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Melanoma is an immunogenic neoplasm infiltrated by T cells, although these adaptive T cells usually fail to eradicate the tumor. Plasmacytoid dendritic cells (PDCs) are potent regulators of the adaptive immune response and can eliminate melanoma cells via TLR-mediated effector functions. The PDC compartment is maintained by progressively restricted bone marrow progenitors. Terminally differentiated PDCs exit the bone marrow into the circulation, then home to lymph nodes and inflamed peripheral tissues. Infiltration by PDCs is documented in various cancers. However, their role within the melanoma immune contexture is not completely known. We found that in locoregional primary cutaneous melanoma (PCM), PDC infiltration was heterogeneous, occurred early, and was recurrently localized at the invasive margin, the site where PDCs interact with CD8+ T cells. A reduced PDC density was coupled with an increased Breslow thickness and somatic mutations at the NRAS p.Q61 codon. Compared with what was seen in PCM, high numbers of PDCs were found in regional lymph nodes, as also identified by in silico analysis. In contrast, in metastatic melanoma patients, PDCs were mostly absent in the tumor tissues and were significantly reduced in the circulation, particularly in the advanced M1c group. Exposure of circulating PDCs to melanoma cell supernatant (SN-mel) depleted of extracellular vesicles resulted in significant PDC death. SN-mel exposure also resulted in a defect of PDC differentiation from CD34+ progenitors. These findings indicate that soluble components released by melanoma cells support the collapse of the PDC compartment, with clinical implications for refining TLR agonist–based trials.

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Melanoma is an immunogenic neoplasm infiltrated by T cells, although these adaptive T cells usually fail to eradicate the tumor. Plasmacytoid dendritic cells (PDCs) are potent regulators of the adaptive immune response and can eliminate melanoma cells via TLR-mediated effector functions. The PDC compartment is maintained by progressively restricted bone marrow progenitors. Terminally differentiated PDCs exit the bone marrow into the circulation, then home to lymph nodes and inflamed peripheral tissues. Infiltration by PDCs is documented in various cancers. However, their role within the melanoma immune contexture is not completely known. We found that in locoregional primary cutaneous melanoma (PCM), PDC infiltration was heterogeneous, occurred early, and was recurrently localized at the invasive margin, the site where PDCs interact with CD8+ T cells. A reduced PDC density was coupled with an increased Breslow thickness and somatic mutations at the NRAS p.Q61 codon. Compared with what was seen in PCM, high numbers of PDCs were found in regional lymph nodes, as also identified by in silico analysis. In contrast, in metastatic melanoma patients, PDCs were mostly absent in the tumor tissues and were significantly reduced in the circulation, particularly in the advanced M1c group. Exposure of circulating PDCs to melanoma cell supernatant (SN-mel) depleted of extracellular vesicles resulted in significant PDC death. SN-mel exposure also resulted in a defect of PDC differentiation from CD34+ progenitors. These findings indicate that soluble components released by melanoma cells support the collapse of the PDC compartment, with clinical implications for refining TLR agonist–based trials.

Monoclonal antibodies (Ab) targeting immune checkpoints like CTLA-4 or PD-1 have come of age in the treatment of metastatic melanoma and further approvals are expected for other malignancies like lung and renal cell cancer as well. However, the majority of patients still do not experience clinical benefit upon these therapies. Moreover, immune-related side effects and the costs of these therapies prompt the search for their precise mode of action and for biomarker discovery. Here, we describe different classes of immunologic correlates such as pharmacodynamic changes observed in all treated patients, correlates with response during treatment (surrogate markers) or at the time-point of tumor assessment, as well as predictive markers for response and for immune-related adverse events. This review gives an overview of available data about correlates analyzed in the serum, all in immune cell subsets in the peripheral blood or in tumor-infiltrating lymphocytes. We will discuss how to prospectively validate and integrate these parameters for routine assessment of patients in daily clinical practice and give an outlook on promising future directions of biomarker research.

Compelling experimental evidences point to monitoring of the absolute number of circulating ICOS-positive T cells as an early predictive marker of clinical activity of anti-CTLA-4 antibodies in cancer patients. Here, we report available data focusing on this issue and operative procedures to detect ICOS expression on circulating peripheral blood mononuclear cells during CTLA-4 blockade therapy.

Compelling experimental evidences point to monitoring of the absolute number of circulating ICOS-positive T cells as an early predictive marker of clinical activity of anti-CTLA-4 antibodies in cancer patients. Here, we report available data focusing on this issue and operative procedures to detect ICOS expression on circulating peripheral blood mononuclear cells during CTLA-4 blockade therapy.

Thymosin α1 (Tα1) is an immunomodulatory peptide released by the thymus gland in mammals. It was first described in 1977 as a potential agent for the treatment of immune deficiencies and cancer. Among solid tumors, a number of clinical trials have investigated the activity of Tα1 in melanoma. In particular, a large randomized phase II trial that evaluated the safety and efficacy of combining Tα1 with dacarbazine and interferon alpha in metastatic melanoma patients provided the rationale for further clinical applications. The main findings emerging from clinical trials and that support the therapeutic use of Tα1 in human melanoma are summarized and discussed.

CD40 is a costimulatory molecule widely expressed by immune cells and by neoplastic cells of different histotypes. Engagement of surface CD40 mediates different effects depending on cell type and microenvironment. In particular, CD40 expression on immune cells regulates humoral and cellular immunity, while it has apoptotic and antiproliferative activity on selected neoplastic cells. Thus, CD40 targeting may indirectly affect tumor growth through the activation of immune cells and/or directly by mediating cytotoxic effects on neoplastic cells. Preliminary findings emerging from clinical trials indicate that antibodies to CD40 can induce immune modulation and clinical responses in cancer patients.

