Immunologic response to primary cryoablation of high-risk prostate cancer

Cryobiology 57 (2008) 66–71

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Immunologic response to primary cryoablation of high-risk prostate cancer q Tongguo Si, Zhi Guo *, Xishan Hao * Tianjin Medical University Cancer Hospital and Institute, Interventional Therapy Department, Huanhuxi Road, Hexi District, Tianjin, China

a r t i c l e

i n f o

Article history: Received 25 March 2008 Accepted 5 June 2008 Available online 13 June 2008 Keywords: Prostate Tumor Cryoablation Immune

a b s t r a c t Objective: To assess whether a specific cytotoxic T-cell response can be induced in patients with prostate cancer after cryoablation. Material and Methods: Twenty Patients with high-risk prostate cancer underwent cryoablation. Blood was sampled prior to, 4 and 8 weeks after treatment. Serum cytokine levels were analyzed by ELISA, and the Th1/Th2 ratio was estimated from the IFN-c/IL-4 ratio. Peripheral blood mononuclear cells (PBMC) were stimulated with autologous prostate cancer-derived protein lysates, and frequency of tumor-specific Tcells was tested ex vivo in an IFN-c ELISPOT assay. To assess cytolytic activity, T-cells were co-incubated with human prostate cancer cells, LNCaP, or with renal cancer cells, GRC-1, and release of cytosolic adenylate kinase was measured by a luciferase assay. Result: 4 weeks after cryoablation significantly higher levels of TNF-a and IFN-c were observed compared to before treatment, and to 8 weeks after treatment. No changes in IL-4 or IL-10 were observed. The Th1/ Th2 ratio (10.47 ± 0.80), 4 weeks after treatment, was increased compared to before treatment (3.98 ± 0.45), but decreased 8 weeks later (7.65 ± 0.64). Tumor-specific T-cell responses were evident after cryosurgery in PBMC. Cytolytic activity against LNCaP was increased 4 weeks after treatment compared to before treatment (594.49 ± 154.84 versus 4.20 ± 0.68, P < 0.01), but was decreased 8 weeks later (79.70 ± 18.73). No response was found in cytolytic activity against GRC-1. Conclusion: Cryoablation of prostate cancer can improve tumor-specific cytotoxic T-cell stimulation with a dramatically increased tumor specific cytolytic activity. However, the response is not sufficiently maintained to prevent cancer relapse. Ó 2008 Elsevier Inc. All rights reserved.

Introduction Cryoablation is an alternative treatment strategy for prostate cancer (CaP) that destroys tumor and prostate gland tissue by freezing. It was introduced in the 1960s by Gonder et al. [14], but it was later abandoned, due to the inability to control the freezing process, which lead to high complication rates of rectal injury, and urethral obstruction [28]. Only since the 1990s, with the introduction by Gary Onik of a real-time ultrasound visualization, in combination with temperature sensors [23], has cryoablation become a safer, and acceptable option for treatment of prostate cancer patients. The development of cryotherapy for localized prostate cancer provides a potentially curative option for patients with primary or recurrent disease, with less morbidity than with radical surgery [36,3]. Cryoablation leaves tumor proteins and tumor-associated antigens intact. The presence of residual tumor antigens in an inflammatory microenvironment can stimulate anti-tumor immune q

This article was not supported by any funding. * Corresponding authors. E-mail address: [email protected] (Z. Guo).

0011-2240/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.cryobiol.2008.06.003

responses [18]. The majority of the evidence for cryoimmunologic responses comes from reports of distant disease resolving after ablation of a primary tumor [29]. Several isolated studies from the 1980s attempted to characterize the immunologic changes in patients undergoing cryoablation of oral cavity cancers [9,8] or breast cancer [30]. Although they demonstrated increases in nonspecific markers of immune response, they were limited by the available immunologic assays. More recently, Ravindranath et al. [25] demonstrated that cryoablation of colorectal metastases to the liver led to the augmentation of serum antibodies against cancer associated gangliosides. The authors suggest that cryosurgery is similar to autologous vaccines because it releases autologous tumor-antigens, thereby augmenting the immune response to the tumor itself. But Osada et al. [24] report that after cryoablation for hepatic primary or metastatic tumor, a cryoimmune response was only found in limited number of patients. In the present study, our aim was to verify whether cryoablation could activate an anti-tumor specific T-cell response in prostate cancer patients. Therefore we studied the alterations in serum cytokine levels, T-cell responses to prostate cancer derived antigens, and the cytolytic activity of peripheral blood mononuclear cells (PBMCs) against the human prostate cancer line, LNCaP.

