Clinical experience with ipilimumab 3 mg/kg: real-world efficacy and safety data from an expanded access programme cohort.

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.

Tremelimumab for patients with chemotherapy-resistant advanced malignant mesothelioma: an open-label, single-arm, phase 2 trial.

BACKGROUND: Monoclonal antibodies to cytotoxic T-lymphocyte antigen 4 (CTLA4) have therapeutic activity in different tumour types. We aimed to investigate the efficacy, safety, and immunological activity of the anti-CTLA4 monoclonal antibody, tremelimumab, in advanced malignant mesothelioma. METHODS: In our open-label, single-arm, phase 2 study, we enrolled patients aged 18 years or older with measurable, unresectable malignant mesothelioma and progressive disease after a first-line platinum-based regimen. Eligible patients had to have a life expectancy of 3 months or more, an Eastern Cooperative Oncology Group performance status of 2 or less, and no history of autoimmune disease. Patients received tremelimumab 15 mg/kg intravenously once every 90 days until progressive disease or severe toxicity. The primary endpoint was the proportion of patients who achieved an objective response (complete or partial response), with a target response rate of 17% according to the modified Response Evaluation Criteria in Solid Tumors (RECIST) for pleural malignant mesothelioma or standard RECIST 1.0 for peritoneal malignant mesothelioma. Analyses were done according to intention to treat. This trial is registered with EudraCT, number 2008-005171-95, and ClinicalTrials.gov, number NCT01649024. FINDINGS: Between May 27, 2009, and Jan 10, 2012, we enrolled 29 patients. All patients received at least one dose of tremelimumab (median two doses, range one to nine). No patients had a complete response and two patients (7%) had a durable partial response (one lasting 6 months and one lasting 18 months); one partial response occurred after initial progressive disease. Thus, the study did not reach its primary endpoint. However, we noted disease control in nine (31%) patients and a median progression-free survival of 6·2 months (95% CI 1·3-11·1) and a median overall survival of 10·7 months (0·0-21·9). 27 patients (93%) had at least one grade 1-2 treatment-emergent adverse event (mainly cutaneous rash, pruritus, colitis, or diarrhoea), and four patients (14%) had at least one grade 3-4 treatment-emergent adverse event (two gastrointestinal, one neurological, two hepatic, and one pancreatic). INTERPRETATION: Although the effect size was small in our phase 2 trial, tremelimumab seemed to have encouraging clinical activity and an acceptable safety and tolerability profile in previously treated patients with advanced malignant mesothelioma. FUNDING: Associazione Italiana per la Ricerca sul Cancro, Istituto Toscano Tumori, Pfizer, and Fondazione Buzzi Unicem.

Long-term survival and immunological parameters in metastatic melanoma patients who responded to ipilimumab 10 mg/kg within an expanded access programme.

BACKGROUND: Ipilimumab can result in durable clinical responses among patients with advanced melanoma. However, no predictive marker of clinical activity has yet been identified. We provide preliminary data describing the correlation between immunological parameters and response/survival among patients with advanced melanoma who received ipilimumab 10 mg/kg in an expanded access programme. METHODS: Patients received ipilimumab 10 mg/kg every 3 weeks (Q3W) for four doses (induction) and Q12W from week 24 (W24) as maintenance therapy. Tumor assessments were conducted Q12W. Expression of inducible T cell costimulator (ICOS) on CD4(+) and CD8(+) T cells was assessed at baseline, W7, W12 and W24, and the ratio between absolute neutrophils (N) and lymphocytes (L) determined at baseline, W4, W7 and W10. RESULTS: Median overall survival among 27 patients was 9.6 months (95 % CI 3.2-16.1), with 3- and 4-year survival rates of 20.4 %. Five patients survived >4 years. Patients with an increase in the number of circulating ICOS(+) T cells at W7 were more likely to experience disease control and have improved survival. An N/L ratio below the median at W7 and W10 was also associated with better survival compared with an N/L ratio above the median. CONCLUSIONS: Ipilimumab can induce long-term survival benefits in heavily pretreated patients with metastatic melanoma. Changes in the number of circulating ICOS(+) T cells or N/L ratio during ipilimumab treatment may represent early markers of response. However, given the limited sample size, further investigation is required.