Surgical resection represents the treatment of choice for primary melanoma, providing the best results, in terms of long-term survival, for thin lesions. However, surgery has limited efficacy in the management of locoregional and distant metastases, as do chemotherapy and radiotherapy. In this context, immunotherapy is a promising and additional therapeutic option to cure melanoma patients and to prevent relapse of the disease. The identification and characterization of immunogenic tumor-associated antigens (TAAs) has prompted the design and development of a plethora of therapeutic vaccines to drive the host immune system to specifically recognize and eradicate neoplastic cells. Although encouraging, clinical results obtained with TAA-based vaccination are not yet satisfactory. In this regard, a great number of experimental data indicate that neoplastic cells are capable of escaping the host’s immune surveillance and impairing the efficacy of immunotherapeutic approaches by different mechanisms. Among the...

Clinical activity was observed in metastatic melanoma (MM) patients treated with ipilimumab (IPI) combined with fotemustine (FTM) in the phase II NIBIT-M1 study. Peripheral blood mononuclear cells (PBMCs) and serum were collected from MM patients at pre- and at weeks 12 and 24 post-treatment. A comprehensive phenotypic and functional immunomonitoring of circulating T cells, and the detection of soluble immunoregulatory molecules was carried out and correlated with clinical outcome. The frequency at baseline and along the treatment of circulating T central memory cells expressing activation/differentiation markers, such as CD3+CD4+CD45RO+BTLA+, CD3+CD4+4-1BB or Th17 lymphocytes correlated with the clinical outcome of MM patients. Moreover, either the absence or the presence of soluble NKG2D ligands (ULBP-1 or -2) at baseline in the serum of MM patients enabled to discriminate subjects with long-term survival (median overall survival, (OS) = 33.6 mo for ULBP-1 and -2) from poor survivors (OS = 9.8 or 6.6 mo, respectively). Conversely, no significant association between the levels of soluble MICA, MICB and ULBP-3 and the clinical outcome of patients was observed. An inverse correlation between circulating levels of these molecules at baseline and frequency of either CD3+CD4+CD45RO+BTLA+ or Th17 or CD3+CD4+4-1BB+ T cells occurred in patients with a favorable clinical outcome. The simultaneous monitoring of different immune parameters, though validation in a large cohort of patients is needed, allowed to identify an association between phenotypic and soluble markers representing a possible predictive immunological signature for the clinical activity of IPI plus FTM.

Long-term analysis of the NIBIT-M1 trial continues to demonstrate efficacy of the combination of ipilimumab and fotemustine in metastatic melanoma patients with or without brain metastases. The addition of fotemustine to ipilimumab does not impair its immunomodulatory activity. Background In the NIBIT-M1 study, we reported a promising activity of ipilimumab combined with fotemustine in metastatic melanoma (MM) patients with or without brain metastases. To corroborate these initial findings, we now investigated the long-term efficacy of this combination. Patients and methods This analysis captured the 3-year outcome of MM patients who received ipilimumab combined with fotemustine as first- or second-line treatment. Median overall survival (OS), 3-year survival rates, immune-related (ir) progression-free survival (irPFS), brain PFS, and ir duration of response (irDOR) for the entire population and for patients with brain metastases were assessed. Clinical results were correlated with circulating CD3+CD4+ICOS+CD45RO+ or CD45RA+ T cells, neutrophil/lymphocyte (N/L) ratios, and tumorBRAF-V600 mutational status. Results Eighty-six MM patients, including 20 with asymptomatic brain metastases that had been pre-treated with radiotherapy in 7 subjects, were enrolled in the study. With a median follow-up of 39.9 months, median OS and 3-year survival rates were 12.9 months [95% confidence interval (CI) 7.1–18.7 months] and 28.5% for the whole study population, and 12.7 months (95% CI 2.7–22.7 months) and 27.8% for patients with brain metastases, respectively. Long-term ir adverse events consisting of G1 rush and pruritus occurred in 21% of patients. The absolute increase from baseline to week 12 in ‘memory’ but not in ‘naive’ T cells identified patients with a better survival (P = 0.002). The N/L ratio correlated with a significantly better survival at early time points.BRAF status did not correlate with clinical outcome. Conclusions Long-term analysis of the NIBIT-M1 trial continues to demonstrate efficacy of ipilimumab combined with fotemustine in MM patients. Fotemustine does not seem to impair the immunologic activity of ipilimumab. EudraCT number 2010-019356-50. CinicalTrials.gov NCT01654692.

