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Of 93 patients with pretreated, BRAFV600 mutation-positive advanced melanoma who received vemurafenib or dabrafenib before (n = 45) or after (n = 48) treatment with ipilimumab 3 mg/kg, median overall survival (mOS) from first treatment was 9.9 and 14.5 months, respectively. Among patients treated with a BRAF inhibitor first, mOS from the end of BRAF inhibition was 1.2 months for those who did not complete ipilimumab treatment as per protocol, compared with 12.7 months for those who did (p < .001). Prospective, randomized studies are required to determine the optimal sequencing of ipilimumab and BRAF inhibitors in patients with BRAF-mutated metastatic melanoma.

Follow-up is managed internally in 94% of centers and is programmed according to international guidelines in 52% of high-volume hospitals (>25 melanoma diagnoses per year); the remainder use internal guidelines; fewer low-volume centers (≤25 diagnoses per year) have internal guidelines (25%, p = 0.001). Instrumental examinations for stage III and IV disease are similar, while the examination interval changes from 3/4 months for stage III to 2/3 months for stage IV, and use of PET/CT increases from 44 to 54%. Overall, thoracic and abdominal CT is used most for follow-up in stage III (83%), while bone scintigraphy is used more commonly in low-volume centers (41 vs. 19%, p = 0.003), despite similar use of PET/CT (48 vs. 41%). Brain CT or MRI is more common in high-volume centers (63 vs. 39%, p > 0.0001), as is echography of draining lymph nodes (71 vs. 52%, p = 0.01). Hepatic/abdominal echography and thoracic radiography are used in about 50% of centers, regardless of type. In stage IV, use of bone scintigraphy is similar among groups (ca. 40%); brain CT/NMR use increases from 51 to 64% and is more common in high-volume centers (p = 0.03). Lymph node echography is more common in high-volume centers (56 vs. 39%, p = 0.03).

Ipilimumab is a human monoclonal antibody, that blocks cytotoxic T-lymphocyte antigen-4 (CTLA-4), normally expressed in activated T cells. CTLA-4 downregulates T-cell activation pathways and induces progressive immune tolerance. Blocking this downregulation, determines an enhancement of antitumor T-lymphocyte response. A recent randomized Phase III trial using ipilimumab (MDX020-010) demonstrated, for the first time, a statistically significant improvement in overall survival in patients with metastatic melanoma, prolonging median survival from 6.4 to 10.1 months. It is noteworthy that the study population was composed of previously treated patients, of whom more than 70% had poor prognostic factors and that the adverse events experienced were reversed with appropriate treatment. Further trials are now needed to evaluate the real impact of ipilimumab on survival and on response duration in treatment-naive patients. Clinical research is also warranted into the association of ipilimumab with chemoimmunother...

Ipilimumab is a fully human, monoclonal antibody that blocks cytotoxic T-lymphocyte antigen-4 to potentiate antitumor T-cell responses. In a phase III trial, ipilimumab monotherapy at 3 mg/kg demonstrated an improvement in overall survival (OS) in patients with previously treated, metastatic melanoma. Here, we conducted a retrospective analysis of efficacy and safety data from a phase II clinical trial in which treatment-naive and previously treated patients with metastatic melanoma received ipilimumab at an investigational dose of 10 mg/kg. Patients were randomized 1:1 to receive oral budesonide or placebo, and ipilimumab at 10 mg/kg every 3 weeks for 4 doses, to determine whether prophylactic budesonide affected the rate of grade ≥2 diarrhea. One hundred fifteen patients were randomized and treated: 62 had received prior systemic therapy for metastatic disease and 53 had not. No efficacy endpoint was affected by budesonide therapy, and the efficacy data were therefore pooled for budesonide and placebo subgroups. Median OS was 30.5 months for treatment-naive patients who received ipilimumab, with survival rates of 69.4%, 62.9%, and 56.9% at 12, 18, and 24 months. In previously treated patients who received ipilimumab, median OS was 13.6 months, with survival rates of 50.0%, 37.7%, and 28.5% at 12, 18, and 24 months. There were no meaningful differences in the number of objective responses or rate of grade ≥2 diarrhea between groups. These retrospective analyses are the first to provide survival data for ipilimumab in treatment-naive and previously treated patients within the same clinical trial.

Dendritic cells (DCs) are unique specialized antigen-presenting cells capable of priming naive T cells and inducing antigen-specific cytotoxic T lymphocytes. This study presents an update of clinical results from a DC-based phase I-II clinical vaccine trial in stage IV melanoma. From 2003 to 2010, 27 patients with metastatic melanoma were treated with mature DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin and with subcutaneous low-dose interleukin-2. Delayed-type hypersensitivity (DTH) tests for in-vivo immunomonitoring were performed at baseline and every four vaccinations thereafter. Two complete, two mixed and six partial responses, and five stable diseases were observed (overall response, 37.0%; clinical benefit, 55.5%). All 15 responders showed DTH positivity. A median overall survival of 22.9 months [95% confidence interval (CI): 13.4-61.3] for DTH-positive patients (19) and 4.8 months (95% CI: 3.9-11.9) for DTH-negative patients (8; log rank=7.26; P=0.007) was observed. The overall median overall survival was 16 months (95% CI: 9-33). Our results would seem to highlight a relationship between positive-DTH test and an improved survival.

The liver is the primary site of metastases in most uveal melanoma patients. We retrospectively investigated intraarterial chemotherapy (IAC) as treatment for patients with hepatic melanoma metastases.Twenty-three patients (18 with uveal melanoma) received fotemustine (14 patients, 61.9%) or carboplatin (9 patients, 31.1%) via hepatic IAC delivery. The catheter was introduced through percutaneous access to the femoral artery with drugs delivered directly to the hepatic artery, and was removed at the end of each treatment cycle. A total of 3 cycles was planned, repeated every 21 days. However, patients with a clinical response could receive more than 3 cycles, provided that the toxic effects were acceptable.IAC was well tolerated and no catheter-related complications or grade 4 toxicities were reported. Considering only uveal melanoma patients, the overall response rate and disease control rate was 16.7% and 38.9%, respectively. Median time to progression was 6.2 months (95% CI 3.7-10.5) and median overall survival was 21 months (95% CI 8-39).IAC is well tolerated and is a valid choice for patients with a poor prognosis since median survival rates are among the longest reported.

Background: This study compared the central nervous system (CNS) metastasis incidence between a temozolomide- and a dacarbazine-based regimen in untreated stage IV melanoma patients. Methods: A total of 150 patients were randomly assigned to receive either oral temozolomide (200 mg m−2 per day; days 1–5) or intravenous dacarbazine (800 mg m−2; day 1), in combination with intravenous cisplatin (75 mg m−2; day 1) and subcutaneous interleukin-2 (3 MU twice daily; days 9–18), every 28 days (CTI and CDI). Results: A total of 149 patients were eligible for an intention-to-treat analysis (CTI: n=74, CDI: n=75). The 1-year cumulative CNS incidence failure was 20.6% for CTI and 31.1% for CDI (P=0.22). In all 24 patients in CTI (32%) and 34 (45%) in CDI developed CNS metastases; 31 patients died of early systemic progression, before CNS evaluation. Median survival time was 8.4 months in the CTI and 8.7 in the CDI arm; in patients with CNS metastases the median survival time was 13.5 months in the CTI and 11.5 in the CDI arm. No difference in toxicity was observed between the two arms. Conclusion: The incidence of CNS failures in metastatic melanoma was not significantly reduced and the clinical course was not modified substituting a dacarbazine-based regimen with a temozolomide–based regimen. Patients who developed CNS metastases did not have a worse prognosis than patients progressing in other sites and should not be excluded from new investigational studies.