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and interferon-gamma (IFN-c) were measured in peripheral blood by ELISA kits (R&D systems, Minneapolis, MN). In brief, sera were added to cytokine-specific monoclonal antibodies coated onto microplates. After 2 h at room temperature, the microplates were washed four times with wash buffer, and then incubated with cytokine conjugates for 2 h. After washing, substrate solution was added and incubated for 30 min in the dark. The optical density of each well at 450 nm was determined with a microplate reader, and values were calculated relative to those of a standard control. The Th1/Th2 ratio was estimated from the IFN-c/IL-4 ratio.

Patients and method Patients Twenty men, with a median age of 67 years, were identified who underwent cryoablation of the prostate for clinically localized, T1–T3 CaP with high-risk features. Inclusion criteria were patients with pre-hormone therapy high-risk features of CaP, which were defined as either a PSA level P10 ng/mL, or a pathology report indicating a Gleason sum score P8, or both of these features. Patients who had undergone prior radical prostatectomy or radiation treatment for CaP were excluded from this analysis. Patients were required to have no evidence of metastatic disease by bone scan, computer-assisted tomography, or magnetic resonance imaging. Patient characteristics are summarized in Table 1.

Peripheral blood mononuclear cells PBMCs were isolated from fresh heparinized blood by Ficoll– Hypaque density-gradient centrifugation, and resuspended at 1  106/mL in RPMI 1640 supplemented with 25 mmol/L HEPES, 2 mmol/L L-glutamine, 50 mg/mL gentamicin, and 10% FCS (complete medium). CD8 cell depletion (>96%) was done with the use of antibody-coated MACS microbeads (Miltenyi Biotec, Auburn, CA) followed by magnetic separation according to the manufacturer’s instructions.

Treatment All patients received a rectal enema the night before the procedure. Under spinal anesthesia, patients were placed in a lithotomy position. Cystoscopy was undertaken, and a supra-pubic bladder catheter was placed under direct vision. All procedures were performed according to the modified Onik technique using an ENDOCare unit with argon, and helium gas for the freezing and thawing, respectively, for a total of two freeze–thaw cycles. Under transrectal ultrasound guidance, five to seven cryoprobes were introduced into the prostate, and four thermoprobes were located bilaterally in the neurovascular bundles, one in the Denonvilliers’ fascia, and the other at the sphincter. The freezing process was monitored in real time by transrectal ultrasound, and by thermo probes to enable direct visualization of the ice ball and to avoid lesions to adjacent tissues. During the procedure, the urethra was protected with a warming device at 37 °C degrees that was kept in place until the patients left the operating room. All patients were discharged within 24 h, with the supra-pubic catheter removed after a week.

Preparation of tumor lysate A sample of prostate cancer and non-tumor tissue was obtained from the patients using ultrasound guided transcutaneous biopsy. The biopsy cores were divided into minute pieces. A small volume of normal saline was added, and the mixture was passed several times through a 19G needle, attached to a 5 ml syringe, until the passage of the mixture occurred without difficulty. The process was repeated with 21G, 23G, and if possible 25G needles. The entire mixture was placed in liquid nitrogen until frozen, and then thawed in a water bath at 42 °C. The freezing and thawing was repeated for a total of five times. The sample was passed through another 23G or 25G needle to disperse any clumps. The tube was sealed and irradiated at 10,000 rads, then stored at 140 °C. Enzyme-linked immunospot assay

Measurement of serum factors Polyvinylidene plates (96 wells; Millipore, Bedford, MA) were pre-coated with 5 lg/mL anti-IFN-c monoclonal antibody (mAb), 1-DIK (Mabtech, Nacka, Sweden) overnight at 4 °C. Plates were then washed four times with PBS and blocked with RPMI/10% FCS for 2 h

Systemic blood samples were centrifuged at 1000g for 15 min at 4 °C. Serum was stored at 80 °C until assays were performed. Interleukin (IL) 4 and 10, tumor necrosis factor-alpha (TNF-a), Table 1 Patient characteristics and treatment outcome Patient