Ipilimumab in pretreated patients with metastatic uveal melanoma: safety and clinical efficacy.

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.

Ipilimumab experience in heavily pretreated patients with melanoma in an expanded access program at the University Hospital of Siena (Italy).

AIM OF STUDY: To evaluate the feasibility of ipilimumab treatment for metastatic melanoma outside the boundaries of clinical trials, in a setting similar to that of daily practice. METHODS: Ipilimumab was available upon physician request in the Expanded Access Programme for patients with life-threatening, unresectable stage III/IV melanoma who failed or did not tolerate previous treatments and for whom no therapeutic option was available. Induction treatment with ipilimumab 10 mg/kg was administered intravenously every 3 weeks, for a total of 4 doses, with maintenance doses every 12 weeks based on physicians' discretion and clinical judgment. Tumors were assessed at baseline, Week 12, and every 12 weeks thereafter per mWHO response criteria, and clinical response was scored as complete response (CR), partial response (PR), stable disease (SD), or progressive disease. Durable disease control (DC) was defined as SD at least 24 weeks from the first dose, CR, or PR. RESULTS: Disease control rate at 24 and 60 weeks was 29.6% and 15%, respectively. Median overall survival at a median follow-up of 8.5 months was 9 months. The 1- and 2-year survival rates were 34.8% and 23.5%, respectively. Changes in lymphocyte count slope and absolute number during ipilimumab treatment appear to correlate with clinical response and survival, respectively. Adverse events were predominantly immune related, manageable, and generally reversible. One patient died from pancytopenia, considered possibly treatment related. CONCLUSION: Ipilimumab was a feasible treatment for malignant melanoma in heavily pretreated, progressing patients. A sizeable proportion of patients experienced durable DC, including benefits to long-term survival.

Tremelimumab plus durvalumab retreatment and 4-year outcomes in patients with mesothelioma: a follow-up of the open label, non-randomised, phase 2 NIBIT-MESO-1 study.

BACKGROUND: The NIBIT-MESO-1 study demonstrated the efficacy and safety of tremelimumab combined with durvalumab in patient with unresectable mesothelioma followed up for a median of 52 months [IQR 49-53]. Here, we report 4-year survival and outcomes after retreatment, and the role of tumour mutational burden (TMB) in identifying patients who might have a better outcome in response to combined therapy. METHODS: NIBIT-MESO-1 was an open-label, non-randomised, phase 2 trial of patients with unresectable pleural or peritoneal mesothelioma who received intravenous tremelimumab (1 mg/kg bodyweight) and durvalumab (20 mg/kg bodyweight) every 4 weeks for four doses, followed by maintenance intravenous durvalumab at the same dose and schedule for nine doses. In this follow-up study, patients with disease progression following initial clinical benefit-ie, a partial repsonse or stable disease-were eligible for retreatment and with the same doses and schedules for tremelimumab and durvalumab as used in the NIBIT-MESO-1 trial. The primary endpoint, immune-related objective response rate, was evaluated per immune-related modified Response Evaluation Criteria in Solid Tumors (RECIST) or immune-related RECIST 1.1 criteria for patients with pleural or peritoneal malignant mesothelioma, respectively. Key secondary endpoints were overall survival and safety, and TMB was also evaluated post hoc in patients who had tumour tissue available before treatment. The intention-to-treat population was used for analysis of all efficacy endpoints. This study is registered with ClinicalTrials.gov, number NCT02588131. FINDINGS: 40 patients were enrolled in the NIBIT-MESO-1 study between Oct 30, 2015, and Oct 12, 2016. At data cut-off, April 30, 2020, five (13%) of 40 patients were alive, and 35 (88%) patients had died of progressive disease. At a median follow-up of 52 months (IQR 49-53), median overall survival was 16·5 months (95% CI 13·7-19·2). Survival was 20% (eight of 40 patients) at 36 months and 15% (six of 40 patients) and 48 months. 17 (43%) of 40 patients met the criteria for enrolment in the retreatment study and were retreated with at least one dose of tremelimumab and durvalumab. No immune-related objective responses were observed in the 17 retreated patients. Seven (41%) of 17 patients achieved immune-related stable disease. From the start of retreatment to a median follow-up of 24 months (22·0-25·0), median overall survival was 12·5 months (95% CI 0·0-25·8), and survival at 12 months was 52·9%, at 18 months was 35·3%, and at 24 months was 23·5%. There were no grade 3-4 immune-related adverse events in the retreatment cohort. In a post-hoc analysis of 28 patients for whom tumour tissue before treatment was available, patients with a TMB higher than the median value of 8·3 mutations per Mb had a higher median overall survival compared with patients with TMB below the median value, but this difference was non-significant. Moreover, when patients were additionally stratified for ICI retreatment (n=13), there was a significant difference in survival between those with a TMB higher than the median of 8·3 mutations per Mb and those with TMB lower than the median in the retreated cohort (41·3 months vs 17·4 months; p=0·02). INTERPRETATION: Tremelimumab combined with durvalumab was associated with long-term survival in patients with mesothelioma. Retreatment was safe and resulted in clinically meaningful outcomes, thus suggesting its potential application in the clinical practice of mesothalioma patients. FUNDING: NIBIT Foundation, Fondazione AIRC, AstraZeneca.