PURPOSE: Anthracyclines and fluoropyrimidines are very active in breast cancer, while liposomal doxorubicin has low cardiotoxicity. We conducted a dose-finding study of the combination of liposomal doxorubicin and capecitabine in patients with pretreated metastatic breast cancer. PATIENTS AND METHODS: Patients received liposomal doxorubicin 60 mg/m2 on day 1 plus capecitabine 825 mg/m2 bid (level 0) or 1,000 mg/m2 bid (level 1) on days 1-14 of each 21-day cycle to establish the maximum tolerated dose (MTD) and cardiac safety. RESULTS: Nine patients were enrolled and a total of 52 courses were delivered (median 6 cycles per patient [range 4-7]). Grade 4 neutropenia occurred in 15% of cycles, with one episode of febrile neutropenia; most nonhematological toxicities were mild or moderate. No formal MTD was established, and the study was closed because two cardiac events were observed at dose level 1 and another at dose level 0 in patients pretreated with epirubicin > or = 560 mg/m2. CONCLUSIONS: The recommended dose for phase II studies is liposomal doxorubicin 60 mg/m2 on day 1 plus capecitabine 825 mg/m2/bid on days 1-14 of each 21-day cycle. Despite the lower cardiotoxicity of liposomal doxorubicin, the risk of cardiac damage persists in anthracycline-pretreated individuals and mandates close cardiac monitoring and careful evaluation of the overall cumulative dose.

When the cytotoxic T-lymphocyte-associated protein (CTLA)-4 molecule, present on the surface of a lymphocyte after immune cell activation, binds B7 surface molecules, T-lymphocyte anergy occurs, resulting in cell proliferation and inhibition of IL-2 secretion. Treatment with the anti-CTLA-4 monoclonal antibody ipilimumab (MDX-010) seems to prevent this anergy and permits specific T-lymphocyte activation against tumor antigens. Two Phase I trials on metastatic melanoma have been conducted using doses ranging from 3 to 20 mg/kg, followed by several Phase II trials. Although immune-related adverse events were reported in most of the clinical trials, these were generally manageable with systemic steroids or symptomatic treatments, and anti-tumor efficacy did not appear to be affected. Durable objective response rates were obtained in 4.6–15% of patients, with a further increment in late responses after long-term stable disease or after initial progression. Early and late responses, or stabilizations that are ...

A patient with resected stage III nodular melanoma treated with high-dose interferon-α-b2 adjuvant therapy went on to develop generalized lymphadenopathy and splenomegaly. The total body positron emission tomography showed a high 18 F-fluorodeoxyglucose uptake (standardized uptake values > 9), indicating possible lymph node and spleen malignancies. Histologic examinations of an axillary lymph node biopsy and an osteomedullar biopsy were negative, excluding both melanoma metastases and hematopoietic tumors. The symptoms completely regressed after suspension of treatment and a follow-up positron emission tomography was negative. It remains to be seen whether this unusual event can be ascribed to an autoimmune phenomenon linked to potential treatment efficacy and survival.

Purpose The PRAME tumour antigen is expressed in several tumour types but in few normal adult tissues. A dose-escalation phase I/II study (NCT01149343) assessed the safety, immunogenicity and clinical activity of the PRAME immunotherapeutic (recombinant PRAME protein (recPRAME) with the AS15 immunostimulant) in patients with advanced melanoma. Here, we report the phase I dose-escalation study segment. Patients and methods Patients with stage IV PRAME-positive melanoma were enrolled to 3 consecutive cohorts to receive up to 24 intramuscular injections of the PRAME immunotherapeutic. The RecPRAME dose was 20, 100 or 500 µg in cohorts 1, 2 and 3, respectively, with a fixed dose of AS15. Adverse events (AEs), including predefined dose-limiting toxicity (DLT) and the anti-PRAME humoral response (ELISA), were coprimary end points. Cellular immune responses were evaluated using in vitro assays. Results 66 patients were treated (20, 24 and 22 in the respective cohorts). AEs considered by the investigator to be causally related were mostly grade 1 or 2 injection site symptoms, fatigue, chills, fever and headache. Two DLTs (grade 3 brain oedema and proteinuria) were recorded in two patients in two cohorts (cohorts 2 and 3). All patients had detectable anti-PRAME antibodies after four immunisations. Percentages of patients with predefined PRAME-specific-CD4+T-cell responses after four immunisations were similar in each cohort. No CD8+ T-cell responses were detected. Conclusions The PRAME immunotherapeutic had an acceptable safety profile and induced similar anti-PRAME-specific humoral and cellular immune responses in all cohorts. As per protocol, the phase II study segment was initiated to further evaluate the 500 µg PRAME immunotherapeutic dose. Trial registration number NCT01149343, Results.

Background and Objectives Conventional chemotherapy has not proven effective in improving long-term results of surgery for liver metastases from colorectal cancer. We assessed the usefulness of immunotherapy with tumor infiltrating lymphocytes (TIL) plus Interleukin-2 (IL-2) as adjuvant treatment. Methods Between 1995 and 1998, 47 patients were enrolled onto a prospective protocol; 25 entered the treatment group (A) and 22 entered the control group (B). All patients had undergone radical liver resection. TIL obtained from surgical specimens from group A patients were cultured and activated in vitro with IL-2, then reinfused into the patients with IL-2. We investigated pre- and post-IL-2 stimulation expression of T cell receptor (TCR) ζ- and e-chains, p56lck, Fas, and Fas-L by TIL immunostaining. Results Fourteen patients from group A (56%) received immunotherapy; 14 from group B (60%) underwent conventional chemotherapy, and the remaining 19 patients did not receive any treatment. No significant differences between the two groups were found in the actuarial and disease-free survival (DSF) rates after 1, 3, and 5 years. After IL-2 exposure, TCR ζ-chain expression significantly increased (P = 0.001); An increase in TCR e-chain expression (P = 0.04), and p56lck (P = 0.03) was detected; TCR e-chain expression was significantly increased in disease-free patients compared to those who relapsed (P = 0.04). Fas-L expression was correlated with the TCR e-chain and p56lck levels (P = 0.05). Conclusions Our data suggest that we are still a long way from being able to propose TIL + IL-2 treatment as an effective adjuvant therapy. However, the results confirm that the biological indicators examined could play an important role in modulating immunitary response against tumor cells. J. Surg. Oncol. 2004;87:46–52. © 2004 Wiley-Liss, Inc.

Alterations in the expression of signal activation molecules, such as the T-cell receptor (TCR) zeta and epsilon chains and p56lck tyrosine kinase, are described in tumor-infiltrating lymphocytes (TIL). The aim of this study was to ascertain if such molecules were present in near-tumor-tissue lymphocytes (NTTL) and peripheral blood lymphocytes (PBL), as well as TIL, of renal cell carcinoma patients, to verify whether this tumor induces immunosuppression only locally or affects distant lymphocytes as well. Tissue from the tumor and from healthy nearby sites, as well as blood samples, were obtained from 27 consecutive patients who had undergone radical nephrectomy for renal cell carcinoma. Phenotype analysis and immunohistochemical staining of the TCR zeta and epsilon chains and p56lck were performed with standard techniques on TIL, NTTL, and PBL, and values were compared for each patient. Low expression of the TCR zeta chain and an almost complete absence of TCR epsilon chain and p56lck expression was observed in TIL (median values: 10% for zeta chain and 0% for epsilon and p56lck). In NTTL, these signal transduction molecules were expressed by a higher percentage of cells (60%, 50%, and 60%, respectively; p=0.000 vs. TIL), whereas PBL showed an almost normal expression of zeta and epsilon chains (80% and 90%, respectively; p=0.000 vs. TIL). Conversely, p56lck was detected in a greater proportion of NTTL than PBL (50% vs. 10%; p=0.001). The absence or the very low expression of signaling activation molecules in TIL compared with NTTL and PBL in renal cancer patients suggest that tumor-induced immunosuppression generally occurs or starts locally.