Age

PSA (ng/ml) before

Gleason score

Clinical stage

PSA (ng/ml) 3 months later

PSA (ng/ml) 6 months later

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

72 64 68 78 63 60 67 57 68 76 70 68 72 75 58 60 62 68 72 64

25.1 30.0 14.8 22.4 9.6 26.2 21.7 26.4 17.8 12.6 10.4 8.9 19.8 12.6 25.4 23.8 25.2 20.5 14.4 24.6

6 8 7 6 8 9 6 7 8 8 7 9 6 8 8 7 8 7 6 8

T2a T3a T2a T2a T2a T2b T2a T2a T2a T2a T2b T2b T2a T2b T3a T2a T2a T2a T2a T2b

0 0.2 0 0 0 0.3 0.2 0 0 0 0.1 0 0 0 0 0.2 0.2 0 0 0.2

0.1 0.2 0 0 0 0.3 0 0.1 0 0 0.2 0.1 0.4 0 5.6* 0.1 0 0 0.3 0

*

Prostate cancer recurrent proved by biopsy.

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at 37 °C. Cryopreserved PBMCs were thawed and plated at 200,000 per well in the presence or absence of 25 lg/mL protein lysates, and 1 lg/mL phytohemagglutinin as positive control, in a total volume of 100 lL. PBMCs were seeded in duplicate for antigenic stimulation, and in triplicate for controls. The plates were incubated overnight at 37 °C, 5% CO2, and washed seven times with PBS/ 0.05% Tween before addition of the second, biotinylated anti-IFNcmAb, 7-B6-1 biotin (Mabtech) at 1 lg/mL, and incubated for 3 h. After a further washing step, anti-biotin alkaline phosphatase-conjugated antibody (Mabtech) was added for 90 min. Following another washing step, individual cytokine producing cells were detected as dark spots after a 5–10 min reaction with 5-bromo-4chloro-3-indolyl phosphate and nitro blue tetrazolium using an alkaline phosphatase-conjugated substrate (Bio-Rad Laboratories). Spots were enumerated as IFN-c spot forming units using an enzyme-linked immunospot (ELISPOT) plate reader (AID). The number of specific IFN-c secreting T-cells was calculated by subtracting the unstimulated control value from the stimulated sample. Wells were considered as positive if they were at least double background levels, with at least 10 spots per well. Cytotoxicity assay Cytolytic activity of T-cells was measured by an adenylate kinase (AK) release assay. Ten thousand target cells were incubated with 1000 effector cells in a final volume of 200 lL fetal calf serum growth medium (FCS-GM) in round-bottom 96-well microtiter plates. After incubation for 4 h at 37 °C 100 lL of supernatant were harvested and stored at 20 °C for further analysis. The human prostate cancer cell line, LNCaP, and the renal cancer cell line, GRC-1, which had previously been HLA matched (ABO-system), served as target cells. Maximum AK release was obtained by incubating target and effector cells with 1% v/v Triton X-100, and baseline AK release with medium alone. Baseline release from T-cells and tumor cells was <10% of maximal release in all experiments and subtracted from each value. The activity of AK was determined by detection of auto-luminescence using a luciferase assay (ToxiLight Kit, Cambrex Corporation, NJ, USA). Twenty microliters of supernatant were incubated with AK-detection reagent (Cambrex Corporation, NJ, USA) for 5 min at room temperature. The bioluminescence was measured by a luminometer (BD Monolight 3096 Microplate Luminometer, BD Biosciences, Heidelberg, Germany) and expressed as relative luminescence units (RLU).