Epigenetically regulated clonal heritability of CTA expression profiles in human melanoma.

The intratumoral heterogeneity of cancer testis antigens (CTA) expression, which is driven by promoter methylation status, may hamper the effectiveness of CTA-directed vaccination of melanoma patients. Thus, we investigated whether the intratumoral heterogeneity of CTA expression is inherited at cellular level, or evolves throughout cellular replication, leading to a phenotypically unstable tumor cell population with reduced immunogenicity and/or able to escape immune control. Utilizing a previously characterized ex vivo clonal model of intratumoral heterogeneity of CTA expression in melanoma, Mel 313 MAGE-A3-low clone 5 (clone 5(M3-low)) and MAGE-A3-high clone 14 (clone 14(M3-high)) were sub-cloned and analyzed for CTA profile. Molecular assays demonstrated that levels of MAGE-A3 expression were highly conserved among generated sub-clones, as compared to parental clones. A similar behavior was identified for an extensive panel of other CTA investigated. Inherited levels of MAGE-A3 expression correlated with the extent of promoter methylation among clone 5(M3-low) and clone 14(M3-high) sub-clones analyzed. Treatment of clone 5(M3-low) with a DNA hypomethylating agent (DHA) resulted in an up-regulated expression of MAGE-A3, which was inherited at single cell level, being still detectable at day 60 in its sub-clones. Bisulfite sequencing demonstrated that also MAGE-A3 promoter methylation status was inherited among sub-clones generated from DHA-treated clone 5(M3-low) and strictly correlated with MAGE-A3 expression levels in investigated sub-clones. Similar results were obtained for additional CTA studied. Altogether our findings demonstrate that constitutive and DHA-modified CTA profiles of melanoma cells are clonally inherited throughout cellular replications, thus providing relevant insights to improve the effectiveness of CTA-based immunotherapy.

Immune Checkpoint Inhibitors for Cancer Therapy in the COVID-19 Era.

The potential immune intersection between COVID-19 disease and cancer therapy raises important practical clinical questions and highlights multiple scientific gaps to be filled. Among available therapeutic approaches to be considered, immune checkpoint inhibitors (ICI) seem to require major attention as they may act at the crossroads between cancer treatment and COVID-19 disease, due to their profound immunomodulatory activity. On the basis of available literature evidence, we suggest guidance to consider for treating physicians, and propose areas of clinical and preclinical investigation. Comprehensively, although with the necessary caution, ICI therapy seems to remain a suitable therapeutic option for patients with cancer during the COVID-19 pandemic.

Therapeutic efficacy of ipilimumab, an anti-CTLA-4 monoclonal antibody, in patients with metastatic melanoma unresponsive to prior systemic treatments: clinical and immunological evidence from three patient cases.