The most recent advances in immunology bear witness to the fact that tumors, in particular melanoma, escape recognition by the host's immune system and can locally inactivate its effectors, T-cells and antigen presenting cells. There is, however, preclinical evidence that the immune system, opportunely stimulated, is capable of recognizing and killing tumor cells. It has been verified that the activation of autologous dendritic cells, derived from peripheral blood and pulsed with tumor antigens, results in the specific stimulation of T-cells against the tumor. Preliminary data from dendritic cell vaccination trials, mainly of advanced melanoma, show unequivocal evidence of immunization and of the first clinical responses. Many questions remain to be answered before more effective and widespread use of this type of vaccination is possible, especially in the early stages of the disease.

Background Vaccination with dendritic cells (DC) loaded with tumor antigens elicits tumor-specific immune responses capable of killing cancer cells without inducing meaningful side-effects. Patients with advanced melanoma enrolled onto our phase II clinical studies have been treated with autologous DC loaded with autologous tumor lysate/homogenate matured with a cytokine cocktail, showing a clinical benefit (PR + SD) in 55.5% of evaluable cases to date. The beneficial effects of the vaccine were mainly restricted to patients who developed vaccine-specific immune response after treatment. However, immunological responses were only induced in about two-thirds of patients, and treatments aimed at improving immunological responsiveness to the vaccine are needed. Methods/Design This is a phase II, “proof-of-principle”, randomized, open-label trial of vaccination with autologous DC loaded with tumor lysate or homogenate in metastatic melanoma patients combined with immunomodulating RT and/or preleukapheresis IFN-α. All patients will receive four bi-weekly doses of the vaccine during the induction phase and monthly doses thereafter for up to a maximum of 14 vaccinations or until confirmed progression. Patients will be randomized to receive: (1.) three daily doses of 8 Gy up to 12 Gy radiotherapy delivered to one non-index metastatic field between vaccine doses 1 and 2 and, optionally, between doses 7 and 8, using IMRT-IMAT techniques; (2.) daily 3 MU subcutaneous IFN-α for 7 days before leukapheresis; (3.) both 1 and 2; (4.) neither 1 nor 2. At least six patients eligible for treatment will be enrolled per arm. Daily 3 MU IL-2 will be administered subcutaneously for 5 days starting from the second day after each vaccine dose. Serial DTH testing and blood sampling to evaluate treatment-induced immune response will be performed. Objective response will be evaluated according to immune-related response criteria (irRC). Discussion Based upon the emerging role of radiotherapy as an immunologic modifier, we designed a randomized phase II trial adding radiotherapy and/or preleukapheresis IFN-α to our DC vaccine in metastatic melanoma patients. Our aim was to find the best combination of complementary interventions to enhance anti-tumor response induced by DC vaccination, which could ultimately lead to better survival and milder toxicity.

Aim and background Recent data in the literature indicate that antigen-presenting cells (APC) are inactive in tumour tissue because of local immunosuppression. Tumour-infiltrating lymphocyte (TIL) signal activation transducing mechanisms are also seriously impaired. Administration of granulocyte macrophage-colony stimulating factor (GM-CSF) may lead to APC recovery and interleukin (IL)-2 may restore local TIL activation. Moreover, IL-2 increases the systemic lymphocyte population, an event that seems to correlate with a better prognosis. Study design The present phase I–II study was carried out to examine whether intralesional injection of GM-CSF followed by subcutaneous IL-2 would induce a clinical response in advanced, pretreated elderly melanoma patients. Methods Sixteen patients over 60 years of age received intralesional GM-CSF (150 ng per lesion on day 1), generally divided between the two largest cutaneous lesions, followed by perilesional subcutaneous IL-2 (3 000 000 IU) for 5 days (days 3–7 inclusive) every 3 weeks. Results Four clinical responses [two partial (PR) and two minimal (MR)] (25%), which also involved lesions that had not been directly treated, and nine cases of stable disease were observed. The response duration for PR and MR was 9, 4, 4 and 2.5+ months, respectively. Stable disease (56%) recorded in the nine patients was short-term (3–6 months). Three patients rapidly progressed after two, two and one therapy cycles, respectively. The patient who reached the best PR had a fairly high absolute lymphocyte count (1600–2400/mm3). The second one, who reached complete remission after subsequent locoregional chemotherapy and hyperthermia, however, had a low absolute lymphocyte count that had doubled by the end of treatment. Blood lymphocyte values in the other patients were too varied to allow any correlation with clinical response. Therapy was well tolerated and only mild fever was observed, with the exception of one patient who had grade 3 fever, with muscle pain and arthralgia. Conclusions Considering the very low toxicity observed, this treatment might be indicated in elderly patients for whom systemic therapy is no longer a viable option. Improved scheduling and timing could result from further studies.

Malignant pleural mesothelioma is often unresectable at diagnosis, is refractory to cytotoxic agents and is frequently complicated by pleural effusion. The expected survival range for patients with or without involvement of visceral pleura is respectively 1–9 and 9–12 months; mesothelioma-related pleural effusion severely impairs the patients’ quality of life and easily relapses after conservative treatments. Intrapleural administration of IL-2 is reported to be effective both in tumor-associated malignant pleurisy and on primary mesothelioma, whereas few data exist about IL-2 systemic administration. In order to assess the palliative and therapeutic activity of IL-2 in unresectable pleural malignant mesothelioma with pleural effusion, we performed a phase II study on 31 consecutive patients (M/F 16/15; median age 61 years, range 40–84; PS ECOG 0 n=7; ECOG 1 n=15; ECOG 2 n=9; stage IA n=13; IB n=9; II n=7; IV=2) who received first-line therapy with intrapleural repeated instillation of 9 000 000 I.U. IL-2 twice/weekly for 4 weeks, after needle thoracenthesis. In nonprogressing patients, 3 000 000 I.U. IL-2 were subcutaneously administered thrice weekly for up to 6 months. Toxicity (WHO criteria) with intrapleural IL-2 consisted of grade 3 fever in 6/31 (19%) patients and of cardiac toxicity (failure) grade 3 in one patient (3%); toxicity during subcutaneous treatment was mild to moderate, mainly a flu-like syndrome. In 28/31 (90%) of patients there was no further or minimal (asymptomatic) pleural fluid collection (according to Paladine criteria); pleurisy relapsed only in 1/28 patients after 19 months. Tumor objective response (WHO criteria), evaluated by CT, occurred in seven patients (one CR and six PR; ORR 22%); ten patients achieved SD and 14 patients progressed. Median overall survival was 15 months (range 5–39) in all patients. IL-2 intrapleural administration followed by low-dose IL-2 subcutaneously in pleurisy-complicated malignant mesothelioma is feasible and active both in palliation of pleural effusion and on primary tumor, with manageable toxicity. The overall survival observed in nonprogressing patients warrants further randomized studies with IL-2 aimed to the patient outcome.

Aims and background Adoptive immunotherapy with tumor infiltrating lymphocyte (TIL) reinfusion plus continuous interleukin-2 (IL-2) infusion could represent an innovative way of treating immunogenic tumors. This study therefore recruited melanoma, colorectal and renal carcinoma patients whose metastases had been surgically removed. Study design The treatment was initially given to 22 patients with advanced disease and more recently to 39 disease-free (DF) patients after radical metastasectomy. The latter group was selected in view of a theoretically better lymphocyte/tumor cell ratio and with the aim to improve disease-free and overall survival (DFS-OS) in very high risk patients. The starting IL-2 dose was 12 MlU/day (West's schedule); doses were modulated on the bases of toxicity parameters. Even though patients received different total amounts of IL-2, all of them completed the treatment. Results The treatment was offered to 22 advanced-stage cancer patients (12 melanomas, 9 colorectal carcinomas, 1 kidney carcinoma). Few and short stabilizations were observed with a median survival of 12 months (range, 3-29). Subsequently, another 39 patients were treated in an adjuvant setting after radical metastasectomy (18 melanomas, 19 colorectal carcinomas, 2 kidney cancers). Eleven out of 17 DF melanoma patients (64.7%) are still free of disease after a median of 37+ months (range, 5+ - 69+). In the group of DF colorectal cancer patients eight (44.4%) are still DF after a median of 21+ months (range, 7+ - 67+ months). One of the two patients with kidney cancer is still DF after 28+ months. Two patients (1 melanoma and 1 colorectal cancer) had just been treated and were therefore not evaluable. Severe toxicity occurred in three cases but was rapidly resolved. There was a great diversity in IL-2 doses administered; comparison of the total IL-2 dose administered between the patients who are still DF and those who progressed revealed no difference between the two groups of colorectal cancer patients, whereas melanoma patients who progressed rebeived an average IL-2 dose of 6.5 MlU/day versus 15.8 MlU/day in DF patients. No differences were observed in any of the groups between the number of TILs reinfused and clinical response. Conclusions The study is still ongoing; it has been decided to focus on DF melanoma patients after radical metastasectomy, for whom the data seem to be encouraging. Further endpoints of the study are the role of IL-2 dosage in the adjuvant setting, and the possibility to make correlations between biological parameters and clinical results.