of rectal injury, nor any need for blood transfusion. Of the 14 previously potent patients, erectile dysfunction was found in 8 (57%). Urinary incontinence, defined as the need for one or more pads a day, was present in two patients. Serum levels of TNF-a and of IFN-c were increased 4 weeks after treatment, but were significantly decreased 8 weeks after treatment. IL-4 and IL-10 showed no remarkable changes after treatment (Table 2). The Th1/Th2 ratio (10.47 ± 0.80) was increased 4 weeks after treatment compared to that before treatment (3.98 ± 0.45). However, 8 weeks after treatment the Th1/Th2 ratio had decreased (7.65 ± 0.64) (Table 2). Significant differences between the mean values of Th1/Th2 before treatment, and 4 weeks after treatment or between 4 weeks and 8 weeks after treatment were observed (P < 0.01). Frequency of tumor-specific T-cells was tested ex vivo in an IFNc ELISPOT assay. Before treatment, three patients showed a specific T-cell stimulation response to tumor protein lysate and one the patients showed a T-cell response to non-tumor prostate tissue. 4 weeks after treatment, a T-cell response to autologous tumor tissue was detected in 15 patients. T-cell responses were also detected against autologous non-tumor prostate tissue in four patients. 8 weeks after treatment, specific T-cell responses were detected against tumor tissue in eight patients and against non-tumor tissue in two patients (Table 3). Although no significant difference was observed between the mean values of spots generated with non-tumor tissues and tumor tissues before treatment, after treatment this difference was highly significant (P < 0.01). Significant differences between the mean values of spots generated with tumor tissues before treatment and 4 weeks after treatment or between 4 weeks and 8 weeks after treatment were also observed (P < 0.01). Comparison of the mean values of spots generated with non-tumor tissue also showed statistically significant difference between before and 4 weeks after treatment and between 4 weeks and 8 weeks after treatment (P < 0.05). This may be due to a common antigen T-cell response between non-tumor and tumor prostate tissue. 4 weeks after cryoablation, PBMCs presented a highly elevated cytotoxic activity against LNCaP cells, with a mean release of AK (as represented by RLU) of 594.49 ± 154.84, while the baseline value before treatment was 4.20 ± 0.68. The response, was however, significantly decreased 8 weeks after treatment with RLU values of 79.70 ± 18.73. There was no significant change in the cytotoxic activity against GRC-1 cells. The spontaneous release of AK was always <2% of the maximum release (Table 4). Discussion

Statistical analyses Data were summarized as the mean ± SD unless stated otherwise. The statistical software package SPSS 13.0 for Windows (SPSS Incorporated, Chicago, IL) was used for all statistical analyses. Statistical analysis of differences among the mean values of the spots was done using Kruskal–Wallis H test, and the statistical analysis for the other results was done using an ANOVA with a post-hoc test. P-values less than 0.05 were considered significant. Results PSA scores were all significantly decreased 4 weeks after treatment, and there was no PSA failure case within three months. After 6 months an elevated PSA score was found in patient 15, and cancer recurrence was proved by biopsy (Table 1), hormone therapy was recommended. Complications were mostly minor, with 20% (4/20) of mild hematuria and 15% (3/20) of perineal/scrotum hematoma with no need for further treatment. There was no case

Organ-confined CaP has traditionally been treated with radical surgery, external beam radiation therapy, and brachytherapy. However, these treatments can result in a substantial risk of significant morbidity. Since the first published application of cryosurgery to the prostate by Gonder et al. [14], cryoablation has undergone significant advancements to make it available as a legitTable 2 Alterations in serum factor levels induced by cryoablation therapy (pg/ml) Cytokine

Before treatment

4W after treatment

8W after treatment

IL-4 IL-10 TNF-a INF-c Th1/Th2 (INF-c/IL-4)

9.01 ± 0.59 32.68 ± 1.65 42.82 ± 2.32 35.72 ± 2.94 3.98 ± 0.45

8.45 ± 0.62 31.18 ± 1.72 75.17 ± 2.88* 88.12 ± 3.38* 10.47 ± 0.80*

8.57 ± 0.54 31.55 ± 1.58 61.90 ± 2.09 65.31 ± 3.14 7.65 ± 0.64

* Significant difference was observed between 4W after treatment and before treatment or 8W after treatment (P < 0.01).

