November 1997
Vladimir Mouraviev

1. BACKGROUND AND RATIONALE
Cryosurgical ablation of prostate (CSAP) has gained popularity as alternative treatment for localized prostate cancer with excellent local tumor control, being performed at more than 100 centers in USA. But there are only isolated reports with CSAP using for palliation of stage D prostate cancer. Remarkable recent advances in our understanding of the immune system and the advent of new immunostimulants, especially recombinant cytokines, now permit direct activation and regulation (direction) of immune responses as well as ways of obtaining more long-lasting immunity. Many investigators on the basis of the wide range of tissue effects that can be attained by cryonecrosis, expressed great expectations for the cryoimmunologic (both a specific humoral and a cellular immune response) augmentation of tumor destruction. Moreover, in advanced-stage disease- where tumor heterogeneity confounds treatment by conventional therapy and patients succumb to the direct effects of metastasis and complications associated with treatment- cryoimmune response may additionally be tumoricidal to metastases.

Cryoimmunisation may result in a response that may be tumoricidal as well as tumor enhancing -bidirectional. Primary tumor-antigen liberation lies not merely in augmentating an antitumor response but in directing (modulating) the response toward that will be tumoricidal. However further antigenic stimulation actually attenuates the immune response by an intrictic biochemical feedback mechanism. This one decreases the T cell activity, thereby self-regulating the production of interleukin 1 (IL-1) and other cytokines. This idea further explains a prolongation of immune response. This a "booster" response was successfully achieved by multiple freezing of the prostate at intervals of 30 days or more.

Therefore, from the bidirectional nature of the cryoimmune response exhibited, evaluation and elucidation of the role of local and systemic cytokinotherapy before and after cryosurgery and their possible regulation should prove most enlightening.

A novel "biochemotherapy" approach includes a combination of standard chemotherapy regimens and biological agents appear to work well together when given simultaneously to patients with several different types of cancer. As it is known, IL-1 is as main cytokine vital to the proliferation and maturation of whole immune cascade, with a wide range of biological activities related to host response to infectious, immunological, antinflamatori stimuli. Previously it has been reported that recombinant human interleukin 1 (rHu IL-1) shows antitumor activity against syngenic tumors in mice.

Antitumor drugs are considered to be cytotoxic and probably inhibit tumor growth through their direct activation against the tumor cells. On the other hand, IL-1 is a cytokine with multiple antitumor mechanisms, i.e. the enhancement of cytotoxic activity of monocytes, of natural killer cells, and of cytotoxic T-lymphocytes, the production of lymphokines such as interleukin 2 and gamma-interferon, which have been demonstrated to have antitumor activity, and the direct cytotoxic action against some tumor cells. It is uncertain which mechanisms are important for the antitumor action of IL-1, although there is some evidence showing that the action of IL-1 appears through the induction of cytotoxic T-lymphocytes and its adjuvant activity potentiating antitumor immunity. But Nakamura S. et al. (1991) demonstrated that rHu Il-1 alone are not sufficient for curing of tumors even at high doses but optimal combinations of the frequently bring about complete cure of tumors.
Moreover, in an inflammatory response, such as that initiated by cryonecrosis, the target tissue will be subjected to the action of not just only rHu IL-1 but several cytokines as a result of chain-reaction, that may act synergistically to promote one arm of the immune response while inhibiting the other. Cytokines are pleiotropic and may function in autocrine, paracrine and endocrine manner. Developing data suggest that locally produced cytokines may arouse immunogenicity and facilitate cell-to-cell interactions and antigen presentation by inducing or enhancing expression of select membrane proteins.

One important clinical consideration is the fact that nearly all prostate cancers are at least initially sensitive to a variety of anti-neoplastic drugs. Treatment regimens incorporating combinations of these drugs currently are the mainstay of therapy, and can often achieve substantial reductions in tumor burden and prolongation of survival.

