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Association of DNA hypomethylating agents (DHA) with immune-checkpoint inhibitors (ICI) is a promising strategy to improve efficacy of ICI-based therapy. Here we report the five-year clinical outcome and an integrated multi-omics analysis of pre- and on-treatment lesions from advanced melanoma patients enrolled in the phase Ib NIBIT-M4 study, a dose-escalation trial of the DHA agent guadecitabine combined with ipilimumab. With a minimum follow-up of 45 months the median OS was 25.6 months; the 5-year OS rate was 28.9% and the median DoR was 20.6 months. Specific genomic features and extent of T and B cellmediated immunity discriminated lesions of responding compared to non-responding patients. Enrichment for proliferation and EMT-related gene programs, and immune escape mechanisms characterized lesions from non-responding patients. Integration of a genetic immunoediting index (GIE) with an adaptive immunity signature (ICR) stratified patients/lesions into four distinct subsets and discriminated 5-year OS and PFS. These results suggest that coupling of immunoediting with activation of adaptive immunity is a relevant requisite for achieving long term clinical benefit by epigenetic immunomodulation in advanced melanoma patients.

DNA hypomethylating agents (DHA) combined with immune-checkpoint inhibitors (ICI) is a promising strategy to improve the effect of immunotherapy. Providing initial support to this notion, the phase Ib NIBIT-M4 study, a dose-escalation trial of the DHA guadecitabine plus ipilimumab, has shown this regimen to be safe, tolerable and with promising immunomodulatory and anti-tumor activity in advanced melanoma. Here we report the 5-year clinical outcome of NIBIT-M4 patients and its correlation with an integrated multi-omic analysis. The NIBIT-M4 enrolled unresectable Stage III or IV cutaneous melanoma patients. Median Overall Survival (OS), Progression Free Survival (PFS), 5-year OS and PFS rate, and median Duration of Response (DoR) were assessed. Tumor biopsies were performed at baseline and at week 4 and 12 on-treatment. RNA-seq, whole exome sequencing (WES), RRBS methylation profiling, and tumor immune contexture data generated by immunohistochemistry (IHC), were integratively analyzed and correlated with clinical outcome. Patients were classified as responder (R) or non-responder (NR) based on Disease Control. Nineteen patients were enrolled in the NIBIT-M4 study between October 2015, and August 2018. As of July 1, 2022, median OS was 25.6 months (mo) (95% CI, 0.0-52.9), median PFS was 5.2 mo (95% CI, 4.0-6.4); 5-year OS rate was 28.9% and 5-year PFS rate was 5.3 %; median DoR was 20.6 mo (95% CI, 12.4-28.8). Tumor biopsies for correlative analyses were available from 14 patients. Compared to NR, R patients showed higher mutation frequency in NRAS, while NR patients harbored more frequent somatic alterations in genes involved in Epithelial to Mesenchymal Transition (EMT) and chromatin organization pathways. By tumor transcriptome analysis, NR patients showed enrichment for proliferation/differentiation/EMT pathways associated with suppression of INF-ɣ/HLA-II/immune-related transcriptional modules. R patients displayed a dynamic pattern of activated immune signatures related to CD8+ Tex cells, B cells, tertiary lymphoid structures, TFH cells and IFN-ɣ, reaching highest expression levels in week 12 biopsies. Deconvolution of transcriptomic data showed higher CD8+ and NK cell content and higher correlation of TCRB clonality with CD8+ content in R patients. By integration of a genetic immunoediting index (GIE) with an adaptive immunity signature (ICR) lesions were stratified into four distinct subsets. Interestingly, the ICR/GIE classification discriminated 5-year OS and PFS while the classification based on response groups did not. Moreover, patients with a “high ICR/non-GIE” (i.e., without immunoediting) showed lower expression of antigen presentation and processing-related genes associated with defective HLA-class I expression in the lesions, in spite of high CD8+ content. Citation Format: Teresa Noviello, Anna Maria Di Giacomo, Francesca Pia Caruso, Alessia Covre, Giovanni Scala, Luigi Ferraro, Sandra Coral, Wolf H. Fridman, Catherine Sautes-Fridman, Roberta Mortarini, Elena Simonetti, Maria Fortunata Lofiego, Davide Bedognetti, Andrea Anichini, Michele Ceccarelli, Michele Maio. Guadecitabine plus ipilimumab in unresectable melanoma: Five-year follow-up and correlation with integrated, multi-omic analysis in the NIBIT-M4 trial [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3273.