Adoptive tumour infiltrating lymphocytes (TIL) in combination with a modulated dosage of interleukin-2 (IL-2) can be used with acceptable toxicity in the treatment of immunogenic tumours. Following an experience of reinfusion in advanced melanoma, colorectal and renal cancer patients, treatment was given to disease-free patients after metastasectomy. The high risk of relapse and favourable ratio between reinfused TIL and possible microscopic residual disease determined this choice of adjuvant treatment. A group of 12 patients with advanced disease (7 melanoma, 4 colorectal carcinoma, 1 kidney carcinoma) were treated with TIL (median 5.8 x 10(10) cells) and IL-2 (West's schedule) modulated towards a lower dosage (from 12 to 6 MIU/day) in order to maintain an acceptable level of toxicity. As treatment was well tolerated, it was offered to another 22 patients in an adjuvant setting after metastasectomy (11 melanoma, 10 colorectal carcinoma, 1 renal cancer), the median dose of TIL reinfused being 4.95 x 10(10) cells. No objective response was observed in advanced patients: all patients progressed after a median of 1.5 months (0-8 months) and median survival was 8 months (3-22+ months). Thirteen patients from the second group are still disease-free after a median of 23+ months (9+ - 47+ months). The remaining 9 patients relapsed after a median of 5 months (3-18 months). Toxicity was moderate as clinical and hepatic/renal function parameters were used to assess the need for dose reductions. Consequently, there was great diversity in IL-2 dosages administered. In particular, there seemed to be a difference in IL-2 doses administered between disease-free cases and those who progressed (17.5 MIU/day versus 7 MIU/day in melanoma patients; 11.2 MIU/day versus 7.1 MIU/day in colorectal cancer patients). By contrast, no differences were observed between number of TIL reinfused and clinical response. Phenotypical characteristics of reinfused TIL were similar to those reported in the literature: 97% were CD3 and 92% were CD8. Aspecific cytolytic activity was evaluated on 12 cases whereas, in 2 melanoma cases, autologous tumour tissue was available for the specific cytotoxicity test. Perforin levels in TIL measured at the end of culture were generally high or very high. Cytokine levels were measured on the supernatant at the end of culture, with an estreme variability in results. Finally, delta chain and p56lck were histologically assessed on the resected tissue from which TIL were cultivated. There were virtually none of the former and a complete absence of the latter, which concurs with data reported in the literature. The same immunocytochemical analysis was carried out on TIL at the end of culture. This time an almost complete restoration of both functions was seen, especially in melanoma patients, who are still free from disease. The study is on-going and it has been decided to focus on disease-free patients after metastasectomy in order to increase the number and possibility of clinical and histological correlations.

BACKGROUND: Tumor cells killed by radiation therapy (RT) are a potentially good source of antigens for dendritic cell (DC) uptake and presentation to T-cells. RT upregulates cell death receptors such as Fas/CD95 and MHC-I, induces the expression of co-stimulatory molecules on tumor cells, and promotes production of pro-inflammatory cytokines. High-dose interleukin-2 (HD-IL-2) bolus has been shown to obtain objective response rates ranging from 15% to 17% in patients with metastatic melanoma or renal cell carcinoma (RCC), with 6% to 8% of cases experiencing a durable complete response. However, HD-IL-2 is also associated with severe side-effects; if it is to remain a component of the curative treatment strategy in patients with metastatic melanoma or RCC, its therapeutic efficacy must be improved and patients who are most likely to benefit from treatment must be identified a priori. We designed a clinical study combining immunomodulating RT and HD-IL-2 to evaluate their clinical and immunological efficacy and to explore the predictive and prognostic value of 1) tumor-specific immune response and 2) serum levels of proangiogenic cytokines. METHODS/DESIGN: The primary endpoint of this proof-of-principle phase II study is immune response. Secondary endpoints are the identification of biomarkers potentially predictive of response, toxicity, response rate and overall survival. Three daily doses of booster radiotherapy (XRT) at 6–12 Gy will be administered to at least one metastatic field on days −3 to −1 before the first and third cycle. Treatment with IL-2 (dose 18 MIU/m2/day by continuous IV infusion for 72 hours) will start on day +1 and will be repeated every 3 weeks for up to 4 cycles and then every 4 weeks for a further 2 cycles. Immune response against tumor antigens expressed by melanoma and/or RCC will be evaluated during treatment. Circulating immune effectors and regulators, e.g. cytotoxic T lymphocytes and regulatory T cells, as well as serum levels of proangiogenic/proinflammatory cytokines will also be quantified. DISCUSSION: This study aims to evaluate the potential immunological synergism between HD-IL-2 and XRT, and to identify biomarkers that are predictive of response to IL-2 in order to spare potentially non responding patients from toxicity. TRIAL REGISTRATION: EudraCT no. 2012-001786-32 CLINICALTRIALS.GOV IDENTIFIER: NCT01884961

Background Ipilimumab improves survival in patients with advanced melanoma. The activity and safety of ipilimumab outside of a clinical trial was assessed in an expanded access programme (EAP). Methods Ipilimumab was available upon physician request for patients aged 16 or over with pretreated stage III (unresectable)/IV melanoma, for whom no other therapeutic option was available. Patients received ipilimumab 3 mg/kg every 3 weeks for four doses. Patients with stable disease or an objective response to ipilimumab were eligible for retreatment upon disease progression. Tumour assessments were conducted at baseline and week 12. Patients were monitored for adverse events (AEs) within 3 to 4 days of each scheduled visit. Results Of 855 patients participating in the EAP in Italy, 833 were evaluable for response. Of these, 13% had an objective immune response, and the immune-related disease control rate was 34%. Median progression-free survival and overall survival were 3.7 and 7.2 months, respectively. Efficacy was independent of BRAF and NRAS mutational status. Overall, 33% of patients reported an immune-related AE (irAE). The frequency of irAEs was not associated with response to ipilimumab. Conclusions Outside of a clinical trial setting, ipilimumab is a feasible treatment option in patients with pretreated metastatic melanoma, regardless of BRAF and NRAS mutational status. Data from this large cohort of patients support clinical trial evidence that ipilimumab can induce durable disease control and long-term survival in patients who have failed to respond to prior treatment.