Most notable are the estramustine phosphate (EMP or "Estracyt" by Pharmacia&Upjohn) combinations, which combine microtubule inhibitors with different mechanisms to achieve superadditive or synergistic effects. EMPis estradiol attached to nitrogen mustard via a carbamate ester linkage. This linkage is a very stable, and its antineoplastic activity is predominantly attributable to its two main metabolites, estramustine and estromustine. Their cytotoxic effects are exerted through binding to microtubule-associated proteins, and this produces microtubule disruption and prevents microtubule assembly during metaphase. Recently phase II trials yielded an average overall response rate of EMP-monotherapy up to 37% in over 600 men with hormon-unresponsive prostate cancer (Benson R. et al., 1990).
Unfortunately, for many of the tumor cases, recurrence with drug-resistant tumors often follows chemo-and hormonal therapy-induced remissions. It seems likely that our new approach that attempt to generate T-cell mediated anti-tumor immunity will need to occur in the context of existing remission including systemic chemo- and hormonal therapy. Many of these agents are themself highly immunosuppressive.
With recognition that EMP was a microtubule inhibitor, it was theorized that its combination with matural IL-1with different sites of action might result in an additive or synergistic antitumor effect. Our pilot-study results have demonstrated that in the setting of cryoimmunnotherapy by rHu IL-1b with basic adjuvant therapy by estracyt resulting in a combined efficacy that exceeds either modality. Additionally, we have noted the protective action of rHu IL-1b to the whole cascade of humoral and cellular immunity to possible side-effects of basic palliative therapy- significantly myelosuppression and other adverse effects of estracyt, allowing to increase its doses up to maximum efficacy.

During last year we have been treating six prostate cancer patients with stage D2 under this protocol of cryoimmunotherapy. In 3 cases we have investigated the clinical remission of metastatic lesions, biochemical significant failure of PSA (from 83 to 125 ng/ml initially to 15 - 45 consequently), improvement of quality of life, indirect features of increasing of tumor-specific immunity (increasing of quantitative parameters of antigen activation- CD25, HLA-2, total lymphocytes, cytotoxic lymphocytes and natural killers; enhancing of functional activity of lymphocytes, stimulation of IL-2, IL-8 etc.). It was a very important event to support this specific immune response not by means of consequential cryotherapy, but the repeated immunotherapy by rHuIL-1-b. In 1 case we have reached the partial immune response with regression of some metastases from inner organs (not osteolytic origin).

In 1 case with terminal stage of D2 patient was died due to the polyorgan failure as the result of the toxic complications of the intensive scheme of chemotherapy.

2. OBJECTIVES
The purpose of this study is to use the combination of CSAP with cytokinotherapy by IL-1b as more guided and accordingly more effective approach to treat patients with metastatic (stage D2) prostate cancer. There will be attempt to eradicate the prostate cancer or treat any metastases or other symptoms arising from this disease.
These objectives of the trial are to determinate the safety and efficacy of cryoimmunotherapy in patients as measured by:
2.1. Time to treatment failure
2.2. Time to progression
2.3. Time to survival
2.4. Quality of life
3. STUDY DESIGN
The study is designed to treat 24 patients with D2 prostate cancer.. In other randomized group including 16 similar patients we plan to use the conventional cytoreductive CSAP without immunotherapy as palliative treatment, solely as attempt to eliminate or cease the local symptoms (persistent bleeding from prostatic urethra or locally referrable pain notcontrolled by analgetics) and make the patient more comfortable.
4. SUBJECT POPULATION
Inclusion criteria
Patients will be included in this study if they meet the following criteria:
4.1.Biopsy proven prostate cancer
4.2.Positive bone scan, chest x-ray, abdominal/pelvic CT or MRI
4.3.Local voiding problems, bleeding, rectal obstructive problems or perineal/pelvic pain
4.4. Ability to grant informed consent
Exclusion criteria
Patients will be excluded from this study if they have any inherent non- correctable bleeding problems.