The management of unresectable metastatic melanoma is a major clinical challenge because of the lack of reliably effective systemic therapies. Blocking cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) has recently been proposed as a strategy to enhance cell-mediated immune responses to cancer, and clinical trials have demonstrated that anti-CTLA-4 therapy can produce durable outcomes with different response patterns than cytotoxic chemotherapy. We enrolled eight out of 155 patients with advanced melanoma in a multicentre phase II trial that evaluated the activity and tolerability of ipilimumab, a fully human, anti-CTLA-4 monoclonal antibody ( www.clinicaltrials.gov ; NCT00289627; CA184-008). Here we report our experience with three of these patients, who experienced progressive disease after a variety of previous therapies, including prior immunotherapies, and who achieved good outcomes with ipilimumab. One patient had a partial response ongoing at 17+ months on ipilimumab despite failure with four prior therapies, and the other two patients showed durable stable disease, both still ongoing at 17+ and 20+ months, respectively. The patient achieving a partial response experienced no side effects while receiving ipilimumab. The other two patients developed immune-related adverse events (irAEs) including rash (one case; grade 2) and diarrhoea (both cases; grades 1 and 2, respectively); the histopathology of colon biopsy samples from both was suggestive of colitis, with an abundant CD8+ T-cell infiltrate. Nausea, vomiting and acute pancreatitis were also observed in one patient. In addition, immunohistochemical findings of a dense CD8+, TIA1+ and granzyme B+ lymphoid infiltrate within a biopsied lesion provide indirect evidence of functional T-cell activation induced by treatment. These case reports highlight the potential for anti-CTLA-4-based therapy in previously treated patients with advanced melanoma. Moreover, because the patterns of response to ipilimumab differ from chemotherapy, we need to understand how and when patients may respond to treatment so that appropriate clinical decisions can be made.

Functional up-regulation of human leukocyte antigen class I antigens expression by 5-aza-2'-deoxycytidine in cutaneous melanoma: immunotherapeutic implications.

PURPOSE: To investigate the potential of the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-aza-CdR) to improve the effectiveness of immunotherapeutic approaches against melanocyte differentiation antigens. EXPERIMENTAL DESIGN: The effect of 5-aza-CdR on the constitutive expression of gp100 was investigated in 11 human melanoma cell lines by real-time reverse transcription-PCR and indirect immunofluorescence (IIF) analyses. 5-aza-CdR-mediated changes in the levels of expression of human leukocyte antigen (HLA) class I antigens and HLA-A2 allospecificity, intercellular adhesion molecule-1 (ICAM-1), and leukocyte-function-associated antigen-3 were investigated by IIF analysis on melanoma cells under study. The recognition of gp100-positive Mel 275 melanoma cells, treated or not with 5-aza-CdR, by HLA-A2-restricted gp100((209-217))-specific CTL was investigated by (51)Cr-release assays, IFN-gamma release and IFN-gamma ELISPOT assays. RESULTS: The constitutive expression of gp100 was not affected by 5-aza-CdR on all melanoma cells investigated. Compared with untreated cells, the exposure of Mel 275 melanoma cells to 5-aza-CdR significantly (P < 0.05) up-regulated their expression of HLA class I antigens and of ICAM-1. These phenotypic changes significantly (P < 0.05) increased the lysis of 5-aza-CdR-treated Mel 275 melanoma cells by gp100-specific CTL and increased their IFN-gamma release. 5-aza-CdR treatment of Mel 275 cells also induced a higher number of gp100-specific CTL to secrete IFN-gamma. CONCLUSIONS: Treatment with 5-aza-CdR improves the recognition of melanoma cells by gp100-specific CTL through the up-regulation of HLA class I antigens expression; ICAM-1 also contributes to this phenomenon. These findings highlight a broader range of therapeutic implications of 5-aza-CdR when used in association with active or adoptive immunotherapeutic approaches against a variety of melanoma-associated antigens.

Long-term follow up of metastatic melanoma patients treated with Thymosin alpha-1: investigating immune checkpoints synergy.

