Radio frequency ablation combined with interleukin-2 induces an antitumor immune response to renal cell carcinoma in a murine model.

PURPOSE: Immune based therapy has gained renewed interest in the search for treatment strategies for metastasized renal cell carcinoma. We determined whether radio frequency ablation combined with interleukin-2 would induce a tumor specific immune response and serve as an in situ vaccine against renal cell carcinoma. MATERIALS AND METHODS: Mice with orthotopic renal tumors were treated with radio frequency ablation combined with interleukin-2, radio frequency ablation only, interleukin-2 only or no treatment as the control. Immunohistochemistry was done to determine which cells were present in the tumor after treatment. In vitro tumor specific cytotoxicity assays were performed with CD8+ T and natural killer cells derived from the spleen of treated mice. To study whether treatment could prevent metastasis or affect the growth of established metastases we induced lung metastasis intravenously before or after treatment and subsequently quantified it. RESULTS: The number of natural killer, CD4+ and CD8+ T cells was significantly increased in the tumor tissue of radio frequency ablation/interleukin-2 treated mice (p <0.0001). Natural killer and CD8+ T cells showed strong antitumor activity in vitro after combination treatment. Lung metastatic formation was significantly prevented in mice that received combination therapy (p <0.0001). Lung metastases were significantly smaller after combination treatment (vs interleukin-2 p = 0.025). CONCLUSIONS: Radio frequency ablation of the primary tumor combined with interleukin-2 induces a systemic antitumor immune response to renal cell carcinoma, which is much stronger than that of interleukin-2 monotherapy. This effect appears to be mediated by natural killer and CD8+ T cells. It may have an important role in the development of new immunotherapy strategies for renal cell carcinoma.

Cytokine therapy response as a selection criterion for cytoreductive nephrectomy in metastatic renal clear-cell carcinoma of intermediate prognosis. Results and conclusions from a combined analysis.

AIMS: To assess the strategy of using an absence of progression at metastatic sites following initial cytokine therapy outcome as a selection criterion for nephrectomy in patients with synchronous metastatic renal carcinoma and an intermediate prognosis according to the Memorial Sloan Kettering prognostic index classification. MATERIALS AND METHODS: A combined retrospective analysis of patients with clear-cell subtype from studies of initial cytokine treatment response to assist with selection of patients for nephrectomy. We analyzed survival times, UCLA integrated staging system scores, number of nephrectomies and risk of progression to unresectability of the primary tumor during treatment. RESULTS: There were 33 patients in total. Nephrectomies were not performed in 10 (30%) patients whose cancers had progressed at metastatic sites. Median survival time was 4 months with none of the patients dying of local tumor progression. The median survival time of the 21 patients with nonprogressive cancer and the primary removed was 17 months. Of those, 8 had a survival time < or =1 year (median 8.5 months) and a progression-free survival time of 4 months and 13 had a survival time >1 year (median 25 months). The median progression-free survival time was 7 months (4-57 months). Four of the 5 objective responses at metastatic sites (5/33, 14%) occurred in those surviving >1 year. CONCLUSIONS: We propose that progression at metastatic sites during initial immunotherapy may be used to identify patients with a short survival time and who are unlikely to benefit from nephrectomy.

Immunogenicity, including vitiligo, and feasibility of vaccination with autologous GM-CSF-transduced tumor cells in metastatic melanoma patients.