Purpose: The immunomodulatory activity of DNA hypomethylating agents (DHAs) suggests they may improve the effectiveness of cancer immunotherapies. The phase Ib NIBIT-M4 trial tested this hypothesis using the next-generation DHA guadecitabine combined with ipilimumab. Patients and Methods: Patients with unresectable stage III/IV melanoma received escalating doses of guadecitabine 30, 45, or 60 mg/m2/day subcutaneously on days 1 to 5 every 3 weeks, and ipilimumab 3 mg/kg intravenously on day 1 every 3 weeks, starting 1 week after guadecitabine, for four cycles. Primary endpoints were safety, tolerability, and MTD of treatment; secondary were immune-related (ir) disease control rate (DCR) and objective response rate (ORR); and exploratory were changes in methylome, transcriptome, and immune contextures in sequential tumor biopsies, and pharmacokinetics. Results: Nineteen patients were treated; 84% had grade 3/4 adverse events, and neither dose-limiting toxicities per protocol nor overlapping toxicities were observed. Ir-DCR and ir-ORR were 42% and 26%, respectively. Median CpG site methylation of tumor samples (n = 8) at week 4 (74.5%) and week 12 (75.5%) was significantly (P < 0.05) lower than at baseline (80.3%), with a median of 2,454 (week 4) and 4,131 (week 12) differentially expressed genes. Among the 136 pathways significantly (P < 0.05; Z score >2 or ←2) modulated by treatment, the most frequently activated were immune-related. Tumor immune contexture analysis (n = 11) demonstrated upregulation of HLA class I on melanoma cells, an increase in CD8+, PD-1+ T cells and in CD20+ B cells in posttreatment tumor cores. Conclusions: Treatment of guadecitabine combined with ipilimumab is safe and tolerable in advanced melanoma and has promising immunomodulatory and antitumor activity.

Background: Therapeutic targeting of the PD-1/PDL-1 axis significantly increases the survival of melanoma (MM) and non-small cell lung cancer (NSCLC) patients. Despite this unprecedented efficacy, a sizeable proportion of MM and NSCLC patients fails to benefit from therapy due to primary or secondary resistance to treatment. In this scenario, deepening the knowledge on mechanism(s) underlying treatment failure allows to design novel, mechanism-based, therapeutic approaches to overcome resistance to anti-PD-1/PDL-1 therapy. Along this line, we have extensively characterized the immunomodulatory properties of DNA hypomethylating agents (DHA) in different human malignances. Exposure of neoplastic cells to DHA efficiently improved antigen-restricted and -unrestricted T cell recognition of cancer cells in vitro through the upregulation/induction of the expression of epigenetically regulated tumor associated antigens, HLA class I and/or accessory/co-stimulatory molecules by neoplastic cells. Supporting these ex vivo data, combining the systemic administration of DHA (ie, decitabine or guadecitabine) with antibodies to different immune checkpoints significantly reduced tumor growth of murine syngeneic grafts, compared to single-agent therapy. Supporting these pre-clinical findings, our phase Ib NIBIT-M4 study has been the first clinical trial demonstrating that systemic administration of guadecitabine followed by CTLA-4 blockade with ipilimumab is safe and tolerable in MM patients, shows a promising anti-tumor, and induces a significant modulation of immune related pathways in tumor samples (Di Giacomo AM, Clin Cancer Res 2019). Prompted by these results and to further explore the efficacy of DHA combined with immune-checkpoints blockade we have designed and activated the NIBIT-ML1 trial. This study aims at investigating the efficacy of guadecitabine combined with ipilimumab and nivolumab in reverting the resistance to PD-1/PDL-1 therapy of MM and NSCLC patients. Methods: The NIBIT-ML1 is a randomized, phase II study designed according to the two stages optimal design by Simon, in unresectable Stage III or Stage IV MM (Cohort A) or NSCLC (Cohort B) patients who failed therapy with anti-PD-1/PDL-1 as last treatment. Primary objective of the study is immune (i)-ORR according to iRECIST criteria. Secondary objectives include safety, DCR, PFS, median OS, and survival rate at 1 and 2-years. Extensive cellular and molecular immune correlates will also be explored. Following a safety run-in phase in 6 subjects per Cohort, eligible patients will be randomized to receive: guadecitabine plus ipilimumab and nivolumab (ARM A) or ipilimumab and nivolumab (ARM B). Sample size will range from 6 to 92 patients per Cohort. The first patient first visit is foreseen by March 2020 (NCT04250246) Citation Format: Anna Maria Di Giacomo, Luana Calabrò, Riccardo Danielli, Monica Valente, Elisabetta Gambale, Sandra Coral, Giovanni Amato, Harold Keer, Diana Giannarelli, Mohammad Azab, Andrea Anichini, Alessia Covre, Michele Maio. A randomized, multi-center, phase II study of nivolumab combined with ipilimumab and guadecitabine or nivolumab combined with ipilimumab in melanoma and NSCLC patients resistant to anti-PD-1/-PD-L1: The NIBIT-ML1 Study [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT270.