BACKGROUND: Immune checkpoint blockade antibodies (imAbs), such as the anti Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) ipilimumab (IPI) raised overall survival (OS) in metastatic melanoma (MM). Further, long-term OS is a crucial endpoint in MM. Thymosin alpha-1 (Tα1) with dacarbazine (DTIC) showed activity in a phase II trial and a compassionate use program (EAP). We report on long-term follow-up of patients treated with Tα1 to investigate the preconditioning role of Tα1 in imAbs-treated patients. METHODS: Records of patients with melanoma treated with Tα1 within a phase II trial and EAP program were reviewed comparing median OS among patients that sequentially received anti-CTLA-4 imAb and Tα1. Further, the effect of Tα1 on IPI long-term survivor patients was investigated. RESULTS: Among patients treated with Tα1, 21/61 patients received sequentially even anti CTLA-4 imAbs. Median OS at the data cut-off was 57.8 and 7.4 months in patients treated sequentially with anti-CTLA-4 imAbs or not, respectively. Moreover, pretreatment with Tα1 in all (95) IPI-evaluable patients confirmed a significant increase in long-term OS. CONCLUSION: This is the first report on long-term follow-up of Tα1-treated patients. Moreover, an advantage in OS in patients sequentially treated with Tα1 and IPI was seen that suggests a synergistic effect.

Tremelimumab combined with durvalumab in patients with mesothelioma (NIBIT-MESO-1): an open-label, non-randomised, phase 2 study.

BACKGROUND: Tremelimumab, an anti-CTLA4 monoclonal antibody, initially showed good activity when used alone in patients with mesothelioma, but did not improve the overall survival of patients who failed on first-line or second-line chemotherapy compared with placebo in the DETERMINE study. We aimed to investigate the efficacy and safety of first-line or second-line tremelimumab combined with durvalumab, an anti-PD-L1 monoclonal antibody, in patients with malignant mesothelioma. METHODS: In this open-label, non-randomised, phase 2 trial, patients with unresectable pleural or peritoneal mesothelioma received intravenous tremelimumab (1 mg/kg bodyweight) and durvalumab (20 mg/kg bodyweight) every 4 weeks for four doses, followed by maintenance intravenous durvalumab at the same dose and schedule for nine doses. The primary endpoint was the proportion of patients with an immune-related objective response according to the immune-related modified Response Evaluation Criteria in Solid Tumors (RECIST; for pleural mesothelioma) or immune-related RECIST version 1.1 (for peritoneal mesothelioma). The primary analysis was done by intention to treat, whereas the safety analysis included patients who received at least one dose of study drug. This trial is registered with the European Clinical Trials Database, number 2015-001995-23, and ClinicalTrials.gov, number NCT02588131, and is ongoing but no longer recruiting patients. FINDINGS: From Oct 30, 2015, to Oct 12, 2016, 40 patients with mesothelioma were enrolled and received at least one dose each of tremelimumab and durvalumab. Patients were followed-up for a median of 19·2 months (IQR 13·8-20·5). 11 (28%) of 40 patients had an immune-related objective response (all partial responses; confirmed in ten patients), with a median response duration of 16·1 months (IQR 11·5-20·5). 26 (65%) patients had immune-related disease control and 25 (63%) had disease control. Median immune-related progression-free survival was 8·0 months (95% CI 6·7-9·3), median progression-free survival was 5·7 months (1·7-9·7), and median overall survival was 16·6 months (13·1-20·1). Baseline tumour PD-L1 expression did not correlate with the proportion of patients who had an immune-related objective response or immune-related disease control, with immune-related progression-free survival, or with overall survival. 30 (75%) patients experienced treatment-related adverse events of any grade, of whom seven (18%) had grade 3-4 treatment-related adverse events. Treatment-related toxicity was generally manageable and reversible with protocol guidelines. INTERPRETATION: The combination of tremelimumab and durvalumab appeared active, with a good safety profile in patients with mesothelioma, warranting further exploration. FUNDING: Network Italiano per la Bioterapia dei Tumori Foundation, Associazione Italiana per la Ricerca sul Cancro, AstraZeneca, and Istituto Toscano Tumori.

Soluble NKG2D ligands are biomarkers associated with the clinical outcome to immune checkpoint blockade therapy of metastatic melanoma patients.