PURPOSE: To determine the feasibility, toxicity, and immunologic effects of vaccination with autologous tumor cells retrovirally transduced with the GM-CSF gene, we performed a phase I/II vaccination study in stage IV metastatic melanoma patients. PATIENTS AND METHODS: Sixty-four patients were randomly assigned to receive three vaccinations of high-dose or low-dose tumor cells at 3-week intervals. Tumor cell vaccine preparation succeeded for 56 patients (88%), but because of progressive disease, the well-tolerated vaccination was completed in only 28 patients. We analyzed the priming of T cells against melanoma antigens, MART-1, tyrosinase, gp100, MAGE-A1, and MAGE-A3 using human leukocyte antigen/peptide tetramers and functional assays. RESULTS: The high-dose vaccination induced the infiltration of T cells into the tumor tissue. Three of 14 patients receiving the high-dose vaccine showed an increase in MART-1- or gp100-specific T cells in the peripheral blood during vaccination. Six patients experienced disease-free survival for more than 5 years, and two of these patients developed vitiligo at multiple sites after vaccination. MART-1- and gp100-specific T cells were found infiltrating in vitiligo skin. Upon vaccination, the T cells acquired an effector phenotype and produced interferon-gamma on specific antigenic stimulation. CONCLUSION: We conclude that vaccination with GM-CSF-transduced autologous tumor cells has limited toxicity and can enhance T-cell activation against melanocyte differentiation antigens, which can lead to vitiligo. Whether the induction of autoimmune vitiligo may prolong disease-free survival of metastatic melanoma patients who are surgically rendered as having no evidence of disease before vaccination is worthy of further investigation.

Normal values of serum S-100B predict prolonged survival for stage IV melanoma patients.

The value of normal S-100B levels to predict survival was evaluated in 145 patients with stage IV melanoma. Treatment consisted of temozolomide given alone or was followed by combined cytokine immunotherapy, given every three to four weeks, with an evaluation of response following two treatment-cycles. S-100B values were measured prior to and following each cycle of systemic therapy and regularly thereafter. Patients with normal initial S-100B values (n=32) had higher response rates and fewer and more favourable metastatic sites with better overall survival rates than patients with elevated S-100B levels (median 14.0 versus 6.6 months). Normal S-100B values increased in nearly all patients (28/31) after a median of 7.9 months. In addition, patients with rapid normalisation of their serum level (n=12) following systemic treatment experienced prolonged survival. However, upon multivariable analysis S-100B prior to treatment lost its independence as a prognostic factor, whereas lactate dehydrogenase (LDH) remained. When measured after treatment, both markers had independent value.

The timing of immunotherapy and nephrectomy in multimodality treatment of metastatic renal cell carcinoma.

Multimodality treatment of metastatic renal cell carcinoma with immunotherapy and cytoreductive surgery is controversial. Especially the benefit of removing asymptomatic primary tumors in synchronous metastatic renal cell cancer has been debated since several non-randomized, retrospective studies revealed an improved response to immunotherapy and prolonged survival following initial nephrectomy. Two recent randomized prospective trials both demonstrated a prolonged survival in those who were randomly assigned to undergo nephrectomy of the primary tumor prior to treatment with interferon alfa-2b than in those who were assigned to undergo treatment with interferon alfa-2b alone. In these trials the survival benefit was limited and strongly influenced by overall performance score. The timing of immunotherapy, either as neoadjuvant (prior to nephrectomy) or adjuvant treatment (following nephrectomy) in the multimodality approach of synchronous metastatic renal cell carcinoma remains controversial. Selection of patients, the possible mechanisms underlying the survival advantage of the combination of nephrectomy and immunotherapy, and the timing of the treatment modalities are discussed herein.

Long-term persistence of T cell memory to HBsAg after hepatitis B vaccination.

AIM: To determine if the T cell memory to HBsAg can persist for a long time after hepatitis B (HB) vaccination. METHODS: Thirty one vaccine recipients who were healthcare workers (18 females and 13 males aged 34-58 years) from Utrecht University Hospital, Netherlands, and had previously received a standard course of vaccination for hepatitis B were investigated and another 9 unvaccinated healthy volunteers from the same hospital were used as the control. Blood samples were taken just before the experiment to test serum anti-HBs levels and the subjects were classified into different groups according to their serum titers of anti-HBs and vaccination history. Their peripheral blood mononuclear cells (PBMC) were isolated from freshly heparinized venous blood and the proliferative response of T lymphocytes to the recombinant hepatitis B surface antigen (HBsAg) was investigated. RESULTS: Positive serum anti-HBs was found in 61.3% (19/31) vaccine recipients and a significant in vitro lymphocyte proliferative response to recombinant HBsAg was observed in all the vaccinees with positive anti-HBs. Serum anti-HBs level < or =10 IU/L was found in 38.7% (12/31) subjects. In this study, we specially focused on lymphocyte proliferative response to recombinant HBsAg in those vaccine recipients with serum anti-HBsAg less than 10 IU/L. Most of them had received a standard course of vaccination about 10 years before. T lymphocyte proliferative response was found positive in 7 of the 12 vaccine recipients. These results confirmed that HBsAg-specific memory T cells remained detectable in the circulation for a long time after vaccination, even when serum anti-HBs level had been undetectable. CONCLUSION: The T cell memory to HBsAg can persist for at least 10 years after HB vaccination. Further booster injection is not necessary in healthy responders to HB vaccine.