Rationale: Glioblastoma multiforme (GBM) is the most common and deadliest primary brain tumour in adults, with a very dismal prognosis and limited effective therapies. Immunotherapy may play a role in treating GBM, though the initial clinical results are still poor, and novel combined treatment(s) could possibly improve its efficacy. Manipulating the epigenome has been considered a therapeutic opportunity in GBM, since epigenetic modifications regulate glioma proliferation, metastasis, invasion, and prognosis. In addition, epigenetic alterations may negatively regulate the cross-talk between GBM and immune cells in GBM microenvironment (TME), thus limiting the efficacy of immunotherapy. We previously demonstrated the immunomodulatory potential of epigenetic drugs in cancer cells, and translated this notion in the clinic to improve efficacy of immunotherapy of melanoma patients (Di Giacomo AM, CCR 2019). Accordingly, this study aimed to evaluate the immunomodulatory potential of epigenetic drugs in GBM, and to provide the scientific rationale for new combinatorial therapeutic approaches in GBM patients. Methods: Patient-derived GBM (pGBM) cell lines (n=10) were established from surgically-removed GBM lesions and treated with the DNA hypomethylating agent guadecitabine (1 µM); commercially available human GBM (cGBM) cell lines (n=3) were treated with guadecitabine (1 µM) and/or with the EZH2-inhibitor GSK126 (10 µM). Expression of HLA class I and ICAM-1 by GBM cells was investigated by flow-cytometry; cancer testis antigens (CTA, i.e., NY-ESO, MAGE-A1 and MAGE-A3) and the epithelial-mesenchymal transition (EMT)-regulating cadherins (i.e., CDH1 and CDH2) expression was analyzed by quantitative Real-Time PCR. Results: A positive modulation of immune profile was observed in all investigated guadecitabine-treated GBM cells. Specifically, in untreated vs guadecitabine-treated cells, MFI mean values ± SD of HLA class I were 402±326 vs 442±303 in pGBM cells, and 277±127 vs 381±138 in cGBM cells; MFI mean values ± SD of ICAM-1 were 53±72 vs 92±116 in pGBM cells, and 5±6 vs 26±17 in cGBM cells. Though a slight increment was induced by GSK126 for HLA class I (312±169) and ICAM-1 (9±11) expression in cGBM, an additive effect was detected with GSK126+guadecitabine (405±261 for HLA class I and 35±22 for ICAM-1) in cGBM cells. Guadecitabine induced NY-ESO-1, MAGE-A1, and MAGE-A3 expression in 9/10, 6/10, and 5/8 pGBM cells, respectively and in all cGBM cells. Levels of CTA expression were increased in cGBM by GSK126+guadecitabine vs guadecitabine with mean Fold Change (FC) of 1.5, 1.6 and 1.6 for NY-ESO-1, MAGE-A1 and MAGE-A3 expression. Finally, CDH1 expression was induced in 1/3 and in 2/3 cGBM cells by guadecitabine and guadecitabine+GSK126, respectively; CDH2-positive expression was upregulated by guadecitabine (mean FC=2.45) but not by guadecitabine+GSK126. Conclusions: Immunomodulatory properties of epigenetic drugs in GBM should be exploited to improve the efficacy of GBM immunotherapy. Citation Format: Maria Fortunata Lofiego, Francesca Piazzini, Carolina Fazio, Ornella Cutaia, Carla Chiarucci, Sara Cannito, Elisabetta Gambale, Monica Valente, Anna Maria Di Giacomo, Sandra Coral, Clelia Miracco, Giuseppe Oliveri, Michele Maio, Alessia Covre. Immunomodulatory activity of epigenetic drugs: Laying the ground for new combined immunotherapeutic strategies for glioblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4488.