Summary Background CTLA4 blockade by tremelimumab 15 mg/kg every 90 days provided preliminary evidence of activity in patients with pretreated malignant mesothelioma; however, retrospective exposure–response analysis of data from patients with melanoma suggested that this schedule could result in underexposure to tremelimumab. We therefore investigated the efficacy and safety of an intensified schedule of tremelimumab in patients with advanced malignant mesothelioma. Methods In this open-label, single-arm, phase 2 study, participants aged 18 years or older with unresectable, advanced malignant mesothelioma (measurable in accordance with the Response Evaluation Criteria in Solid Tumors [RECIST]), a life expectancy of 3 months or more, an Eastern Cooperative Oncology Group performance status of 2 or less, and who had failed a first-line platinum-based regimen were enrolled at the University Hospital of Siena, Siena, Italy. Participants received tremelimumab 10 mg/kg once every 4 weeks for six doses, then every 12 weeks until disease progression, unacceptable toxic effects, or refusal to continue treatment. The primary endpoint was the proportion of patients achieving an immune-related objective response (complete or partial), assessed in all patients who received at least one dose of the study drug. This study is registered with the European Union Clinical Trials Register, number 2012-002762-12, and ClinicalTrials.gov, number NCT01655888. Findings Between July 30, 2012, and July 15, 2013, we enrolled 29 patients with a median age of 65 years (range 42–78), stage III (n=11) or IV (n=18) disease, and an Eastern Cooperative Oncology Group performance status of 0–1 (n=23) or 2 (n=6). Malignant mesothelioma histology was epithelioid (n=21, including one peritoneal), biphasic (n=6), sarcomatoid (n=1), or undefined (n=1). Patients received a median of six doses of tremelimumab (range 1–13). After a median follow-up of 21·3 months (IQR 18·7–25·9), four immune-related-partial responses were recorded, one at the first tumour assessment (after about 12 weeks) and three at the second tumour assessment (about 24 weeks), with two responses occurring after initial progressive disease and one response after initial stable disease. 15 (52%) of patients achieved disease control, with a median duration of 10·9 months (95% CI 8·2–13·6). According to modified RECIST, one patient (3%) achieved a partial response and 11 (38%) patients achieved disease control rate. Grade 1–2 treatment-related adverse events occurred in 26 (90%) patients and grade 3–4 adverse events in two (7%) patients. The most common treatment-related adverse events were gastrointestinal, dermatological, and fever. Interpretation Our results suggest that the intensified schedule of tremelimumab investigated seems to have clinical and immunological activity in patients with advanced malignant mesothelioma, and a good safety profile. The same intensified schedule is now being investigated in an ongoing randomised, double-blind, placebo-controlled, phase 2b study. Funding Associazione Italiana per la Ricerca sul Cancro, Istituto Toscano Tumori, and MedImmune.

Purpose: Total loss of surface presentation of human leukocyte antigen (HLA) class I molecules, protecting tumor cells from the recognition by cytotoxic host CD8+ T cells, is known to be caused by mutations in the β2-microglobulin (β2m) gene. We asked whether abnormalities of chromosome 15, harboring the β2m gene on 15q21, in addition to β2m gene mutations, are causative for the HLA class I–negative phenotype of melanoma cells.Experimental Design: To answer this, we established primary cell lines from the β2m-negative metastatic melanoma tissues of four different patients and analyzed them for β2m gene mutations and chromosome 15 aberrations, the latter by loss of heterozygosity analysis, fluorescence in situ hybridization (FISH), and multicolor FISH.Results: Mutations at the β2m gene level were detected in all cell lines. The loss of heterozygosity analysis of microsatellite markers located on chromosome 15 in three of the four cell lines pointed to an extensive loss of chromosome 15 material. Subsequent molecular cytogenetic analysis revealed the coexistence of apparently normal and rearranged versions of chromosome 15 in three cell lines whereas the fourth cell line solely showed rearranged versions. Two of the four cell lines exhibited a special type of intrachromosomal rearrangement characterized by FISH signals specific for the subtelomeric region of 15q at both ends of the chromosome and one centromeric signal in between.Conclusions: Our data indicate that the complete loss of HLA class I expression in melanoma cells is due to the coincidence of the following mutational events: (a) chromosome 15 instability associated with an extensive loss of genetic material and (b) β2m gene mutations.

Primary effusion lymphoma (PEL) is a large B-cell neoplasm with an unfavorable prognosis and limited therapeutic options. In this study, cancer testis antigens (CTA) were investigated as potential immunotherapeutic targets in patients with PEL. Baseline expression of a panel of 11 CTA was highly heterogeneous among five PEL cell lines. In particular, the investigated CTA were not expressed in BC-2 and BC-3 cells, while BC-1, HBL-6, and BCBL-1 cells tested positive for 6, 8, and 9 CTA, respectively. The DNA hypomethylating agent 5-aza-2'-deoxycytdine (5-AZA-CdR) invariably induced or up-regulated the expression of all investigated CTA in all cell lines analyzed. The de novo expression of CTA was still detectable at mRNA and protein level at least 2 months after the end of 5-AZA-CdR treatment. These findings, and the concomitant up-regulation of HLA-class I antigens and ICAM-1 by 5-AZA-CdR, support its clinical use to set-up innovative chemo-immunotherapeutic approaches in PEL.

Angiogenesis is crucial for tumor development and progression, and antiangiogenetic therapy represents a promising approach for cancer treatment. Thus, the in-depth understanding of the molecular mechanism(s) regulating angiogenesis, together with the characterization of molecules expressed by endothelial cells and involved in distinct steps of the angiogenetic process, will greatly improve the design of new and more effective therapeutic strategies in human malignancies. Endoglin (CD105), a cell membrane glycoprotein predominantly expressed on cellular lineages within the vascular system, and over-expressed on proliferating endothelial cells, is involved in blood vessels development and represents a powerful marker of neovascularization. CD105 binds several factors of the Transforming Growth Factor (TGF)-beta superfamily, a pleiotropic cytokine that regulates different cellular functions including proliferation, differentiation and migration. In human malignancies of different histotype, CD105 is highly expressed on endothelial cells of both peri- and intra-tumoral blood vessels, while it is weakly expressed or absent on neoplastic cells. This unique tissue distribution strongly suggests for a prognostic, diagnostic and therapeutic potential of CD105 in neoplastic diseases. In this review we will summarize the structural and functional features of CD105, as well as its tissue distribution in normal and neoplastic tissues. Furthermore, the practical implications of CD105 in human malignancies will also be discussed.