5. METHODS
The treatment design may be started using EMP 600mg/m2 intravenously daily weeks 1-4 and 6-10 with consequent changing to enteral treatment (orally) of similar dose day 1-42, cycles repeated every 56 days. The regime of proposed immunotherapy may be as follow: rHu IL-1b (betaleukin), which has already got an approval from Russian National Pharmacological Comittie and now is produced by St.Petersburg State Research Institute of Highly Pure Biopreparations, in dose 5ng/kg subcutaneously daily during 3-5 days with 1-2 days interval of resting before cryoablation.
Then CSAP will be performed utilising the Spembly Cryosurgical Systems (LCS 3000, manufactured by Candela ,Inc.) with a standard five probe of single freeze technique.
This procedure is carried out as a minimal invasive cytoreductive surgery without cystostomy, using the 'home-made' urethral warmer similar to that described by Cohen and Miller, consisting of a double lumen urethral catheter attached to a pump that recirculates warmed saline (approximately 43C). Then we are leaving a Foley catheter in the urethra for 3 weeks after cryosurgery.

Based on the changes of local and systemic immunity rates, repeated systemic IL- 1b courses may be needed of every 1--3 months after cryoablation. Additionally for enhancement of tumor destruction, we have proposed to inject IL-1b (up to 10 ng) into tumor just at the end of CSAP, i.e. after complete thawing of prostate tissue. An evaluation of this new therapeutic approach against advanced prostate carcinoma will be conducted with routine clinical evaluation (physical examination, sexual story, laboratory findings, urodynamic pressure-flow studies) as well as stated markers of tumor progression- bone scan, chest X-ray, lymphscintigraphy, prostate-specific antigen (PSA), acid and alkaline phosphatase, CT or MRI, prostate biopsy. Also the newer diagnostic immunocytochemical and molecular-biological methods especially for chemo-and hormonal relapsed prostate cancer- multidrug resistance (P- glycoprotein expression by Western blotting cell lysates using specific antibodies) with determination the sensivity to Estracyt, level of androgen receptors ( exon 2-8 amplification of androgen receptors gene by polymerase-chain reaction) and apoptosis index (tumor supressor gene p53 from paraffin-embedded prostate specimens using monoclonal antibody do7- Dako UK Ltd.).

5. ASSESSMENT OF TUMOR RESPONSE
5.1. Specific tests for assessing response will be the digital rectal examination, serum PSA, TRUS, repeat bone scan, acid phosphatase and prostate biopsy.
5.2. Complete objective response: remission of metastatic lesions and no new lesions, no progression of residual cancer (controlled local tumor) as demonstrated by reduction of serum PSA more than 50% , constant local tumor as assessed by digital rectal examination. Persistent tumor may be observed on biopsy, but often with the evidance of "downstaging" and "downgrading".
5.3. Partial objective response: reduction of serum PSA by at least 75% without trend to local control but with partial regression of inner organs metastases and and stable systemic disease.
5.4. Treatment failure: less than 50% reduction in serum PSA, uncontrolled local tumor growh and instable systemic disease.
5.5. Disease progression: rise in serum PSA by more than 25%, systemic progression of tumor with new distant metastatic lesions. These patients are taken off study and may be offered alternative palliative therapy.

6. ANALYSIS OF RESULTS
PSA values will be analyzed by using a repeated measures analysis of variance with Greenhouse-Geisser corrections and post-hoc comparisons. The patients' survival data will be expressed as a percentage of patients surviving from one month to two years.
In addition, survival curves will be generated using the Kaplan-Meier method.
Patients will also be divided into groups based on the above assessment of tumor categories. Analyses between groups will use the generalized Wilcoxon test of Gehan and log-rank test. Survival and success rates from this study will be compared with historical populations consisting of patients who have undergone more traditional treatments (maximal hormonal blocade, different design of chemotherapy etc.).

7. TESTING SCHEDULE