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T. Si et al. / Cryobiology 57 (2008) 66–71 Table 3 T-cell response to prostate cancer and non-tumor prostate tissue by ELISPOT assay before and after cryoablation Patient

Before treatment

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Patient 10 Patient 11 Patient 12 Patient 13 Patient 14 Patient 15 Patient 16 Patient 17 Patient 18 Patient 19 Patient 20 Median

4W after treatment

8W after treatment

Non-tumor

tumor

Non-tumor

Tumor

Non-tumor

Tumor

5 15 10 35 10 20 10 10 20 20 15 15 25 10 15 20 10 25 10 5 15

5 10 5 12 40 20 45 20 15 15 30 10 20 5 15 20 10 20 20 10 15

15 25 25 60 20 15 20 10 10 40 25 20 35 25 20 15 20 40 20 15 20*

25 205 105 30 385 25 90 60 75 210 500 80 305 110 90 30 70 420 20 80 85*

10 15 10 40 25 15 20 10 10 30 25 20 30 15 15 10 15 35 20 15 15

15 40 20 30 85 20 45 30 25 40 60 20 45 30 55 30 20 60 30 30 30

Note: Average values of duplicates spots per 106 PBMCs after subtracting responses to medium alone. In bold are average spots values twice above background (medium). * Significant difference was observed between 4W after treatment and before treatment or 8W after treatment (P < 0.01).

Table 4 Relative luminescence units (RLU) of cytosolic adenylate kinase released from the prostate cancer cell line LNCaP and renal cancer cell line GRC-1 Patient

Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Patient Mean SD

Before treatment

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

4W after treatment

8W after treatment

GRC-1

LNCaP

GRC-1

LNCaP

GRC-1

LNCaP

2.30 3.14 6.31 3.60 5.20 4.12 3.24 5.46 5.04 2.31 2.46 3.54 3.04 5.84 6.02 4.31 5.24 6.34 6.54 0.35 4.22 1.69

3.24 4.13 5.10 4.61 3.62 5.23 3.24 2.64 3.06 5.62 0.46 2.14 4.68 5.74 5.64 6.24 6.47 5.10 4.32 2.71 4.20 1.54

3.01 4.32 2.14 3.32 4.35 6.31 6.56 7.34 6.36 5.32 0.31 2.14 1.30 4.20 4.34 2.63 6.45 5.63 7.13 6.53 4.48 2.07

389.62 421.55 754.12 890.56 625.01 364.23 590.46 685.25 768.23 463.52 652.31 524.63 712.30 801.24 398.74 487.64 572.31 468.32 556.34 763.45 594.49* 154.84

3.62 4.32 5.10 2.01 3.42 2.50 3.12 6.54 5.34 6.42 4.38 6.50 7.02 1.31 4.62 1.03 3.54 6.35 4.31 5.34 4.34 1.78

79.65 95.34 86.52 79.41 82.04 96.32 123.42 63.42 76.48 64.56 105.46 89.35 74.31 38.54 68.79 75.94 68.34 74.56 53.64 97.82 79.70 18.87

Cells were co-incubated with PBMC isolated from each patient taken before, 4 and 8 week after cryoablation. * P < 0.01 vs bioluminescence before treatment or 8W after treatment.

imate treatment strategy for CaP. Compared to surgical excision, the main advantages of cryoablation are that it is a less invasive procedure, resulting in reduced mortality and morbidity, and the ablative procedures can be performed on outpatients, which decreases the treatment cost. To date, cryoablation has been favored as a primary or salvage treatment for prostate cancers. For primary treatment, longer-term follow-up had been provided by Bahn et al. [3], who reported on 590 patients. Surprisingly, there was no difference in the 7-year actuarial biochemical disease-free survival (bDFS) (PSA < 0.5 ng/ mL) rate among low-, medium-, and high-risk patients: 61%, 68%, and 61%, respectively. The rate of positive biopsy was 13% (the majority detected within six months post-operatively). However,

one prospective report [17] including 100 patients who had salvage cryoablation therapy for recurrent prostate cancer after radiotherapy, showed the 5-year bDFS was 73%, 45%, and 11% for the low-, intermediate- and high-risk groups, respectively. In a multicenter study of 975 patients that compared cryosurgery with published results of radiotherapy, using similar risk-stratification and failure criteria, cryosurgery yielded results similar to both external-beam radiation therapy and brachytherapy. The 5-year bDFS rate for cryosurgery was comparable to brachytherapy, and conformal radiation therapy [20]. The mechanisms of cryoablation are multi-factorial, yet they culminate in necrotic cell death secondary to (a) direct cellular damage by ice crystals, and (b) vascular and endothelial injury