The introduction of immune checkpoint blockade into the clinical practice resulted in improvement of survival of a significant portion of melanoma patients. Consequently, predictive biomarkers of response are needed to optimize patient's stratification and the development of combination therapies. The aim of this study was to determine whether levels of soluble NKG2D ligands (MICA, MICB, ULBP1, 2 and 3; sNKG2DLs) in the serum of melanoma patients can serve as useful predictors of response to the treatment with immune checkpoint blockade. sNKG2DLs were measured by ELISA in baseline and post-treatment serum and these results were correlated with the clinical outcome of melanoma patients (N = 194). The same determinations were performed also in a cohort of patients (N = 65) treated with either chemotherapy, radiotherapy, or mutated BRAF inhibitors (BRAFi). Absence of soluble MICB and ULBP-1 in baseline serum correlated with improved survival (OS = 21.6 and 25.3 mo and p = 0.02 and 0.01, respectively) of patients treated with immunological therapies while detectable levels of these molecules were found in poor survivors (OS = 8.8 and 12.1 mo, respectively). Multivariate analysis showed that LDH (p <0.0001), sULBP-1 (p = 0.02), and sULBP-2 (p = 0.02) were independent predictors of clinical outcome for the cohort of melanoma patients treated with immune checkpoint blockade. Only LDH but not sNKG2DLs was significantly associated with the clinical outcome of patients treated with standard or BRAFi regimens. These findings highlight the relevance of sNKG2DLs in the serum of melanoma patients as biomarkers for patients' stratification and optimization of immune checkpoint inhibition regimens.

Intratumor heterogeneity of cancer/testis antigens expression in human cutaneous melanoma is methylation-regulated and functionally reverted by 5-aza-2'-deoxycytidine.

Cancer/testis antigens (CTA) are suitable targets for immunotherapy of human malignancies, and clinical trials are mainly focusing on MAGE-A3. However, the heterogeneous intratumor expression of CTA may hamper the effectiveness of CTA-directed vaccination through the emergence of CTA-negative neoplastic clones. We investigated the intratumor heterogeneity of CTA in human melanoma and the underlying molecular mechanism(s) at clonal level using 14 single cell clones generated from the melanoma lesion Mel 313. Reverse transcription-PCR revealed a highly heterogeneous expression of MAGE-A1, -A2, -A3, -A4, -A6, GAGE 1-6, SSX 1-5, and PRAME among melanoma clones. Only nine clones expressed MAGE-A3 and competitive reverse transcription-PCR identified relative differences in the number of mRNA molecules of up to 130-fold between clones 5 and 14. This clonal heterogeneity of MAGE-A3 expression correlated with the methylation status of specific CpG dinucleotides in MAGE-A3 promoter: i.e., hypomethylated CpG dinucleotides at positions -321, -151, -19, -16, -5, -2, +21, and +42 were found in clones expressing high but not low levels of MAGE-A3. Supporting the role of DNA methylation in generating the intratumor heterogeneity of CTA, the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA-dCyd) invariably induced their expression in all CTA-negative clones. Furthermore, 5-AZA-dCyd-treatment reduced to 6 folds the differential expression of MAGE-A3 between clones 5 and 14, which became recognized to a similar extent by T cells specific for a MAGE-A-encoded peptide. These findings identify promoter methylation as directly responsible for the intratumoral heterogeneity of therapeutic CTA in melanoma and foresee the use of 5-AZA-dCyd to overcome the limitations set by their intratumor heterogeneous expression to CTA-based vaccine therapy.

Targeted therapy of solid malignancies via HLA class II antigens: a new biotherapeutic approach?

Intracellular signals, delivered in professional antigen-presenting cells following the engagement of major histocompatibility complex (MHC) class II molecules, activate a variety of cellular functions that also contribute to efficient antigen presentation. As far as human malignancies, the signaling ability of human leukocyte antigens (HLA) class II molecules is a rather well-characterized event in hematologic tumors; in contrast, very limited evidences are available in solid neoplasias of different histotypes that may constitutively express HLA class II antigens. Among solid malignancies, a significant proportion of human cutaneous melanomas have been shown to express HLA class II molecules, and cutaneous melanoma undoubtedly represents a 'model disease' to investigate tumor immunobiology, to unveil the molecular basis underlying the interactions between neoplastic cells and host's immune system, and ultimately to set up new bio-immunotherapeutic approaches. Upcoming preclinical evidences unveil a signaling potential of HLA-DR antigens expressed on melanoma cells, and suggest for the clinical implication of HLA class II molecules as novel therapeutic targets. Therefore, in this review, we will focus on the emerging role of HLA class II antigens as intracellular signal transducing elements in neoplastic cells of the melanocytic lineage, emphasizing their foreseeable role in targeted therapy of human melanoma and potentially of HLA class II antigens-positive tumors of different histology.