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.

The antibody (Ab) response to allogeneic Me14932 and autologous melanoma cells was analyzed in 13 Stage IV (AJCC) melanoma patients immunized with Me14932 cells transduced with the IL-4 (Me14932/IL-4) ( n=10) or IL-2 (Me14932/IL-2) ( n=3) gene. No Ab response was observed before the 4th vaccination. Among 8 patients that received four vaccinations, 3/5 patients vaccinated with Me14932/IL-4 cells developed Ab (IgG and/or IgM) to Me14932 ( n=3) and to autologous ( n=2) melanoma cells, and 2/3 patients vaccinated with Me14932/IL-2 cells developed Ab (IgG) to Me14932, but not to autologous melanoma cells. Further, among these 5 responding patients, circulating Ab against the HLA-A3 allele, expressed only on vaccinating cells, were identified in the immune sera of 4 patients immunized with Me14932/IL-4 ( n=2) or Me14932/IL-2 ( n=2) cells. These sera mediated antibody-dependent cell cytotoxicity (ADCC) of Me14932 cells, and a direct correlation ( r=0.85; P=0.03) between intensity of staining (IgG) and extent of lysis was found. Immune serum of one of these patients also induced ADCC of autologous melanoma cells, and serum from another patient mediated complement cytotoxicity of Me14932, but not of autologous melanoma cells. Thus, Abs against vaccinating and autologous melanoma cells were generated in 62% of patients after four vaccinations with cytokine-transduced melanoma cells. These findings demonstrate that the identification and titration of alloreactive Ab helps to monitor the extent of immunization against cellular vaccines, while the induction of Ab reactive to antigens shared between vaccinating and autologous melanoma cells may contribute to their therapeutic efficacy.

Protectin (CD59) is a glycosylphosphatidylinositol (GPI)-anchored cell membrane glycoprotein, broadly expressed on melanocytic cells, that represents the main restriction factor of complement (C)-mediated lysis of human melanoma cells. Levels of CD59 expression may impair the clinical efficacy of C-activating monoclonal antibodies (mAb); thus, we investigated the molecular mechanisms underlying the lack of CD59 expression in selected melanoma cells. Serological and biochemical analyses showed that MeWo melanoma cells expressed CD59 neither at cell surface nor at cytoplasmic levels; however, no critical mutations were identified in their CD59 mRNA. Consistently, MeWo CD59 cDNA (MeWo-CD59) was appropriately translated when transfected into the CD59-positive Mel 100 melanoma cells, and into the CD59-negative Nalm-6 pre-B leukemia cells that acquired resistance to C. In contrast, transfection of MeWo cells with CD59 cDNA from Mel 275 melanoma cells did not induce CD59 expression; however, their transfection with the CD59-TM chimeric construct, obtained by replacing the GPI-anchoring signal of MeWo-CD59 with the transmembrane tail of the human low-density lipoprotein receptor, induced the expression of a C-protective transmembrane form of CD59. These data, together with the absent expression of additional GPI-anchored proteins (i.e., CD55), suggest that defects in the biosynthesis and/or processing of GPI-anchored proteins underlie the lack of CD59 expression in MeWo cells. Further unveiling of the molecular mechanism that turns off CD59 expression in human melanoma cells will help to set-up more effective therapeutic strategies utilizing C-activating mAb in melanoma patients.

Endoglin (CD105) is a cell membrane glycoprotein over-expressed on highly proliferating endothelial cells in culture, and on endothelial cells of angiogenetic blood vessels within benign and malignant tissues. CD105 binds several factors of the Transforming Growth Factor (TGF)-β superfamily, and its over-expression modulates cellular responses to TGF-β1. The complex of experimental findings accumulated in the last few years strongly indicate that CD105 is a powerful marker of angiogenesis, and that it might play a critical role in the pathogenesis of vascular diseases and in tumor progression. In this paper, we will review the structural, biological and functional features of CD105, as well as its distribution within normal and neoplastic tissues, emphasizing its foreseeable role as a molecular target for new diagnostic and bioimmunotherapeutic approaches in human malignancies. © 2001 Wiley-Liss, Inc.

The clinical efficacy of therapeutic complement (C)-activating monoclonal antibodies (mAb) to melanoma-associated antigens can be impaired by the levels of expression of C-inhibitory molecules on neoplastic cells. Protectin (CD59) is a glycosylphosphatidylinositol (GPI)-anchored cell membrane glycoprotein, acting as terminal regulator of C cascade, which is heterogeneously expressed in melanomas and represents the main restriction factor of C-mediated lysis of melanoma cells. Thus, we investigated whether the overexpression of CD59 could influence the constitutive susceptibility of distinct melanoma cells to homologous C. Infection of CD59-positive Mel 100 and 70-W melanoma cells by a retroviral vector carrying the CD59 cDNA, significantly (P < 0.05) upregulated their constitutive expression of CD59, whereas it did not affect that of additional C-regulatory molecules. Transduced CD59 was entirely GPI-anchored and showed a molecular weight identical to native CD59. Additionally, higher amounts of soluble CD59 were detected in the conditioned media of CD59-transduced melanoma cells compared with parental cells. CD59-transduced melanoma cells, sensitized by the anti-GD3 disialoganglioside mAb R24, were significantly (P < 0.05) less susceptible to homologous C-lysis than were parental cells; this effect was fully reverted by the masking of CD59 with F(ab′)2 fragments of the anti-CD59 mAb YTH53.1. These results provide conclusive evidence demonstrating that absolute levels of CD59 expression regulate the susceptibility to homologous C of specific melanoma cells, and suggest an additional explanation for the poor clinical results obtained with C-activating mAb in the clinical setting. J. Cell. Physiol. 185:317–323, 2000. © 2000 Wiley-Liss, Inc.