Background Patients with advanced melanoma are faced with a poor prognosis and, until recently, limited treatment options. Ipilimumab, a novel immunotherapy that blocks cytotoxic T-lymphocyte-associated antigen-4, was the first agent to improve survival of patients with advanced melanoma in a randomised, controlled phase 3 trial. We used data from an expanded access programme (EAP) at Italian centres to evaluate the clinical activity and safety profile of ipilimumab 10 mg/kg in patients with advanced melanoma in a setting more similar to that of daily practice. Methods Data were collected from patients enrolled in an ipilimumab EAP across eight participating Italian centres. As per the EAP protocol, patients had life-threatening, unresectable stage III/IV melanoma, had failed or did not tolerate previous treatments and had no other therapeutic option available. Treatment comprised ipilimumab 10 mg/kg every 3 weeks for a total of four doses. If physicians believed patients would continue to derive benefit from ipilimumab treatment, maintenance therapy with ipilimumab 10 mg/kg was provided every 12 weeks. Tumour responses were assessed every 12 weeks using modified World Health Organization criteria and safety continuously monitored. Results Seventy-four pretreated patients with advanced melanoma were treated with ipilimumab 10 mg/kg. Of these, 9 (13.0%) had an objective response, comprising 3 patients with a complete response and 6 with a partial response. Median overall survival was 7.0 months (95% confidence interval, 5.3–8.7) and 16.6% of patients were alive after 3 years. Forty-five patients (60.8%) reported treatment-related adverse events of any grade, which were most commonly low-grade pruritus, pain, fever and diarrhoea. Grade 3 or 4 treatment-related AEs were reported in 8 patients (10.8%). Conclusions The clinical activity and safety profile of ipilimumab 10 mg/kg in the EAP was similar to that seen in previous clinical trials of ipilimumab in pretreated patient populations.

Surgery is the first option for treating melanoma regardless of stage at presentation. We surveyed a representative sample of hospitals to evaluate management and quality of surgical indications for melanoma in Italy. At analysis, hospitals were grouped into high- or low-volume centers, with the population median of 25 diagnoses serving as the cut-off. Surgery for primary melanoma was similar between hospital groups. More high-volume centers were organized to perform sentinel node biopsy (91 vs. 56%). There were no major differences between high- and low-volume centers concerning the surgical approach to stage III and IV disease.

Background Patients with advanced uveal melanoma have a poor prognosis and limited treatment options. Ipilimumab is approved for pre-treated adult patients with advanced melanoma. However, because previous clinical trials with ipilimumab have excluded patients with uveal melanoma, data in this patient population are limited. Patients and methods Pre-treated patients with advanced uveal melanoma received ipilimumab 3 mg/kg through an expanded access programme, every 3 weeks for four doses. Tumour assessments were conducted at baseline and after completion of treatment and patients were monitored throughout for adverse events. Results Among 82 assessable patients, 4 (5%) had an immune-related objective response and 24 (29%) had immune-related stable disease lasting ≥3 months for an immune-related disease control rate of 34%. With a median follow-up of 5.6 months, median overall survival (OS) was 6.0 months and median progression-free survival (PFS) was 3.6 months. The 1-year rates of OS and PFS were 31% and 11%, respectively. The safety profile of ipilimumab was similar to that in patients with cutaneous melanoma. Conclusions These data suggest ipilimumab 3 mg/kg is a feasible option in pre-treated patients with metastatic uveal melanoma. Evidence of disease control and a 1-year survival rate of 31% indicate the need for further investigation in randomised, controlled trials to determine the optimal timing and use of ipilimumab in this patient population.

Melanoma incidence and mortality rates are rising in Italy, indicating that more effective treatments are required both in the adjuvant and metastatic settings. We analyzed clinical practices in the adjuvant and metastatic settings by conducting a nationwide survey of clinicians responsible for managing melanoma treatment and follow-up in a representative sample of Italian hospitals. 95% of participating hospitals completed the panel of questions on adjuvant and metastatic treatment, making it likely that these results give a realistic picture of treatment and follow-up of melanoma patients in Italy. In low-volume hospitals (

A 37-year-old patient with liver metastases from gastric cancer was treated with a double adoptive immunotherapy regimen comprising tumor-infiltrating lymphocytes plus interleukin-2 and subsequently local-regional lymphokine-activated killer cells plus interleukin-2 because of an extremely high in vitro cytotoxic specific activity on established gastric cancer cell lines. The necrosis verified in the center of the hepatic metastasis would appear to demonstrate treatment efficacy, but no clinical response was seen. In vitro cytotoxicity data alone are insufficient to predict the clinical efficacy of adoptive immunotherapy.

Current systemic treatments for metastatic uveal melanoma (UM) have not improved overall survival (OS). The fully human anti-cytotoxic T-lymphocyte antigen-4 (CTLA-4) monoclonal antibody, ipilimumab, improved OS of patients with advanced cutaneous melanoma in a phase 3 trial; however, UM patients were excluded. The aim of this subanalysis, performed by the ipilimumab-ocular melanoma expanded access program (I-OMEAP) study group, was to assess the activity and safety of ipilimumab in patients with UM in a setting similar to daily clinical practice. Patients participating in a multicenter expanded access program (EAP) received induction treatment with ipilimumab 10 mg/kg. Maintenance doses were administered in patients who experienced clinical benefit or at physicians’ discretion. Tumor assessment was evaluated per modified World Health Organization criteria at baseline, Week 12, Week 24, and Week 36. Adverse events (AEs) and immune-related AEs (irAEs) were collected according to Common Terminology Criteria for Adverse Events version 3.0. Thirteen pretreated patients with metastatic UM were treated at 6 European institutions. All patients received at least one dose of ipilimumab. Overall, no objective responses were observed; however, two patients had stable disease (SD), with a third patient achieving SD after initial progressive disease. Median OS as of July 1, 2011, was 36 weeks (range 2–172+ weeks). No grade 3/4 AEs of non-immune origin were reported. Three patients (23%) experienced grade 3 irAEs (1 thrombocytopenia, 1 diarrhea, and 1 alanine/aspartate aminotransferase elevation) that resolved with steroid therapy. The results indicate UM is a potential indication for ipilimumab treatment that should be further investigated in clinical trials.

Summary Background Ipilimumab improves survival of patients with metastatic melanoma, many of whom develop brain metastases. Chemotherapy-induced release of tumour antigens might amplify ipilimumab's antitumour activity. We aimed to investigate the efficacy and safety of ipilimumab plus fotemustine in patients with metastatic melanoma with or without asymptomatic brain metastases. Methods In our open-label, single-arm phase 2 trial, we enrolled patients 18 years or older with measurable, locally advanced, unresectable stage III or stage IV melanoma between July 6, 2010, and April 14, 2011. Eligible patients had a life expectancy of 16 weeks or more and an Eastern Cooperative Oncology Group performance status of 1 or less, and could have received a maximum of one previous line of chemotherapy. Participants received induction treatment of 10 mg/kg intravenous ipilimumab every 3 weeks to a total of four doses, and 100 mg/m 2 intravenous fotemustine weekly for 3 weeks and then every 3 weeks from week 9 to week 24. Patients with a confirmed clinical response were eligible for maintenance treatment from week 24, with ipilimumab every 12 weeks and fotemustine every 3 weeks. The primary endpoint was the proportion of patients with immune-related disease control as established with immune-related response criteria. Analyses were done per protocol. This trial is registered with EudraCT, number 2010-019356-50, and with ClinicalTrials.gov, number NCT01654692. Findings 86 patients were eligible for treatment, of whom 20 had asymptomatic brain metastases at baseline. 40 patients in the study population achieved disease control (46·5%, 95% CI 35·7–57·6), as did ten with brain metastases (50·0%, 27·2–72·8). 47 patients (55%) had grade 3 or 4 treatment-related adverse events, of which the most common was myelotoxicity (thrombocytopenia in 21 [24%] patients and neutropenia in 16 [19%]). The most common grade 3 or 4 immune-related adverse events were hepatic: 21 patients (24%) had grade 3 or 4 increases in concentrations of alanine aminotransferase or aspartate aminotransferase. Interpretation The combination of ipilimumab plus fotemustine has clinical activity in patients with metastatic melanoma, including those with brain metastases. Funding Bristol-Myers Squibb.