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with eventual ischemia [16]. Cryoablation destroys tumor tissue, generating a local necrosis, followed by a marked inflammatory response with a dense T-cell infiltration [12], suggesting the activation of the adaptive T-cell response. Infectious agents can readily activate the innate immune response through interaction of conserved microbial products with receptors on dendritic cells [13]. These pathogen-associated molecular patterns are key elements for a coordinated, and efficient activation of the immune response. Although tumors can express neoantigens or can over-express selfantigens, they lack non-self components of microbial origin, so that tumor cells, like their normal tissue counterparts, are prone to induce tolerance by default. Alternatively, immune responses can be also activated by signals of other origins. Necrotic cell death stimulates dendritic cell maturation [11], and mature dendritic cells direct T-cell differentiation into effector or memory cells, inducing natural killer cell activation and B-cell differentiation into antibody-forming cells [2]. It is still a matter of debate whether the necrotic cancer cells generated by cryoablation can provide sufficient signals to trigger the induction of a tumor-specific T-cell response. The majority of the evidence for cryoimmunologic responses comes from reports of distant disease resolution after ablation of a primary tumor. Suzuki et al. [30] described eight patients with stage IV breast cancer who had advanced primary tumors treated by cryosurgery. In addition to palliation of the primary tumor, two of the eight patients had resolution of distant disease, including regional adenopathy, contralateral metastases and pleural effusion. Clinical observations of remission of metastatic lesions distant from the regions treated by cryosurgery have also been described in other tumor types [10]. But some studies showed no change in the immune status [16], or even suppressed immunity, increased tumor growth and metastasis after cryoablation of primary tumors [34,33]. In several animal models experiment including melanoma [6], colon cancer [32], and breast cancer [26], the tumor debris generated by tumor cryoablation can be captured by dendritic cells, and transported to draining lymph nodes, resulting in a weak, but tumor-specific immune response. For prostate cancer study, Lubaroff et al. [21] demonstrated that cryosurgery alone was not effective in producing an immune response that was protective against rechallenge in Dunning R3327 adenocarcinoma models, and their clinical study showed a diminished immune response after surgical procedure while not differing cryosurgery from radical prostatectomy and transurethral resection of prostate. To date, there have been no special clinical studies to evaluate the immune response after cryoablation of prostate cancers, although the procedure was increasing acceptance. It has been reported that human neoplastic diseases are frequently associated with dysregulation of the equilibrium between the production of certain Th1 and Th2 cytokines [1]. Th1 cytokines (IFN-c in particular) enhance the presentation of antigenic peptides to Th lymphocytes, and activate CTL- and NK-mediated cytolytic functions associated with effective antitumor defense mechanisms [35]. In contrast, Th2 cytokines (IL-10 in particular) were shown to down-modulate tumor-specific immune response by reducing MHC expression on the surface of tumor cells, and inhibiting tumor antigen presentation by antigen-presenting cells [4]. Although increased production of TNF-a and other cytokines induced by cryosurgery inhibits secondary tumor growth, high plasma levels of these factors have been associated with the occurrence of cryoshock [19,31]. In the present study, we examined both Th1 and Th2 cytokines in the serum of prostate cancer patients treated by cryoablation. Levels of TNF-a and IFN-c were significantly increased 4 weeks after cryoablation, while there were no differences in IL-4 and IL-10. Although the cytokine responses were decreased 8 weeks after treatment, these results suggest that cryosurgery promotes the inflammatory cytokines involved in cell-