Targeting of the programmed cell death protein (PD)-1/programmed death-ligand 1 (PD-L1) axis has shown a significant clinical impact in several tumor types. Accordingly, our phase II NIBIT-MESO-1 study demonstrated an improved clinical efficacy in mesothelioma patients treated with the anti-PD-L1 durvalumab combined with the anti-cytotoxic T-lymphocyte antigen (CTLA)-4 tremelimumab, as compared to tremelimumab alone. Due to the promising therapeutic activity of immune check-point inhibitors (ICIs) in mesothelioma patients, the identification of biomarkers predictive of response to treatment is of crucial relevance. The prognostic role of soluble PD-L1 (sPD-L1) proposed in cancer patients prompted us to investigate this protein in sera from mesothelioma patients (n = 40) enrolled in the NIBIT-MESO-1 study. A significant (p &lt, 0.001) increase in sPD-L1 levels was detected in patients after the first cycle and during therapy vs. baseline. A longer overall survival (OS) was observed in patients with sPD-L1 concentrations below (at baseline, d1C2, d1C5 (p &lt, 0.01)) or FC values above (p &lt, 0.05 at d1C2, d1C3, d1C5) their statistically calculated optimal cut-offs. On the basis of these initial results, the specific role of CTLA-4-, PD-L1-, or PD-1-targeting on sPD-L1 release was then investigated in sera from 81 additional ICI-treated solid cancer patients. Results showed a significant (p &lt, 0.001) increase of sPD-L1 levels during therapy compared to baseline only in anti-PD-L1-treated patients, supporting the specific involvement of PD-L1 targeting in the release of its soluble form. Our findings suggest that sPD-L1 represents a predictive biomarker of clinical response to anti-PD-L1 cancer immunotherapy.

DNA hypomethylating agents (DHAs) play a well-acknowledged role in potentiating the immunogenicity and the immune recognition of neoplastic cells. This immunomodulatory activity of DHAs is linked to their ability to induce or to up-regulate on neoplastic cells the expression of a variety of immune molecules that play a crucial role in host-tumor immune interactions. To further investigate the clinical potential of diverse epigenetic compounds when combined with immunotherapeutic strategies, we have now compared the tumor immunomodulatory properties of the first generation DHAs, azacytidine (AZA) and decitabine (DAC) and of the next generation DHA, guadecitabine. To this end, human melanoma and hematological cancer cells were treated in vitro with 1 μM guadecitabine, DAC or AZA and then studied by molecular and flow cytometry analyses for changes in their baseline expression of selected immune molecules involved in different mechanism(s) of immune recognition. Results demonstrated a stronger DNA hypomethylating activity of guadecitabine and DAC, compared to AZA that associated with stronger immunomodulatory activities. Indeed, the mRNA expression of cancer testis antigens, immune-checkpoint blocking molecules, immunostimulatory cytokines, involved in NK and T cell signaling and recruiting, and of genes involved in interferon pathway was higher after guadecitabine and DAC compared to AZA treatment. Moreover, a stronger up-regulation of the constitutive expression of HLA class I antigens and of Intercellular Adhesion Molecule-1 was observed with guadecitabine and DAC compared to AZA. Guadecitabine and DAC seem to represent the optimal combination partners to improve the therapeutic efficacy of immunotherapeutic agents in combination/sequencing clinical studies.