Non-small cell lung cancer (NSCLC) is associated with IL-2-dependent cell-mediated immunodeficiency. As IL-2 is the main lymphocyte growth factor, a phase III randomized multicenter trial was conducted to evaluate the impact of subcutaneous low-dose IL-2 added to standard chemotherapy (CT) on overall survival (OS) in advanced NSCLC patients. Patients (n=241) with histologically confirmed stage IIIb or IV non-operable NSCLC underwent stratified randomization on the basis of center, ECOG PS, stage of disease and percentage of weight loss. Patients received gemcitabine (1000 mg/m2) on days 1 and 8 + cisplatin (100 mg/m2) on day 2 every 21 days for a maximum of 6 cycles [chemotherapy (CT) arm]. In the CT+IL-2 arm, patients also received low-dose subcutaneous IL-2 3,000,000 IU/die on days 3-5, 9-11, 15-17. The study had 90% power to detect a 20% absolute increase in 1-year OS with 118 patients/arm. An overall response (OR) rate of 12.8% (14% in the CT+IL-2 arm and 11.4% in CT arm) was observed. Stable disease was 70 and 66.7%, and progressive disease 16 and 21.8% in the CT+IL-2 and CT arms, respectively. No differences in response were found in any subgroup analysis. At a median follow-up of 32 months, 1-year OS was 45% for the CT+IL-2 arm vs. 51% for the CT arm (p=0.456 log-rank). Median progression-free survival was 6.6 months in the CT+IL-2 arm vs. 6.9 months in the CT arm (p=0.573, log-rank). A higher number of grade 4 toxicities were reported with CT+IL-2. The most common grade ≥3 adverse events were gastrointestinal toxicity (mainly nausea and diarrhea) and myelosuppression. No relevant differences in clinical outcome were observed from the addition of IL-2 to CT. Future studies investigating the role of T-regulators in chemoimmunotherapeutic regimens could be performed.

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer. © 2011 Fox et al; licensee BioMed Central Ltd.

Previous studies have shown that tumor endothelial markers (TEMs 1-9) are up modulated in immunosuppressive, pro-angiogenic dendritic cells (DCs) found in tumor microenvironments. We recently reported that monocyte-derived DCs used for vaccination trials may accumulate high levels of TEM8 gene transcripts. Here, we investigate whether TEM8 expression in DC preparations represents a specific tumor-associated change of potential clinical relevance. TEM8 expression at the mRNA and protein level was evaluated by quantitative real-time RT-PCR and cytofluorimetric analysis in human clinical grade DCs utilized for the therapeutic vaccination of 17 advanced cancer patients (13 melanoma and 4 renal cell carcinoma). The analyses revealed that DCs from patients markedly differ in their ability to up-modulate TEM8. Indeed, mDCs from eight non-progressing patients [median overall survival (OS) = 32 months, all positive to the delayed-type hypersensitivity test (DTH)], had similar TEM8 mRNA expression levels [mDCs vs. immature iDCs; mean fold increase (mfi) = 1.97] to those found in healthy donors (mfi = 2.7). Conversely, mDCs from nine progressing patients (OS < 5 months, all but one with negative DTH) showed an increase in TEM8 mRNA levels (mfi = 12.88, p = 0.0018). The present observations suggest that TEM8 expression levels in DC-based therapeutic vaccines would allow the selection of a subgroup of patients who are most likely to benefit from therapeutic vaccination.

Biological response modifiers (BRMs) have greatly modified the immunotherapy of tumors. Interleukin-2 (IL-2) has brought about metastasis regression in some cases of malignant tumors, however, when given systemically, it results in high toxicity. More recently, the subcutaneous administration of IL-2 (combined with alpha-interferon, alpha-IFN) seems to be capable of offering the same chances of therapeutic response, but this time with a lower level of toxicity. The Authors report an evaluation of toxicity in 22 patients treated with a combination of IL-2 + alpha-IFN i.m. with or without chemotherapy. The side-effects present in the majority of cases were: fever, diarrhea and asthenia. Approximately 50% of the patients had nausea/vomiting, mucositis, skin rashes, and slight leukopenia. The following side-effects were noted to a much lesser degree, thrombocytopenia, alterations in hepatic and dizziness and cystitis. Only one patient reached 4th degree toxicity, with mucositis, asthenia and skin rash. All the other patients received the treatment without suspensions for toxicity. Biological evaluations will enable us to determine in the future, the cases which can benefit from therapeutic intensification and thus it would seem opportune at this time to use therapy with acceptable toxicity.

The Authors report their experiences on the treatment of 13 consecutive cases of gastro-intestinal carcinoid tumors observed over the last 11 years. The primary sites were as follows: intestine (5 cases), appendix (3 cases), colon (1 case) and peritoneum (4 cases); only 3 patients presented systemic signs. Ten patients in advanced phase were treated with a chemotherapeutic regimen containing 5-fluorouracil (5-Fu) and streptozotocin (STZ). One case was excluded from the study because of a concomitant gastric carcinoma. Of the 9 evaluable patients, two achieved partial remission (22%) with a duration of 18+ and 66 months respectively; 4 (44.5%) had stable disease for periods ranging from 7 to 40 months and 3 cases progressed. Severe toxicity (thrombocytopenia and diarrhea) occurred in 2 cases and disappeared with the suspension of therapy. The systemic signs disappeared with treatment and did not appear in 2 cases out of 3. The prospective of the employment of new drugs such as alpha-interferon and, above all, somatostatin provides hope that this uncommon disease may have an improved response rate to treatment in the future.

Background We present our experience of therapeutic vaccination using dendritic cells (DC) pulsed with autologous tumor antigens in patients with advanced melanoma. Methods Twenty-one pretreated advanced melanoma patients were vaccinated with autologous DC pulsed with 100 μg/ml of autologous-tumor-lysate (ATL) or – homogenate (ATH) and 50 μg/ml of keyhole limpet hemocyanin (KLH). The first 8 patients were treated subcutaneously or intradermally with immature-DC (iDC) (range 4.5 – 82 × 106) and the remaining 13 intradermally with in vitro matured DC (mDC) (range 1.2–26 × 106). Subcutaneous interleukin-2 (3 × 106 IU) was administered from days 3 to 7 of each treatment cycle. Results Three of the 8 iDC patients obtained stabilizations (SD), each of 6 months' duration. The 13 mDC patients showed 1 complete response (8 months), 1 partial response (3 months), 2 mixed responses (6 and 12 months) and 3 SD (9, 7+, and 3+ months). Overall responses (OR) were observed in 4/21 (19%) patients, or 4/13 (30.7%) considering mDC treatment only. 10/21 (47.6%) patients showed non progressive disease (NPD), with 7/13 (53.8%) cases of NPD for mDC-treated patients. No major toxicities were observed. The positive delayed-type hypersensitivity (DTH) test to ATL/ATH and/or KLH correlated with increased overall survival (OS). Median OS was 24 months (range 3 – 45) for the 10 DTH-positive (1 iDC and 9 mDC) and 5 months (range 3–14) for the 11 DTH-negative patients (P < 0.001). The in vitro evaluation of gamma IFN-secreting T-cells in 10 patients showed good correlation with both DTH (75%) and clinical outcome (70%). Conclusion Vaccination using DC pulsed with ATL/ATH and KLH in advanced melanoma patients is well tolerated and can induce a clinical response, especially when mDC are used. Successful immunization, verified by positive DTH, leads to longer survival.