To date, no soluble markers can discriminate benign from malignant breast lesions; therefore, to assess the diagnostic potential of circulating intercellular adhesion molecule-1 (sICAM-1), serum concentrations of sICAM-1 were quantitated in 230 consecutive patients that underwent surgery for breast neoplasias, utilizing an enzyme-linked immunosorbent assay. Histological diagnosis revealed that 177 patients had breast cancer and 53 had a benign breast disease. In the cancer patient group, 90 subjects had pT1 tumors without (pT1N0M0, n = 46) or with (pT1N1M0, n = 41; pT1N2M0, n = 3) regional lymph node metastases. Mean levels of serum sICAM-1 of patients with pT1 breast cancer, without or with regional lymph node involvement, were significantly (P0.05) higher than those of patients with benign breast lesions and of 49 age-matched control subjects. Elevated levels of serum sICAM-1 were detected in 27/90 (30%) pT1 breast tumors and in 1/53 (2%) benign breast lesions; thus, among subjects with high levels of sICAM-1, 96% had breast cancer. No significant correlation was found between levels of serum sICAM-1 and breast cancer progression. These observations, altogether, suggest that in the presence of a suspicious breast neoplasm the quantitative analysis of serum sICAM-1 can orient clinical diagnosis towards malignancy.

Protectin (CD59), a glycosylphosphatidylinositol-anchored cell membrane glycoprotein, is differentially expressed on melanocytic cells and represents the main restriction factor of C-mediated lysis of melanoma cells. In this study, we report that CD59-positive melanoma cells constitutively release a soluble form of CD59 (sCD59), and that its levels directly correlate (r = 0.926; P < 0.05) with the amount of membrane-bound CD59. SDS-PAGE analysis showed that the molecular components of sCD59 are similar to those of cellular CD59 expressed by melanoma cells. Melanoma-released sCD59 is anchor positive since it inserts into cell membranes of homologous cells that transiently increase their expression of CD59. Moreover, sCD59 is functional: it blocks the binding of the anti-CD59 mAb YTH53.1 to melanoma cells and reverses its effects on C-mediated lysis. In fact, preincubation of mAb YTH53.1 with scalar doses of conditioned media of CD59-positive but not of CD59-negative melanoma cells reduced significantly (P < 0.05), and in a dose-dependent fashion, the enhancement of C-mediated lysis of anti-GD3-sensitized melanoma cells induced by the masking of cellular CD59 by mAb YTH53.1. Altogether, these data demonstrate that CD59-positive human melanoma cells release a soluble form of CD59 that is structurally similar to cellular CD59, retains its anchoring ability, is functional, and may impair the effectiveness of clinical approaches to humoral immunotherapy for human melanoma.

Meeting abstracts Ipilimumab (IPI) in combination with fotemustine (FTM) has shown a promising clinical activity in metastatic melanoma (MM) patients (pts) enrolled in the NIBIT-M1 trial (Di Giacomo, et al., Lancet Oncology, 2012). This study investigated changes in immunological parameters in the

Summary 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.

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.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.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 survived4 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.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.

Background Prevalence and distribution of pathogenetic mutations in BRAF and NRAS genes were evaluated in multiple melanoma lesions from patients with different geographical origin within the same Italian population. Methods Genomic DNA from a total of 749 tumor samples (451 primary tumors and 298 metastases) in 513 consecutively-collected patients with advanced melanoma (AJCC stages III and IV) was screened for mutations in exon 15 of BRAF gene and, at lower extension (354/513; 69%), in the entire coding DNA of NRAS gene by automated direct sequencing. Among tissues, 236 paired samples of primary melanomas and synchronous or asynchronous metastases were included into the screening. Results Overall, mutations were detected in 49% primary melanomas and 51% metastases, for BRAF gene, and 15% primary tumors and 16% secondaries, for NRAS gene. A heterogeneous distribution of mutations in both genes was observed among the 451 primary melanomas according to patients’ geographical origin: 61% vs. 42% (p = 0.0372) BRAF-mutated patients and 2% vs. 21% (p NRAS-mutated cases were observed in Sardinian and non-Sardinian populations, respectively. Consistency in BRAF/NRAS mutations among paired samples was high for lymph node (91%) and visceral metastases (92.5%), but significantly lower for brain (79%; p = 0.0227) and skin (71%; p = 0.0009) metastases. Conclusions Our findings about the two main alterations occurring in the different tumor tissues from patients with advanced melanoma may be helpful in improving the management of such a disease.