mediated immunity. And in this study, no cryoshock was found, the response was similar to a questionnaire study [27]. Moreover, the ELISPOT results suggested that the rise of INF-c is based on the stimulation of prostate cancer antigens, and the cytotoxicity assay showed the immune response is specific for prostate cancer. Besides, IFN-c could augment natural killer (NK) cell proliferation and activation [15]. In a breast cancer model study, cryoablation induces both a tumor-specific T-cell response in the tumor draining lymph nodes, and an increased systemic NK cell activity, which correlates with rejection of tumors on rechallenge [26]. Although NK cells are not considered to be tumor-specific, a subpopulation, called NKT cells, which express both NK cell receptors and a T-cell receptor, demonstrate antigen specific cytotoxicity [22]. It was our limits not evaluating NK activation and anti-tumor effects effectively induced by cryoablation. In our study, the tumor-specific T-cell response was not maintained for 8 weeks after cryoablation, which may be the reason for cancer recurrence. There were many factors involved in the immune responses, including absence of repeated stimulation by tumor antigen, due to the single cryoablation treatment, or factors that depress immune response, such as regulatory T-cell, and depletion of these cells have been shown to improve anti-tumor immunity [5]. Several animal model studies have demonstrated that the immune response initiated by cryoablation can be amplified to protect from tumor rechallenge by other adjunctive treatment such as injecting BCG into the lesion [21], regulatory T-cell depletion [6], intra-tumor administration of dendritic cells [32] or Toll-like receptor stimulation [7]. One can envision a combination of cryoablation and immune modulation to induce an effective anti-tumor immune response. Further research to determine the optimum methods for prostate cancer treatment is warranted. In conclusion, our results showed the immuno-stimulatory effect induced by cryoablation in human prostate cancer. Although the immune response stimulated by this procedure was transient and may not be sufficient to prevent cancer relapse, the induced immune events may be exploited to design new immunotherapeutic strategies. These novel approaches may potentially be widely applicable to different types of malignancies, because cryoablation can be applied not only to prostate cancer but also to renal cell cancer, breast cancer, lung cancer, and hepatic cancer or to metastasis.

References [1] A.K. Abbas, K.M. Murphy, A. Sher, Functional diversity of helper T lymphocyte, Nature 383 (1996) 787–793. [2] C. Ardavín, S. Amigorena, C. Reis e Sousa, Dendritic cells: immunobiology and cancer immunotherapy, Immunity 20 (2004) 17–23. [3] D.K. Bahn, F. Lee, R. Badalament, et al., Targeted cryoablation of the prostate: 7-year outcomes in the primary treatment of prostate cancer, Urology 60 (2002) 3–11. [4] S. Beissert, J. Hosoi, S. Grabbe, A. Asahina, et al., IL-10 inhibits tumor antigen presentation by epidermal antigen-presenting cells, J. Immunol. 154 (1995) 1280–1286. [5] J. Dannull, Z. Su, D. Rizzieri, et al., Enhancement of vaccine-mediated antitumor immunity in cancer patients after depletion of regulatory T cells, J. Clin. Invest. 115 (2005) 3623–3633. [6] M.H. den Brok, R.P. Sutmuller, S. Nierkens, et al., Efficient loading of dendritic cells following cryo and radiofrequency ablation in combination with immune modulation induces anti-tumour immunity, Br. J. Cancer 95 (2006) 896–905. [7] M.H. den Brok, R.P. Sutmuller, S. Nierkens, et al., Synergy between in situ cryoablation and TLR9 stimulation results in a highly effective in vivo dendritic cell vaccine, Cancer Res. 66 (2006) 7285–7292. [8] R.J. Eastham, J.M. Mason, B.R. Jennings, et al., T-cell rosette test in squamous cell carcinoma of the head and neck, Arch. Otolaryngol. 102 (1976) 171–175. [9] M. Fazio, M. Airoldi, V. Mastromatteo, et al., Cryosurgery as a stimulator of the host’s immune defences in benign and malignant oral cavity tumours, Panminerva Med. 24 (1982) 195–201. [10] A.A. Gage, Cryosurgery for oral and pharyngeal carcinoma, Am. J. Surg. 118 (1969) 669–672. [11] S. Gallucci, M. Lolkema, P. Matzinger, Natural adjuvants: endogenous activators of dendritic cells, Nat. Med. 5 (1999) 1249–1255.