Adoptive immunotherapy with tumor infiltrating lymphocytes (TIL) and interleukin (IL)-2 is reasonably effective in the treatment of patients with advanced melanoma. However, theoretically it should be of greater benefit as adjuvant therapy, especially in high-risk stages (resected stages III and IV). In a preliminary study, 25 patients (aged 23-72 years) with stage III-IV melanoma who underwent resection of metachronous metastases were reinfused with TIL cultivated and expanded in vitro with IL-2 from surgically removed metastases. IL-2 (starting dose 12 x 10 6 IU/m 2 ) was co-administered as a continuous infusion according to West's scheme. A total of 8/22 (36.3%) evaluable patients were disease-free (DF) at a median follow-up of 5 years. DF survival (DFS) and overall survival (OS) rates were 44% and 37%, respectively, at 2 years, and 52% and 45% at 3 years. The CNS was the only site of disease recurrence in 57% of patients who relapsed. DF patients received a higher median dose of IL-2 than those who progressed (total dose 110 x 10 6 versus 86 x 10 6 IU/m 2 , respectively). The progressive reduction in IL-2 dosage allowed all patients to complete treatment without permanent grade 4 toxicity. Analysis of tumor immunosuppression factors in lymphocytes inside the tumor (TCR ζ and e chains, p56 lck , FAS, and FAS-ligand) confirmed that the immunologic potential of TIL, depressed at the time of metastasectomy, was significantly restored after in vitro culture with IL-2. Adoptive immunotherapy with TIL and IL-2 could improve DFS and OS, although further work is required to determine its role in the treatment of patients with high-risk melanoma.

Purpose Clinical studies of ipilimumab have shown improved survival and durable tumour responses across a spectrum of doses and schedules. Here, we evaluate the use of ipilimumab 10 mg/kg outside of clinical trials, in a setting similar to daily practice. Methods Ipilimumab was available upon physician request for patients (pts) aged ≥16 years with unresectable stage III/stage IV melanoma who had either failed systemic therapy or were intolerant to ≥1 systemic treatment and for whom no other therapeutic option was available. Induction therapy with ipilimumab 10 mg/kg was given on Week (wk) 1, 4, 7 and 10 with tumour assessments conducted at baseline, wk 12 and q 12 wks thereafter using modified World Health Organization criteria. Maintenance therapy with ipilimumab was available q 12 wks from wk 24 for pts with evidence of clinical benefit. Pts were monitored for adverse events (AEs) using Common Terminology Criteria for Adverse Events v.3.0. Results In total, 74 pts, including 11 with brain metastasis, received ≥1 dose of ipilimumab and were eligible for analysis. Of these, 43 (58.1%) completed the induction phase and 26 (35.1%) received maintenance therapy (median of 2 cycles; range: 1–13). The disease control rate was 32%, including 3 pts (4%) with a complete response, 6 (9%) with a partial response, and 13 (19%) with stable disease. Median progression-free survival (PFS) and overall survival (OS) was 3 months (mo) (95% CI: 2.3–3.7) and 7 mo (95% CI: 5.3–8.7), respectively. For pts with brain metastases, PFS and OS were 3 mo (95% CI: 2.4–3.6) and 4 mo (95% CI: 2.4–5.6). The 3-yr survival rate was 16.6%, with 11 long survivors pts (>3 yrs). Overall, 45 pts (61%) reported an AE of any grade; most commonly pruritis and diarrhoea. Eight grade 3/4 AEs were reported, including diarrhoea (n = 2), pain (including epigastric; n = 3) and one case each of fever, increased liver enzymes and pancytopenia. Most AEs were manageable through adherence to protocol-specific guidelines. Conclusions Ipilimumab is a feasible treatment option for pts who progressed on, or were unable to tolerate previous therapies, with approximately one-third of pts achieving disease control and a long-term survival benefit. Disclosure M. Maio: Advisory board and honoraria from: BMS and Roche. P. Queirolo: Paola Queirolo served on Advisory Board of Bristol Myers Squibb, Roche-Genentech, GSK, MSD and received honoraria from Brystol Myers Squibb and Roche-Genentech. A. Testori: I have a consultant or advisory relationship to disclose ( BMS, GSK, AMGEM advisory boards); I have honoraria to disclose (from above mentioned advisory boards); I have other remuneration to discloure ( travel expenses from oncovision and Igea). P.A. Ascierto: Consultant or advisory relationship for: Brystol Myers - Squibb Merck Roche GSK Amgen. Honoraria to disclose from BMS, Merck, Roche. V. Chiarion Sileni: Advisory Board and/or Honoraria from: BMS MSD Shering & Plough Roche GSK. All other authors have declared no conflicts of interest.

TPS9105 Background: Preliminary evidence in the literature suggests that interferon receptor subunit IFNAR1 is down-regulated in melanoma cells harboring a mutated active BRAF. This possibility is ...

e20028 Background: Adjuvant high-dose interferon (HDI) treatment of patients (pts) with malignant melanoma (MM) consists of 4 weeks intravenous (IV) induction with 20 MU/m² interferon (IFN) alpha 2b followed by 11 months of 10 Mio IU/m² IFN subcutaneously (sc). It is unclear whether both parts of the regimen are mandatory for the efficacy of HDI treatment. The adjuvant phase III trials DeCOG MM-ADJ-5 and the Italian Melanoma Intergroup (IMI) trial evaluated intermittent IV HDI (iHDI) regimens as compared to standard HDI. In both trials, the experimental arm consisted of iHDI for 3 or 4 cycles, respectively. To gain more insight into the role of iHDI in the adjuvant setting we performed a pooled analysis of both trials. Methods: Based on the intent-to-treat populations, the German DeCOG trial contributed data on 627 MM pts. with stage III (AJCC 2002) resected intransit or lymph node metastasis. From the Italian IMI trial 330 pts. with stage III regional lymph node metastasis excluding intransit metastasis were evaluable. We performed a combined analysis with respect to relapse free survival (RFS) and overall survival (OS), and toxicity using Cox regression models. Results: Median follow-up was 4.6 years in the DeCOG trial and 7.2 years in the IMI trial. There were 320 relapses in the DeCOG trial (51%) and 179 relapses in the IMI trial (54%) resulting in a combined hazard ratio (HR) for RFS of 1.11 (95% CI 0.93; 1.33) comparing iHDI vs.conventional HDI. OS was similar for iHDI vs. conventional HDI (HR 1.04; 95% CI 0.84; 1.29). There was no significant heterogeneity comparing different substages (IIIA vs. IIIB vs. IIIC) or ulcerated versus non-ulcerated primary tumors. In both studies more patients discontinued treatment due to toxicity or impairment of quality of life with conventional HDI as compared to iHDI (DeCOG: 31.0% vs. 16.7%; IMI: 31.2% vs. 23.8%). Conclusions: The pooled analysis of the two pulsed adjuvant HDI treatments showed no significant differences for RFS or OS as compared to conventional HDI. There is a favorable safety profile and less overall toxicity in the pulsed regimens.

Background In cancer immunotherapy, dendritic cells (DCs) play a fundamental role in the dialog between innate and adaptive immune response, but several immunosuppressive mechanisms remain to be overcome. For example, a high number of CD4+CD25++Foxp3+ regulatory T-cells (Foxp3+Tregs) have been observed in the peripheral blood and tumor microenvironment of cancer patients. On the basis of this, we conducted a study on DC-based vaccination in advanced melanoma, adding low-dose temozolomide to obtain lymphodepletion. Methods Twenty-one patients were entered onto our vaccination protocol using autologous DCs pulsed with autologous tumor lysate and keyhole limpet hemocyanin. Patients received low-dose temozolomide before vaccination and 5 days of low-dose interleukin-2 (IL-2) after vaccination. Circulating Foxp3+Tregs were evaluated before and after temozolomide, and after IL-2. Results Among the 17 evaluable patients we observed 1 partial response (PR), 6 stable disease (SD) and 10 progressive disease (PD). The disease control rate (PR+SD = DCR) was 41% and median overall survival was 10 months. Temozolomide reduced circulating Foxp3+Treg cells in all patients. A statistically significant reduction of 60% was observed in Foxp3+Tregs after the first cycle, whereas the absolute lymphocyte count decreased by only 14%. Conversely, IL-2 increased Foxp3+Treg cell count by 75.4%. Of note the effect of this cytokine, albeit not statistically significant, on the DCR subgroup led to a further 33.8% reduction in Foxp3+Treg cells. Conclusions Our results suggest that the combined immunological therapy, at least as far as the DCR subgroup is concerned, effectively reduced the number of Foxp3+Treg cells, which exerted a blunting effect on the growth-stimulating effect of IL-2. However, this regimen, with its current modality, would not seem to be capable of improving clinical outcome.