Epigenetic alterations play a pivotal role in cancer development and progression. Pharmacologic reversion of such alterations is feasible, and second generation "epigenetic drugs" are in development and have been demonstrated to possess significant immunomodulatory properties. This knowledge, together with the availability of new and highly effective immunotherapeutic agents including immune checkpoint(s) blocking monoclonal antibodies, allows us to plan for highly innovative proof-of-principle combination studies that will likely open the path to more effective anticancer therapies.

Continuously improving knowledge of the fine mechanisms regulating cross-talk between immune cells, and of their multi-faceted interactions with cancer cells, has prompted the development of several novel immunotherapeutic strategies for cancer treatment. Among these, modulation of the host's immune system by targeting immunological synapses has shown notable clinical efficacy in different tumor types. Despite this, objective clinical responses and, more importantly, long-term survival are achieved only by a fraction of patients; therefore, identification of the mechanism(s) responsible for the differential effectiveness of immune checkpoint blockade in specific patient populations is an area of intense investigation. Neoplastic cells can activate multiple mechanisms to escape from immune control; among these, epigenetic reprogramming is emerging as a key player. Selected tumor-associated antigens, Human Leukocyte Antigens, and accessory/co-stimulatory molecules required for efficient recognition of neoplastic cells by the immune system have been shown to be epigenetically silenced or down-regulated in cancer. Consistent with the inherent reversibility of epigenetic silencing, "epigenetic" drugs, such as inhibitors of DNA methyltransferases and of histone deacetylases, can restore the functional expression of these down-regulated molecules, thus improving the recognition of cancer cells by both the innate and adaptive immune responses. This review focuses on the immunomodulatory activity of epigenetic drugs and on their proposed clinical use in novel combined chemo-immunotherapeutic regimens for the treatment of solid tumors.

2549 Background: DNA hypomethylating agents show broad immuno-modulatory activity in neoplastic cells, and may improve the effectiveness of cancer immunotherapies. The phase 1b NIBIT-M4 trial investigated a previously unexplored therapeutic strategy using the next-generation DNA hypomethylating agent guadecitabine sequenced with ipilimumab for the treatment of advanced melanoma. Methods: Patients with unresectable Stage III/IV melanoma received escalating doses of guadecitabine 30, 45 or 60 mg/m2 subcutaneously on Days 1–5 every three weeks, and ipilimumab 3 mg/kg intravenously on Day 1 every three weeks, starting one week after guadecitabine, for four cycles. Primary endpoints were the safety, tolerability and maximum tolerated dose of treatment; secondary endpoints included immune-related disease control and objective response. Genome-wide methylation, RNA sequencing, and immunohistochemistry analyses were performed on tumor samples collected at baseline, W4 and W12. (NCT02608437). Results: 19 patients were treated and evaluable for safety and efficacy. The most common treatment-related adverse events of any grade were myelotoxicity (n = 17; 89%) and immune-related adverse events (n = 12; 63%). Grade 3 or 4 myelotoxicity occurred in 15 (79%) patients. There were no dose limiting toxicities. Rates of immune-related disease control and objective response were 8/19 (42%) and 5/19 (26%), respectively. Exploratory analyses of tumour samples (n = 8) showed that median CpG site methylation at Week 4 (74.5%) and Week 12 (75.5%) was significantly lower (p < 0.05) than at baseline (80.3%), with a median of 2454 (Week 4) and 4131 (Week 12) differentially expressed genes identified compared to baseline; among the 136 pathways significantly modulated by treatment, the most frequently activated were immune-related. Tumour immune contexture analysis (n = 11) demonstrated up-regulation of Human Leukocyte Antigen (HLA) class I molecules on melanoma cells, and an increase in CD8+, PD-1+ T cells and in CD20+ B cells in post-treatment tumour core specimens. Conclusions: Sequential guadecitabine and ipilimumab is safe and tolerable in patients with metastatic melanoma, and has promising immunological and anti-tumour activity. Clinical trial information: NCT02608437.