Epigenetic targets for immune intervention in human malignancies.

Emerging evidences suggest that epigenetic events associated with tumor development and progression, such as deregulated methylation of CpG dinucleotides and aberrant histone acetylation, may impair the immunogenic potential of cancer cells. In fact, DNA hypermethylation and/or histone deacetylation contribute to the absent or downregulated expression of different components of the 'tumor recognition complex' (i.e., HLA class I antigens, cancer/testis antigens and accessory/costimulatory molecules) in solid and hemopoietic human malignancies. However, pharmacologic agents that induce DNA hypomethylation or inhibit histone deacetylation can modify these epigenetic phenomena, restoring the defective expression of selected components of the 'tumor recognition complex' in cancer cells. These antigenic modifications positively modulate the immunogenicity and the immune recognition of cancer cells, making epigenetic drugs attractive agents to design new combined chemoimmunotherapeutic strategies for the treatment of cancer patients.

Endoglin (CD105): a powerful therapeutic target on tumor-associated angiogenetic blood vessels.

Among surface molecules expressed on endothelial cells, endoglin (CD105) is emerging as a prime vascular target for antiangiogenetic cancer therapy. CD105 is a cell membrane glycoprotein mainly expressed on endothelial cells and overexpressed on tumor-associated vascular endothelium, which functions as an accessory component of the transforming growth factor -beta receptor complex and is involved in vascular development and remodelling. Quantification of intratumoral microvessel density by CD105 staining and of circulating soluble CD105 has been suggested to have prognostic significance in selected neoplasias. In addition, the potential usefulness of CD105 in tumor imaging and antiangiogenetic therapy has been well documented utilizing different animal models.

Epigenetic modulation of solid tumors as a novel approach for cancer immunotherapy.

Emerging evidence demonstrates that epigenetic events associated with tumor development and progression may impair immunogenicity and immune recognition of cancer cells, possibly favoring their escape also from vaccination-induced antitumor immune responses. In fact, DNA hypermethylation and/or histone deacetylation plays a critical role in the downregulation and/or silencing of several genes involved in the recognition of neoplastic cells by the immune system, including human leukocyte antigens (HLAs), tumor-associated antigens, and accessory/costimulatory molecules. However, as opposed to genetic alterations, epigenetic events can be successfully handled through pharmacologic agents that induce DNA hypomethylation or inhibit histone deacetylation, resulting in a functionally "more efficient" immune profile of cancer cells. In light of the encouraging immunomodulatory results obtained with these "epigenetic drugs," they certainly will be used for the development of combined chemo-immunotherapeutic strategies for the treatment of patients with solid malignancies of different histology.

Epigenetic immunomodulation of hematopoietic malignancies.

Significant progress has been made in the clinical management of hematologic malignancies; nevertheless, a proportion of patients still remains unresponsive to available therapeutic options. Furthermore, patients who respond to specific therapeutic regimens may still require additional treatment to eradicate minimal residual disease. In this scenario, novel immunotherapeutic strategies may significantly impact on the clinical course of hematopoietic tumors in different clinical stages of disease. Among immunotherapeutic approaches under development, promising clinical results are being obtained with vaccination of patients with solid malignancies against cancer testis antigens (CTA), which belong to a growing family of methylation-regulated tumor-associated antigens (TAA) shared among human malignancies of different histologies. Based on these notions, the emerging preclinical and clinical evidence suggest that an immunomodulatory role for epigenetic drugs is highly relevant; in fact, by interfering with DNA methylation, these compounds induce or upregulate the constitutive expression of CTA on actively proliferating neoplastic cells. This novel activity of epigenetic drugs combines with their well-known cytotoxic, pro-apoptotic and differentiating activities in hematopoietic tumors that are extensively described in other chapters of this issue. This review will focus on the expression of CTA in hematopoietic malignancies, on their epigenetic regulation, and on the foreseeable immunotherapeutic implications of DNA hypometylating drugs to design new CTA-based chemo-immunotherapeutic approaches in patients with hematopoietic tumors.