Is a low dose of hepatitis B vaccine enough for a rapid vaccination scheme?

AIM: To determine whether or not a low dose of HB vaccine can be effectively used in the rapid vaccination. METHODS: Rapid vaccination (0, 1, 2 months) with low dose (5 microg) or routine dose (10 microg) HB vaccine was studied in 250 subjects (130 school children and 120 university students). Serum from all the participants was tested for HBsAg, anti-HBs and anti-HBc at 1, 3 and 7 months after the first dose of vaccination and all subjects were serum HBV marks negative before the vaccination. Non-responders to a complete initial vaccination from university students were given an additional vaccination with 10 microg of HB vaccine and their serum anti-HBs was tested again one month later. RESULTS: One month after the third dose of vaccination (third month) sero-conversion rates and geometric mean titer (GMTs) were significantly (P<0.01) higher in the routine dose (resp. 89% and 106.8) than in the low dose group (resp. 72% and 59.5). Sero-conversion rates and GMTs were maintained stable for another 4 months in both groups. After an additional vaccination to non-responders with 10 microg HB vaccine, 17/23 subjects (13/15 from those vaccinated with 5 microg vaccine and 4/8 from those vaccinated with 10 microg vaccine) became anti-HBs positive, yielding similar sero-conversion rates for both dose groups. CONCLUSION: Higher sero-conversion rates and GMTs were reached in those vaccinated with 10 microg HB vaccine than in those vaccinated with 5 microg HB vaccine after a complete vaccination with a 0, 1, 2 month scheme. But the subjects vaccinated with 5 microg vaccine can also reach the similar sero-conversion rate after an additional vaccination.

Lack of elevation of serum S100B in patients with metastatic melanoma as a predictor of outcome after induction with an autologous vaccine of proliferating tumor cells and dendritic cells.

Serum levels of S100B and/or lactate dehydrogenase (LDH) are putative tumor markers in melanoma. Early changes in such markers may correlate with a positive immune response to vaccine therapy. In patients with metastatic melanoma, S100B and LDH serum levels were measured at baseline, and 1 week after 3 weekly subcutaneous injections of investigational, patient-specific vaccines consisting of autologous dendritic cells loaded with antigens from irradiated proliferating autologous tumor cells, and suspended in granulocyte macrophage colony-stimulating factor. There was a poor correlation between S100B and LDH levels at baseline (p = 0.324). Fourteen (14) patients with measurable disease had higher S100B (p = 0.0456) and LDH (p = 0.0013) levels than 31 patients who lacked measurable disease at that time. Fourteen (14) deceased patients (median survival, 13 months) had a mean baseline S100B of 0.62 mug/L (95% confidence interval [CI] 0.00-1.66) and LDH of 815 U/L (95% CI 222-1408); 31 surviving patients (median follow-up, 35.4 months) had mean S100B of 0.07 mug/L (95% CI 0.00-0.23; p = 0.006) and LDH of 442 U/L (95% CI 296-588; p = 0.002). Elevated baseline levels of LDH and S100B were each predictive of inferior progression-free survival (PFS) and overall survival (OS) (both p < 0.0001), but S100B was a better predictor for PFS than LDH. Changes in LDH between baseline and week 4 were not predictive of survival, but an increase in S100B predicted for inferior OS ( p = 0.039). Both LDH and S100B are predictive tumor markers in patients with metastatic melanoma. This is the first study to examine the changes in serum levels of LDH and S100B in response to an autologous tumor-cell vaccine.

Temozolomide in advanced malignant melanoma with small brain metastases: can we withhold cranial irradiation?