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...

Epigenetic regulation allows heritably modulating gene expression profiles without modifying the primary sequence of gDNA. Under physiologic conditions, epigenetic patterns determine tissue-specific gene expression landscapes, gene imprinting, inactivation of chromosome X, and preservation of genomic stability. The most characterized mediators of epigenetic inheritance are gDNA methylation and histone posttranslational modifications that cooperate to alter chromatin state and genome transcription. According to these notions, it is not surprising that cancer cells invariantly deploy epigenetic alterations to achieve gene expression patterns required for neoplastic transformation and tumor progression. In this context, the recently uncovered use of epigenetic alterations by cancer cells to become stealth from the host's immune recognition has significant immunobiologic relevance in tumor progression, and it appears to have potential clinical usefulness. Indeed, immune evasion is among the major obstacles to further improve the efficacy of cancer immunotherapies and to increase long-lasting disease control. Luckily, different “epigenetic drugs” able to revert these “epimutations” are available, some of which have already been approved for clinical use. Here, we summarize the immunomodulatory activities of epigenetic drugs that lead to improved immune recognition of cancer cells and focus on the potential of this class of agents in improving the anticancer activity of novel immunotherapies through combinatorial epigenetic immunotherapy approaches. Clin Cancer Res; 21(18); 4040–7. ©2015 AACR.

Malignant mesothelioma (MM) is a highly aggressive malignancy with a very dismal prognosis. Current treatment for unresectable MM is largely unsatisfactory; therefore, new therapeutic approaches are eagerly awaited. A better understanding of the complex mechanisms of immune escape operated by neoplastic cells and the ability to unleash an efficient anti-tumor immune response by targeting regulatory immune checkpoint(s) with immunomodulatory monoclonal antibodies (mAbs), is leading to very promising clinical results in different tumor types. Herein, we highlight the clinical impact so far identified for these new immunomodulatory agents in MM patients and discuss their prospective use to design novel clinical trials.

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.

In the past two decades, the worldwide steadily rising incidence of melanoma, its dismal prognosis when locally advanced or metastatic, and the absence of clinically effective therapeutic options have prompted studies that generated extensive preclinical knowledge on the biology of melanoma cells and their interaction with the host's immune system. As a consequence, among solid tumors, melanoma represents a “model malignancy” to design and apply in the clinic new bioimmunotherapeutic approaches, that are eventually translated to solid tumors of different histotypes. Despite its waxing and waning appeal as a therapeutic strategy, antibody treatment still represents a promising clinical approach to melanoma. Europe is no exception in the clinical interest for antibodies as therapeutic tools in melanoma patients; European researchers have also focused on preclinical issues that may improve current antibody-based therapeutic approaches on the one hand, while providing novel clinical strategies on the other hand. Semin Oncol 29:471-478. Copyright 2002, Elsevier Science (USA). All rights reserved.

Cancer-testis antigens expressed by different-histotype transformed cells are suitable targets for tumor immunotherapy. However, their heterogeneous expression in neoplastic lesions limits the eligibility of patients for cancer-testis antigen-directed vaccination, and low levels of cancer-testis antigens' expression may impair immune recognition of malignant cells. Because of the primary clinical relevance of cancer-testis antigens' expression in neoplastic tissues, 68 unrelated or sequential metastatic lesions from 56 patients were used to characterize the molecular mechanisms regulating the presence and levels of expression of different cancer-testis antigens of the MAGE family (i.e., MAGE2, 3 and 4) in cutaneous melanoma. Polymerase chain reaction-based methylation analyses showed that methylation status of specific cytosine-guanine dinucleotides in the promoters of investigated cancer-testis antigens correlated with their heterogeneous expression within unrelated metastatic melanoma lesions, and with their homogeneous expression among sequential metastases from three patients with melanoma. Unlike methylated promoters, unmethylated promoters of MAGE2, 3 and 4 genes drove the expression of reporter gene-enhanced green fluorescent protein after transient transfection of cancer-testis antigen-positive Mel 142 melanoma cells. Furthermore, de novo expression of MAGE3 gene induced by the treatment of Mel 195 melanoma cells with the DNA hypomethylating agent 5-aza-2'-deoxycytidine was associated with a 6%-12% demethylation of selected cytosine-guanine dinucleotides in its promoter. Finally, 5-aza-2'-deoxycytidine induced a 16-fold increase of MAGE3 expression in Mel 313 melanoma cells expressing constitutively low levels of the antigen, but did not affect that of Mel 275 melanoma cells expressing high baseline levels of MAGE3. Overall, these findings identify promoter methylation as a shared mechanism directly regulating the expression of therapeutic cancer-testis antigens in metastatic melanomas, and foresee the clinical use of 5-aza-2'-deoxycytidine to design new chemoimmunotherapeutic strategies in patients with melanoma.