8513 Background: Ipilimumab (ipi), an antibody against cytotoxic T-lymphocyte-associated antigen-4, improves survival in patients (pts) with metastatic melanoma (MM); however, objective tumor responses are limited. NIBIT-M1 aims to investigate the efficacy and safety of ipi plus fotemustine (FTM), a cytotoxic alkylating drug, in pts with MM. Methods: Eligible pts, with or without brain metastases, received induction therapy with ipi 10 mg/kg every 3 weeks (Q3W) for four doses and FTM 100 mg/m2 weekly for 3 weeks. Ipi and FTM maintenance therapy was provided Q12W from Week 24 and Q3W from Week 9, respectively. The primary objective was the immune-related (ir) disease control rate (irDCR: pts with complete response [CR], partial response [PR] or stable disease [SD] as determined using the ir response criteria). Secondary objectives included ir objective response rate (ORR), duration of response (DOR) and progression-free survival (PFS); overall survival (OS), and safety. Tumor assessments were performed Q8W from Week 12 to Week 36 and Q12W thereafter. Results: Among 86 pts with unresectable stage III (n=3) or stage IV (n=83) MM treated at 7 NIBIT centers, 42 were previously untreated, 44 had progressed following first-line treatment and 20 had asymptomatic brain metastases. As of December 2011, the irDCR was 46.5% (40/86; 95% CI, 35.7–57.6%); the irORR was 29.1% (95% CI, 19.8–39.8%; 5 CRs and 20 PRs) and with a median 8.3 months follow-up, median irPFS was 5.3 months (95% CI, 3.5–7.1). The 1-year OS rate was 51.8% (95% CI, 37.5–66.1%); median OS was not yet reached. Among all pts, 58.1% and 87% completed ipi or FTM induction, respectively. The most common grade 3/4 drug-related adverse events (AEs) (reported in 54.6% pts overall) were myelotoxicity (43.5%), increased ALT/AST (14.1/10.6%), gastrointestinal (4.7%) and skin-related (2.3%). AEs were generally manageable and reversible per protocol guidance. Conclusions: The study reached its primary objective with 46.5% of pts achieving disease control. The combination of ipi plus FTM is safe; the irDCR, 1-year OS rate and median irPFS warrant its further investigation in MM pts.

Thymosin α1 (Tα1) is an immunomodulatory peptide released by the thymus gland in mammals. It was first described in 1977 as a potential agent for the treatment of immune deficiencies and cancer. Among solid tumors, a number of clinical trials have investigated the activity of Tα1 in melanoma. In particular, a large randomized phase II trial that evaluated the safety and efficacy of combining Tα1 with dacarbazine and interferon alpha in metastatic melanoma patients provided the rationale for further clinical applications. The main findings emerging from clinical trials and that support the therapeutic use of Tα1 in human melanoma are summarized and discussed.

Purpose The prevalence of BRAF, NRAS, and p16CDKN2A mutations during melanoma progression remains inconclusive. We investigated the prevalence and distribution of mutations in these genes in different melanoma tissues. Patients and Methods In all, 291 tumor tissues from 132 patients with melanoma were screened. Paired samples of primary melanomas (n = 102) and synchronous or asynchronous metastases from the same patients (n = 165) were included. Tissue samples underwent mutation analysis (automated DNA sequencing). Secondary lesions included lymph nodes (n = 84), and skin (n = 36), visceral (n = 25), and brain (n = 44) sites. Results BRAF/NRAS mutations were identified in 58% of primary melanomas (43% BRAF; 15% NRAS); 62% in lymph nodes, 61% subcutaneous, 56% visceral, and 70% in brain sites. Mutations were observed in 63% of metastases (48% BRAF; 15% NRAS), a nonsignificant increase in mutation frequency after progression from primary melanoma. Of the paired samples, lymph nodes (93% consistency) and visceral metastases (96% consistency) presented a highly similar distribution of BRAF/NRAS mutations versus primary melanomas, with a significantly less consistent pattern in brain (80%) and skin metastases (75%). This suggests that independent subclones are generated in some patients. p16CDKN2A mutations were identified in 7% and 14% of primary melanomas and metastases, with a low consistency (31%) between secondary and primary tumor samples. Conclusion In the era of targeted therapies, assessment of the spectrum and distribution of alterations in molecular targets among patients with melanoma is needed. Our findings about the prevalence of BRAF/NRAS/p16CDKN2A mutations in paired tumor lesions from patients with melanoma may be useful in the management of this disease.

Background: Epigenetic remodelling of cancer cells is an attractive therapeutic strategy and distinct DNA hypomethylating agents (DHA) are being actively evaluated in patients with hemopoietic or solid tumours. However, no studies have investigated the modulation of gene expression profiles (GEP) induced by DHA in transformed and benign tissues. Such information is mandatory to clarify the fine molecular mechanism(s) underlying the clinical efficacy of DHA, to identify appropriate therapeutic combinations, and to address safety issues related to their demethylating potential in normal tissues. Thus, utilising a syngeneic mouse model, we investigated the remodelling of GEP of neoplastic and normal tissues induced by systemic administration of DHA. Methods: The murine mammary carcinoma cells TS/A were injected s.c. into female BALB/c mice that were treated i.p. with four cycles of the DHA 5-aza-2′-deoxycytidine (5-AZA-CdR) at a fractioned daily dose of 0.75 mg kg−1 (q8 h × 3 days, every week). Whole mouse transcriptomes were analysed by microarrays in neoplastic and normal tissues from control and treated mice. Results were processed by bioinformatic analyses. Results: In all, 332 genes were significantly (P⩽0.05; FC⩾4) modulated (294 up and 38 downregulated) in neoplastic tissues from 5-AZA-CdR-treated mice compared with controls. In decreasing order of magnitude, changes in GEP significantly (P⩽0.05) affected immunologic, transport, signal transduction, spermatogenesis, and G–protein–coupled receptor protein signalling pathways. Epigenetic remodelling was essentially restricted to tumour tissues, leaving substantially unaltered normal ones. Conclusion: The ability of 5-AZA-CdR to selectively target tumour GEP and its major impact on immune-related genes, strongly support the clinical use of DHA alone or combined with immunotherapeutic agents.

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.

