiation of immunologic defenses against carcinoma. This possible benefit is most evident in experimental tumors, but clinical evidence of benefit is not as clear.(ABSTRACT TRUNCATED AT 400 WORDS)