Monoclonal antibodies that target immune checkpoints are undoubtedly changing the therapeutic landscape of different human malignancies. Here we comment on the effects of blocking cytotoxic T lymphocyte-associated protein 4 (CTLA4) by means of the monoclonal antibody tremelimumab in patients with refractory malignant mesothelioma, a deadly disease with no effective therapeutic options.

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.

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.

Prognostic molecular markers are urgently needed for allowing to discriminate the clinical course of disease of melanoma patients, which is highly heterogeneous and unpredictable also within a specific clinicopathological stage and substage of disease. Alterations in DNA methylation have been reported to be widely present in cutaneous melanoma, profoundly impacting its biology. In line with this notion, we have identified methylation markers as independent prognostic factors in stage IIIC melanoma patients. In this chapter we describe the measurement of the methylation of the Long Interspersed Nucleotide Element-1 sequences in laser capture microdissected tumor tissues as a prognostic tool in stage III melanoma patients, which could help in achieving a more appropriate and patient-tailored clinical management of cutaneous melanoma.

Prognostic molecular markers are urgently needed for allowing to discriminate the clinical course of disease of melanoma patients, which is highly heterogeneous and unpredictable also within a specific clinicopathological stage and substage of disease. Alterations in DNA methylation have been reported to be widely present in cutaneous melanoma, profoundly impacting its biology. In line with this notion, we have identified methylation markers as independent prognostic factors in stage IIIC melanoma patients. In this chapter we describe the measurement of the methylation of the Long Interspersed Nucleotide Element-1 sequences in laser capture microdissected tumor tissues as a prognostic tool in stage III melanoma patients, which could help in achieving a more appropriate and patient-tailored clinical management of cutaneous melanoma.

Malignant melanoma is a complex disease that arises and evolves due to a myriad of genetic and epigenetic events. Among these, the interaction between epigenetic alterations (i.e., histone modifications, DNA methylation, mRNA silencing by miRNAs and nucleosome repositioning) has been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, DNA repair, apoptosis, invasion and immune recognition. Differently to genetic lesions, epigenetic changes are potentially pharmacologically reversible by using epigenetic drugs. Along this line, preclinical and clinical findings indicate that these drugs, given alone or in combination therapies, can efficiently modulate the immunophenotype of melanoma cells. The aim of this review is to provide a comprehensive summary of melanoma epigenetics and the current use of epigenetic drugs in the clinical setting.