No treatment prolongs the survival of malignant mesothelioma (MM) patients. Since MM elicits anti-tumor host's immune responses, immunotherapy represents a promising strategy for its control. Immunomodulatory antibodies against components of the B7 family of immunomodulatory molecules that regulate T cell activation are being investigated in human malignancies including MM. The expression of B7-H3, a new component of the B7 family was investigated in primary cultures of human mesothelial cells (HMC) and in MM cell lines by flow cytometry and molecular analyses, and in MM tissues by immunohistochemistry. The role of DNA hypomethylating agents in modulating levels of B7-H3 expression in MM cells was also studied. Reverse transcriptase-polymerase chain reaction (RT-PCR) demonstrated that B7-H3 mRNA was consistently detectable in mesothelial and MM cells investigated; however, real-time quantitative RT-PCR analyses showed highly heterogeneous levels of B7-H3 mRNA among investigated MM cells. The analysis of B7-H3 protein expression indicated that comparable levels of B7-H3 were expressed on both cell types. Treatment with the DNA hypomethylating agent 5-aza-2'-deoxycytidine did not significantly affect the expression of B7-H3 mRNA in MM cells. In vivo, while B7-H3 was expressed in all 13 tumor biopsies of the epithelial variant, with high levels in 54% of cases, it was rarely detectable in spindle type MM in which 1/5 biopsies weakly expressed B7-H3. These findings suggest that B7-H3 is a promising target for new immunotherapeutic strategies in MM, with particular emphasis in the epithelial variant.

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.

Anti-CD40 antibodies are in clinical development in patients with metastatic melanoma, a cancer that has been reported earlier to express CD40. The antitumor activity of anti-CD40 antibodies may be mediated by direct cytotoxic effects on CD40-positive melanoma cells or indirectly through modulation of host cells. In these studies, biopsies of patients with primary and metastatic melanoma, short-term cultures, and established melanoma cell lines were analyzed for CD40 expression using a combination of methods including immunohistochemistry, flow cytometry, and gene expression profiling, and the cytotoxic effects of the anti-CD40 antibody CP-870,893 on melanoma cell lines were tested using cell viability assays. CD40 was expressed at a higher frequency in metastatic melanoma lesions compared with primary melanomas. There was a variable expression of CD40 in synchronous and metachronous melanoma metastases. Expression of CD40 was present in slightly over half of a large panel of short-term primary melanoma cultures, with a wide range of expression levels by flow cytometry. Similar results were obtained in established melanoma cell lines when analyzed at the mRNA level or by surface protein staining. In approximately one-third of cell lines, the expression of CD40 could be up-regulated with a histone deacetylase inhibitor. Treatment with increasing concentrations of CP-870,893 had no antitumor activity against either CD40-positive or negative melanoma cell lines. In conclusion, approximately one-third to one-half of melanomas expresses CD40 at variable levels. Direct exposure to a CD40-activating antibody does not lead to antitumor activity in melanoma cell lines, suggesting that the antitumor effects of these antibodies in the clinic may be indirectly mediated.

Endoglin/CD105 is well acknowledged as being the most reliable marker of proliferation of endothelial cells, and it is overexpressed on tumour neovasculature. Our current knowledge of its structure, physiological role, and tissue distribution suggests that targeting of endoglin/CD105 is a novel and powerful diagnostic and therapeutic strategy in human malignancies, through the imaging of tumour-associated angiogenesis and the inhibition of endothelial cell functions related to tumour angiogenesis. Among biotherapeutic agents, monoclonal antibodies have shown a major impact on the clinical course of human malignancies of different histotypes. Along this line, the potential efficacy of anti-endoglin/CD105 antibodies and their derivatives for clinical purposes in cancer is supported by a large body of available pre-clinical in vitro and in vivo data. In this review, the main findings supporting the translation of antibody-based endoglin/CD105 targeting from pre-clinical studies to clinical applications in human cancer are summarized and discussed.

As for a consolidated tradition, the 5th annual meeting of the Italian Network for Cancer Biotherapy took place in the Certosa of Pontignano, a Tuscan monastery, on September 20–22, 2007. The congress gathered more than 40 Italian leading groups representing academia, biotechnology and pharmaceutical industry. Aim of the meeting was to share new advances in cancer bio-immunotherapy and to promote their swift translation from pre-clinical research to clinical applications. Several topics were covered including: a) molecular and cellular mechanisms of tumor escape; b) therapeutic antibodies and recombinant constructs; c) clinical trials up-date and new programs; d) National Cooperative Networks and their potential interactions; e) old and new times in cancer immunology, an "amarcord". Here, we report the main issues discussed during the meeting.

Tumor-associated angiogenesis is a well-acknowledged therapeutic target for human malignancies, and different markers of tumor neovasculature are actively investigated as potential candidates for antiangiogenetic therapy in cancer. Among these is endoglin (CD105), a homodimeric transmembrane glycoprotein overexpressed on proliferating endothelial cells, which has been identified as a functional component of the transforming growth factor-β (TGF-β) receptor system about a decade ago. The large body of experimental data accumulated so far indicates that CD105 plays a crucial role in angiogenesis and vascular integrity; furthermore, intratumoral microvessel density as evaluated by staining for CD105 represents a strong prognostic factor in solid malignancies of different histology. In this work, we review the biologic features of CD105 and the complex of evidences highlighting its great potentialities as novel target for diagnostic and therapeutic approaches in human malignancies.

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 51Cr-release assays, IFN-γ release and IFN-γ 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-γ release. 5-aza-CdR treatment of Mel 275 cells also induced a higher number of gp100-specific CTL to secrete IFN-γ.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.