BACKGROUND: The efficacy of radiotherapy (RT) in patients who have brain metastases from melanoma is limited. In this study, the authors evaluated the efficacy of treatment with temozolomide in patients with metastatic melanoma, including small brain metastases, who did not require immediate RT and investigated the feasibility of deferring RT. METHODS: Patients with brain metastasis were identified from 3 prospective studies of temozolomide (with or without immunotherapy) for metastatic melanoma. Patients with brain metastasis that measured >2 cm, extensive edema, and localization in the brain stem were excluded from the study. For the current analysis, patients with leptomeningeal metastasis and patients who received previous stereotactic RT were excluded. In patients who achieved a systemic response or stabilization to temozolomide, the response of brain metastasis and the necessity for palliative cranial RT were evaluated. RESULTS: Among 179 patients who received temozolomide for advanced melanoma, 52 patients with brain metastasis were evaluable. Stabilization of systemic metastasis was noted in 7 of 52 patients (13%), and there were 6 responses (5 partial responses and 1 complete response; 11%); thus, in those 13 patients, 6 had stabilization of brain metastasis (11%) and 5 had a response (2 partial responses and 3 complete responses; 9%). Immunotherapy did not influence the neurologic response. The median time to neurologic progression was 7 months (range 2-15, months). RT for cerebral recurrence was required in 2 patients. The median survival of patients with brain metastases was 5.6 months (95% confidence interval, 4.4-6.8 months). Intracranial hemorrhagic complications were not observed. CONCLUSIONS: The current results indicated that it is feasible to treat patients who have advanced melanoma and small brain metastasis with temozolomide as the single treatment. The small subset of patients with systemic response usually showed durable stabilization or a response of brain metastasis. With this approach, neurologic disease can be controlled, and cranial irradiation may be deferred and even withheld in most of patients.

Phase I clinical study with multiple peptide vaccines in combination with tetanus toxoid and GM-CSF in advanced-stage HLA-A*0201-positive melanoma patients.

Successful induction of functional tumor-specific T cells by peptide vaccination in animal models has resulted in many clinical trials to test this approach in advanced-stage melanoma patients. In this phase I clinical trial, 11 end-stage melanoma patients were vaccinated intradermally with 3 peptides: MART-1(26-35) E27L (ELAGIGILTV), tyrosinase(368-376) N375Q (YMDGTMSQV), and gp100(209-217) T210M (IMQVPFSV), admixed with tetanus toxoid and granulocyte-monocyte colony stimulating factor. The peptide vaccine was well tolerated at all tested doses, and led to grade 1-2 toxicity only. Although all patients did show a rise in antitetanus IgG titers, in only 3 patients peptide-specific CD8 T-cells were induced. In 2 cases, the response was directed against MART-1(26-35) and consisted of 0.2% and 3.3% of the CD8 population; however, in both instances these cells did not produce interferon-gamma on stimulation with the unmodified peptide. The third patient mounted a small (0.1%) response against gp100. In a fourth patient, a nonfunctional tyrosinase-specific response (0.6%) was found that was present before vaccination, but was not affected in size nor in function by the vaccine. None of the 11 patients responded clinically according to response evaluation criteria in solid tumors criteria. Although this study is a small scale phase I clinical trial, the efficacy that was observed was disappointingly low. In accordance with previously published peptide vaccination studies, these results add to the increasing evidence that peptide vaccination in itself is not potent enough as an effective melanoma immunotherapy in advanced-stage patients.

A phase-II study of pegylated interferon alfa-2b for patients with metastatic renal cell carcinoma and removal of the primary tumor.

Twenty-two patients with metastatic renal cell carcinoma and removal of the primary tumor were treated with subcutaneous pegylated interferon alfa-2b (PEG-Intron) to evaluate toxicity and efficacy. Start dose was 3.0 microg/kg/week, escalated to 6.0 microg/kg/week. After 2 months, therapy was extended in case of response or stable disease (SD) until progressive disease (PD) or relapse for a maximum of 2 years. National Cancer Institute common toxicity criteria (NCI-CTC) were monitored every 2-4 weeks. After 2 months, nine patients did not continue (8 PD, 1 SD with grade 4 CTC) and 13 extended treatment [three partial response (PR), 10 SD], of these, 11 progressed. One patient with PR developed a durable complete response later. Overall response rate was 13.6% (3/22). Median overall survival is 13 months (range 3-35 months). Dosage was escalated to 6 microg/kg/week in three patients. NCI-CTC grade 2 and 3 required dose attenuation in 12 patients during escalation, and reduction in 10 during the trial. Three patients discontinued because of grade 4 CTC (two fatigue, one hyperglycemia). Fatigue was the major dose-limiting toxicity. These results suggest an efficacy and toxicity of PEG-Intron comparable to standard interferon alfa-2b in patients with mRCC and removal of the primary tumor.