T. Si et al. / Cryobiology 57 (2008) 66–71 [12] S. Gazzaniga, A. Bravo, S.R. Goldszmid, et al., Infammatory changes after cryosurgery-induced necrosis in human melanoma xenografted in nude mice, J. Invest. Dermatol. 116 (2001) 664–671. [13] T.B. Geijtenbeek, S.J. van Vliet, A. Engering, et al., Self- and nonself-recognition by C-type lectins on dendritic cells, Annu. Rev. Immunol. 22 (2004) 33–54. [14] M.J. Gonder, W.A. Soanes, S. Shulman, Cryosurgical treatment of the prostate, Invest. Urol. 3 (1966) 372–378. [15] R.B. Herberman, C.W. Reynolds, J.R. Ortaldo, Mechanism of cytotoxicity by natural killer (NK) cells, Annu. Rev. Immunol. 4 (1986) 651–680. [16] N.E. Hoffmann, J.C. Bischof, The cryobiology of cryosurgical injury, Urology 60 (2002) 40–49. [17] M. Ismail, S. Ahmed, C. Kastner, J. Davies, Salvage cryotherapy for recurrent prostate cancer after radiation failure: a prospective case series of the first 100 patients, BJU Int. 100 (2007) 760–764. [18] J.P. Johnson, Immunologic aspects of cryosurgery: potential modulation of immune recognition and effector cell maturation,Clin. Dermatol. 8 (1990) 39–47. [19] J.J. Joosten, G.N. vanMuijen, T. Wobbes, et al., Cryosurgery of tumor tissue causes endoxin tolerance through an inflammatory response, Anticancer Res. 23 (2003) 427–432. [20] J.P. Long, D. Bahn, F. Lee, et al., Five year retrospective multiinstitutional pooled analysis of cancer related outcomes after cryosurgical ablation of the prostate, Urology 57 (2001) 518–523. [21] D.M. Lubaroff, C.W. Reynolds, L. Canfield, et al., Immunologic aspects of the prostate, Prostate 2 (1981) 233–248. [22] K. Masuda, Y. Makino, J. Cui, et al., Phenotypes and invariant alpha beta TCR expression of peripheral Valpha14+ NK T cells, J. Immunol. 158 (1997) 2076– 2082. [23] G.M. Onik, J.K. Cohen, G.D. Reyes, et al., Transrectal ultrasound-guided percutaneous radical cryosurgical ablation of the prostate, Cancer 72 (1993) 1291–1299. [24] S. Osada, H. Imai, H. Tomita, et al., Serum cytokine levels in response to hepatic cryoablation, J. Surg. Oncol. 95 (2007) 491–498.

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[25] M.H. Ravindranath, T.F. Wood, D. Soh, et al., Cryosurgical ablation of liver tumors in colon cancer patients increases the serum total ganglioside level and then selectively augments antiganglioside IgM, Cryobiology 45 (2002) 10–21. [26] M.S. Sabel, M.A. Nehs, G. Su, et al., Immunologic response to cryoablation of breast cancer, Breast Cancer Res. Treat. 90 (2005) 97–104. [27] J.K. Seifert, D.L. Morris, World survey on the complications of hepatic and prostate cryotherapy, World J. Surg. 23 (1999) 109–114. [28] W.A. Soanes, M.J. Gonder, Use of cryosurgery in prostatic cancer, J. Urol. 99 (1968) 793–797. [29] W.A. Soanes, R.J. Ablin, M.J. Gonder, Remission of metatatic lesions following cryosurgery in prostatic cancer, J, Urol. 104 (1970) 154–159. [30] Y. Suzuki, Cryosurgical treatment of advanced breast cancer and cryoimmunological responses, Skin Cancer 10 (1995) 19–26. [31] B.D. Teague, F.G. Court, C.P. Morrison, et al., Electrolytic liver ablation is not associated with evidence of a systemic inflammatory response syndrome, Br. J. Surg. 91 (2004) 178–183. [32] M. Udagawa, C. Kudo-Saito, G. Hasegawa, et al., Enhancement of immunologic tumor regression by intratumoral administration of dendritic cells in combinationwith cryoablative tumor pretreatment and bacillus calmetteguerin cell wall skeleton stimulation, Clin. Cancer Res. 12 (2006) 7465–7475. [33] M. Urano, C. Tanaka, Y. Sugiyama, et al., Antitumor effects of residual tumor after cryoablation: the combined effect of residual tumor and a proteinbound polysaccharide on multiple liver metastases in a murine model, Cryobiology 46 (2003) 238–245. [34] T. Yamashita, K. Hayakawa, M. Hosokawa, et al., Enhanced tumor metastases in rats following cryosurgery of primary tumor, Gann 73 (1982) 222–228. [35] I.A. York, K.L. Rock, Antigen processing and presentation by the class I major histocompatibility complex, Annu. Rev. Immunol. 14 (1996) 369–396. [36] A. Zisman, A.J. Pantuck, J.K. Cohen, A.S. Belldegrun, Prostate cryoablation using direct transperineal placement of ultra-thin probes through a 17 gauge brachytherapy template—technique and preliminary results, Urology 58 (2001) 988–993.