Background and purpose MM is a chemoresistant cancer with poor prognosis. Further progress is likely to come from novel targeted antiBRAF therapy, available for about 50% of pts, and from immunotherapeutic agents such as the mAb ipilimumab, at present available only in some countries. Preclinical and clinical experiences support the concept that continuous exposure to an alkilating agent can effectively deplete cells of the DNA repair enzyme O6-methylguanine DNA methyltransferare, the primary mechanism of tumor resistance to chemotherapeutic agents like nitrosurea analogs. Our study was finalized to verify this hypothesis using a sequential combination of low dose chemo-modulating TMZ and FM. Primary endpoints were safety and tumor response evaluation. Methods 53 consecutive MM pts were enrolled in the study. The majority of them (80%) were enrolled before the targeted therapy. The main characteristics included: median age 56 years (21-79); ECOG PS 1 (0–2); number of disease sites: 1 in 30%, 2 in 27%, >2 in 43%; M status: M1a 9%, M1b 21%, M1c 70% with 5 pts having brain metastases. The following schedule was used: oral TMZ 100 mg/m2 d 1 and 2; FM iv 100 mg/m2 d 2, 4 h after TMZ. The regimen was repeated every 3 weeks for a maximum total of 9 cycles. Tumour assessments were conducted at baseline and then every 3 cycles. Results 52 pts are evaluable for toxicity and 51 for clinical assessment (1 withdrew consent prior to starting treatment; 2 early). The median number of treatment cycles administered was 7 (range 2-9). There were 13 (25%) responses (1 CR and 9 PRs) with a median duration of 7 months, and 12 (23%) stable disease. Median progression-free survival was 6 months and median overall survival 11+ months (range 2-35+ months). Drug-related toxicities ≥ grade 3 included thrombocytopenia (10%), neutropenia (6%), anemia (2%), and hepatopathy (2%). Approximately 75% of pts were treated without dose reduction. Two patients (4%) discontinued therapy because of toxicities. Conclusions sequential low dose TMZ and FM demonstrated a high activity in our patient population with an acceptable toxicity. This schedule could therefore represent a good alternative for patients not eligible for targeted therapy or in whom previous targeted therapies failed. The study of the correlation between MGMT level and clinical outcomes is ongoing. Disclosure All authors have declared no conflicts of interest.

8529 Background: Patients (pts) with metastatic melanoma (MM) often develop treatment-resistant brain metastases (mets). Treatment includes fotemustine (FTM), which crosses the blood-brain barrier. Ipilimumab (ipi) has shown activity in pts with MM and asymptomatic brain mets (Heller et al. ASCO 2011; abs 8581). In the phase II NIBIT-M1 trial, MM pts with asymptomatic brain mets were eligible for treatment with ipi plus FTM. Here, data from this pt subset are reported. Methods: Eligible pts received induction therapy with ipi 10 mg/kg every 3 wks (Q3W) x4 and FTM 100 mg/m2 weekly for 3 wks, followed by ipi Q12W from Week (W) 24 and FTM Q3W from W9. The primary objective was the immune-related (ir) disease control rate (irDCR: complete/partial response [CR/PR] or stable disease [SD] using the ir response criteria). Secondary objectives included ir objective response rate (ORR) and progression-free survival (PFS); overall survival (OS), and safety. Tumor assessments were performed Q8W from W12 to W36 and Q12W thereafter. Results: Among 86 enrolled pts, 20 had brain mets. Of these, 7 had prior whole brain radiotherapy (n=4) or radiosurgery (n=3). As of December 2011, the irDCR was 50% (10/20; 95% CI, 27.2–72.8%) with an irORR of 40% (95% CI, 19.1–63.9%: 2 CRs and 6 PRs). Pts with irDC also had stability/reduction (n=5) or disappearance (n=5) of brain mets. Among pts with progressive disease, all but one had progression in the brain. With median follow-up of 8.3 months (range: 0.4–16.9), median irPFS was 4.6 months (95% CI, 0.7–12.3). The 1-year OS rate was 52.9% (95% CI, 26.6–79.2); median OS was not reached. Induction with ipi and FTM was completed by 55% and 85% pts, respectively. Grade 3/4 drug-related adverse events (AEs) occurred in 60% pts; most commonly myelotoxicity (50%), increased ALT/AST (5%) and gastrointestinal (5%). AEs were generally manageable and reversible per protocol guidance. CNS AEs of any grade (i.e., haemorrhage, headache and seizure) occurred in 25% pts (grade 3/4 in 2 pts) and were attributed to disease progression. Conclusions: The combination of ipi plus FTM is active and safe in pts with MM and brain mets, regardless of prior treatment, and will be further explored in the phase III NIBIT-M2 trial.

Twenty-one patients with advanced, pretreated colorectal cancer in disease progression were entered in a phase II study to investigate the use of 5-fluorouracil (5FU) + leucovorin with subcutaneous Interleukin-2 + alpha interferon (alpha-IFN). Eighteen of these patients were evaluable for response to treatment: 1 partial response (PR) (duration 8 months), 9 stable disease (SD) (median duration of 6.5 months, range 2-15) and 8 progressive disease (PD). The PR patient survived for 15 months, the SD patients for a median of 11 months and 8 months for PD patients. Toxicity evaluated in the 21 patients reached grade 4 for mucositis in two cases. Grade 3 toxicity was observed more frequently for fever (52.3%) and diarrhea (33.3%) and was most probably the result of the combined side-effect of chemotherapy and the biological response modifiers (BRMs). Treatment was, for the most part, carried out on an out-patient basis as originally planned. In 15 patients tests were carried out to verify whether any immuno-activation had taken place. Significant increases were found during the course of therapy regarding cluster of differentiation activation (HLA-DR, CD71, CS25). Different curves were observed during the course of treatment with respect to the CD8 value, which proved higher in SD patients than in PD patients. Our study would seem to suggest that the addition of BRMs to 5FU + leucovorin could increase survival. The next step, however, must be to determine lower doses of IL-2 for subcutaneous administration in order to reduce toxicity but maintain the same immunostimulation.

8506 Background: High dose IFNalfa2b, according to the ECOG 1684 schedule, is the only approved adjuvant treatment for stage III melanoma. The risk/benefit profile has limited its use, while effica...

e13029 Background: Cytolytic T lymphocytes (CTL) able to kill tumor cells are efficiently induced by Dendritic Cells (DC); however, immunosuppressive factors, as CD4+CD25+FoxP3+ T lymphocytes (T-re...

We reviewed the clinical results of a dendritic cell-based phase II clinical vaccine trial in stage IV melanoma and analyzed a patient subgroup treated with standard therapies after stopping vaccination. From 2003 to 2009, 24 metastatic melanoma patients were treated with mature dendritic cells pulsed with autologous tumor lysate and keyhole limpet hemocyanin and low-dose interleukin-2. Overall response (OR) to vaccination was 37.5% with a clinical benefit of 54.1%. All 14 responders showed delayed type hypersensitivity positivity. Median overall survival (OS) was 15 months (95% CI, 8–33). Eleven patients underwent other treatments (3 surgery, 2 biotherapy, 2 radiotherapy, 2 chemotherapy, and 4 biochemotherapy) after stopping vaccination. Of these, 2 patients had a complete response and 5 a partial response, with an OR of 63.6%. Median OS was 34 months (range 16–61). Our results suggest that therapeutic DC vaccination could favor clinical response in patients after more than one line of therapy.

e19025 Background: MUP are estimated to comprise between 3.7% and 6% of all melanomas. It represents an interesting phenomenon not clearly explained yet and also its natural history is controversia...