Phase I/II study of sequential chemoimmunotherapy (SCIT) for metastatic melanoma: outpatient treatment with dacarbazine, granulocyte-macrophage colony-stimulating factor, low-dose interleukin-2, and interferon-alpha.

A regimen of sequential chemoimmunotherapy (SCIT) was studied in a phase I/II study to analyze toxicity, anti-tumor and immunomodulatory effects in patients with previously untreated metastatic cutaneous melanoma. Treatment consisted of dacarbazine (DTIC) 800 mg/m2 administered intravenously (i.v.) on day 1, followed by subcutaneous (s.c.) molgramostim (GM-CSF), 10 times 2.5 micro g/kg on days 2 to 12, s.c. low-dose interleukin-2 (IL-2), 10 times 1.8 MU on days 8 to 18, and s.c. interferon-alpha-2b (IFN-alpha), 5 times 6 MU on days 15 to 20. Dosages were not escalated. Therapy was given in the form of outpatient treatment. Changes in T-lymphocyte phenotype and in soluble mediators were monitored during treatment. A total of 32 patients with stage IV melanoma were enrolled in the study. Treatment was well tolerated, without serious toxicity. In all cases, it could be given as outpatient treatment. Ten subjects out of the 31 patients evaluated showed an objective response, with 4 complete responses (CR) and 6 partial responses (PR); the response rate amounted to 32% (95% CI: 16-49%). Median survival of all patients was 8 months, with those patients who responded to treatment living longer than the non-responding group. Survival rate at 1 year was 22%. Monitoring of the effects of treatment revealed increased numbers of activated T-lymphocytes, both in the CD4 and in the CD8 subsets. The levels of soluble mediators such as sIL-2R and sCD8 were also increased. Changes were noted as early as the GM-CSF treatment period, and were observed to a further extent during IL-2 treatment. In the present study, it was found that this sequential chemoimmunotherapy regimen consisting of 4 agents (DTIC, GM-CSF, IL-2, IFN-alpha) has acceptable toxicity, can be administered on an outpatient basis, results in increased numbers of activated T-lymphocytes, and induces activity against metastatic melanoma that warrants further investigation.

Preclinical evaluation of anti-CD86 immunotoxin in rhesus monkeys: analysis of systemic toxicity, pharmacokinetics, and effect on primary T-cell responses.

CD86 and CD80 costimulatory antigens are highly overexpressed on Hodgkin/Reed-Sternberg cells in patients with Hodgkin's disease (HD) and are candidate target antigens for immunotoxins in order to eliminate minimal residual disease. In this study we have evaluated the pharmacokinetics (and immunological) toxicity in rhesus monkeys of immunotoxins consisting of gelonin conjugated to anti-CD86 (alphaCD86-IT). Both alphaCD86-IT and alphaCD80-IT inhibited protein synthesis in B cell lines from rhesus monkeys and inhibited the mixed leukocyte reaction. Reactivity of the alphaCD86 antibody with rhesus monkey CD86 (RhCD86) was shown by cloning and transfecting RhCD86, which conferred reactivity of the alphaCD86 antibody used in this study. alphaCD86-IT was administered as single intravenous bolus injection in four rhesus monkeys, and achieved plasma concentration in 50-fold excess able to eliminate cultured Hodgkin/Reed-Sternberg cells up to 6 h. The animals were capable of generating primary immune responses to both gelonin and murine IgG within 9 days after infusion with alphaCD86-IT. No evidence could be found of significant (immunological) toxicity. The results of this study show that alphaCD86-IT can be applied safely in an effective dose.