In the last three decades huge efforts have been made to characterize genetic defects responsible for cancer development and progression, leading to the comprehensive identification of distinct cellular pathways affected by the alteration of specific genes. Despite the undoubtable role of genetic mechanisms in triggering neoplastic cell transformation, epigenetic modifications (i.e., heritable changes of gene expression that do not derive from alterations of the nucleotide sequence of DNA) are rapidly emerging as frequent alterations that often occur in the early phases of tumorigenesis and that play an important role in tumor development and progression. Epigenetic alterations, such as modifications in DNA methylation patterns and post-translational modifications of histone tails, behave extremely different from genetic modifications, being readily revertable by "epigenetic drugs" such as inhibitors of DNA methyl transferases and inhibitors of histone deacetylases. Since epigenetic alterations in cancer cells affect virtually all cellular pathways that have been associated to tumorigenesis, it is not surprising that epigenetic drugs display pleiotropic activities, being able to concomitantly restore the defective expression of genes involved in cell cycle control, apoptosis, cell signaling, tumor cell invasion and metastasis, angiogenesis and immune recognition. Prompted by this emerging clinical relevance of epigenetic drugs, this review will focus on the large amount of available data, deriving both from in vitro experimentations and in vivo pre-clinical and clinical studies, which clearly indicate epigenetic drugs as effective modifiers of cancer phenotype and as positive regulators of tumor cell biology with a relevant therapeutic potential in cancer patients.

Emerging in vitro evidence points to an immunomodulatory activity of DNA hypomethylating drugs in human malignancies. We investigated the potential of 5-aza-2'-deoxycytidine (5-AZA-CdR) to modulate the expression of cancer testis antigens (CTA) and of HLA class I antigens by melanoma xenografts, and the resulting modifications in immunogenicity of neoplastic cells. Three primary cultures of melanoma cells, selected for immune phenotype and growth rate, were grafted into BALB/c nu/nu mice that were injected intraperitoneally with different dose- and time-schedules of 5-AZA-CdR. Molecular analyses demonstrated a de novo long-lasting expression of the CTA MAGE-1, -2, -3, -4, -10, GAGE 1-6, NY-ESO-1, and the upregulation of MAGE-1, MAGE-3, and NY-ESO-1 levels in melanoma xenografts from 5-AZA-CdR-treated mice. Serological and biochemical analyses identified a de novo expression of NY-ESO-1 protein and a concomitant and persistent upregulation of HLA class I antigens and of HLA-A1 and -A2 alleles. Immunization of BALB/c mice with 5-AZA-CdR-treated melanoma cells generated high titer circulating anti-NY-ESO-1 antibodies. Altogether, the data obtained identify an immunomodulatory activity of 5-AZA-CdR in vivo and strongly suggest for its clinical use to design novel strategies of CTA-based chemo-immunotherapy for melanoma patients.

See article by Jerkic et al. [12] (pages 845–854) in this issue. Initial studies carried out at the beginning of the past century identified increased vascularity as a common feature associated with tumor growth; based on these morphological observations, it was then demonstrated that local tumor growth and distant metastasis of neoplastic cells are strictly dependent on adequate blood supply. The latter was subsequently shown to be provided by blood vessels neoformed from pre-existing ones whose growth is directly promoted by neoplastic cells through the local release of angiogenic factors. Altogether, these observations provided a strong scientific rationale to hypothesize the clinical use of an anti-angiogenic approach based on therapeutic tools able to inhibit molecularly characterized regulators of angiogenesis as an effective strategy to treat cancer [1,2]. Nevertheless, among a large array of novel therapeutic agents with anti-angiogenic activity tested in the clinical setting in the past decade, the humanized version of a murine anti-vascular endothelial growth factor (VEGF) monoclonal antibody (mAb) (bevacizumab) is the only such agent that has received approval for the treatment of mestatastic colorectal cancer in association with chemotherapy [2]; thus, plenty of room is still available for the clinical development of new … *Corresponding author. Division of Medical Oncology and Immunotherapy, University Hospital of Siena, Strada delle Scotte 14, 53100 Siena, Italy. Tel.: +39 0577 586335; fax: +39 0577 586303. Email address: mmaio{at}cro.it

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.

Primary effusion lymphoma (PEL) is a large B-cell neoplasm with an unfavorable prognosis and limited therapeutic options. In this study, cancer testis antigens (CTA) were investigated as potential immunotherapeutic targets in patients with PEL. Baseline expression of a panel of 11 CTA was highly heterogeneous among five PEL cell lines. In particular, the investigated CTA were not expressed in BC-2 and BC-3 cells, while BC-1, HBL-6, and BCBL-1 cells tested positive for 6, 8, and 9 CTA, respectively. The DNA hypomethylating agent 5-aza-2'-deoxycytdine (5-AZA-CdR) invariably induced or up-regulated the expression of all investigated CTA in all cell lines analyzed. The de novo expression of CTA was still detectable at mRNA and protein level at least 2 months after the end of 5-AZA-CdR treatment. These findings, and the concomitant up-regulation of HLA-class I antigens and ICAM-1 by 5-AZA-CdR, support its clinical use to set-up innovative chemo-immunotherapeutic approaches in PEL.

The role of human leukocyte antigens (HLA) class II molecules in transducing intracellular signals in immune cells is well established. Solid tumors of different histotype can also express HLA class II antigens; however, their intracellular signaling ability is essentially unknown. Due to the frequent expression of HLA class II molecules in primary and metastatic lesions, cutaneous melanoma was utilized to investigate whether the engagement of HLA-DR molecules transduces functional intracellular signal(s). Triggering of HLA-DR molecules by the anti-HLA-DR monoclonal antibody (mAb) L243 induced a significant (P

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.