Immune checkpoint(s) blockade is becoming the therapeutic mainstay in melanoma and lung cancer. Based on these important clinical achievements, immunotherapy with immunomodulating monoclonal antibodies (mAb) is being explored as a new therapeutic modality in most human malignancies, either alone or in combination with other immunomodulating agents. We have provided extensive evidence that the second generation DNA hypomethylating agent (DHA), guadecitabine (SGI-110), plays a promising role in potentiating the immunogenicity and the immune recognition of human malignancies through the up-regulation of the expression of different immune molecules on cancer cells. These findings led us to hypothesize that DHA could represent ideal partners for immune checkpoint(s) blocking agents to improve their therapeutic efficacy. Here, we studied the expression of programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), and 2 (PD-L2), and cytotoxic T lymphocyte-associated antigen 4 (CTLA4) in peripheral blood mononuclear cells of acute myeloid leukemia (AML) patients (N = 23), enrolled in a phase 1-2 study with guadecitabine. Patients included in this preliminary analysis were selected based on their responsiveness to therapy. Quantitative RT-PCR analyses showed a constitutive expression (gene/β-actin molecules >7.5E-05) of PD1, PD-L1, PD-L2 and CTLA-4 in 82.6%, 100%, 47.8% and 8.7% patients, respectively. Guadecitabine therapy up-regulated (≥2 fold) the expression of PD1, PD-L1, PD-L2 and CTLA-4 in 57.9%, 56.5%, 18.2% and 0% patients, respectively, at any of the time-points investigated during treatment. De novo expression of PD1, PD-L2 and CTLA-4 was observed in 100%, 41.6% and 38.1% of the immune checkpoint(s)-negative patients, respectively. Though preliminary, these results further support the strong link between epigenetics/DNA methylation and immune response, in cancer patients, and strengthen the therapeutic potential of epigenetic immunomodulation, with “consolidated” and emerging immune checkpoint(s) blocking mAb. Citation Format: Carolina Fazio, Alessia Covre, Maria Lofiego, Pietro Taverna, Mohammad Azab, James N. Lowder, Sandra Coral, Michele Maio. Immune checkpoint(s) expression in AML patients enrolled in a phase 1-2 study with guadecitabine. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2325.

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.

Background The prognosis of cutaneous melanoma (CM) differs for patients with identical clinico-pathological stage, and no molecular markers discriminating the prognosis of stage III individuals have been established. Genome-wide alterations in DNA methylation are a common event in cancer. This study aimed to define the prognostic value of genomic DNA methylation levels in stage III CM patients. Methods Overall level of genomic DNA methylation was measured using bisulfite pyrosequencing at three CpG sites (CpG1, CpG2, CpG3) of the Long Interspersed Nucleotide Element-1 (LINE-1) sequences in short-term CM cultures from 42 stage IIIC patients. The impact of LINE-1 methylation on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analysis. Results Hypomethylation (i.e., methylation below median) at CpG2 and CpG3 sites significantly associated with improved prognosis of CM, CpG3 showing the strongest association. Patients with hypomethylated CpG3 had increased OS (P = 0.01, log-rank = 6.39) by Kaplan-Meyer analysis. Median OS of patients with hypomethylated or hypermethylated CpG3 were 31.9 and 11.5 months, respectively. The 5 year OS for patients with hypomethylated CpG3 was 48% compared to 7% for patients with hypermethylated sequences. Among the variables examined by Cox regression analysis, LINE-1 methylation at CpG2 and CpG3 was the only predictor of OS (Hazard Ratio = 2.63, for hypermethylated CpG3; 95% Confidence Interval: 1.21-5.69; P = 0.01). Conclusion LINE-1 methylation is identified as a molecular marker of prognosis for CM patients in stage IIIC. Evaluation of LINE-1 promises to represent a key tool for driving the most appropriate clinical management of stage III CM patients.

Cancer testis antigens (CTA) are a large family of tumor-associated antigens expressed in human tumors of different histological origin, but not in normal tissues except for testis and placenta. This tumor-restricted pattern of expression, together with their strong in vivo immunogenicity, identified CTA as ideal targets for tumor-specific immunotherapeutic approaches, and prompted the development of several clinical trials of CTA-based vaccine therapy. Driven by this practical clinical interest, a more detailed characterization of CTA biology has been recently undertaken. So far, at least 70 families of CTA, globally accounting for about 140 members, have been identified. Most of these CTA are expressed during spermatogenesis, but their function is still largely unknown. Epigenetic events, particularly DNA methylation, appear to be the primary mechanism regulating CTA expression in both normal and transformed cells, as well as in cancer stem cells. In view of the growing interest in CTA biology, the aim of this review is to provide the most recent information on their expression, regulation and function, together with a brief summary of the major clinical trials involving CTA as therapeutic agents. The pharmacologic modulation of CTA expression profiles on neoplastic cells by DNA hypomethylating drugs will also be discussed as a feasible approach to design new combination therapies potentially able to improve the clinical efficacy of currently adopted CTA-based immunotherapeutic regimens in cancer patients.

NIBIT (acronym for the Network Italiano per la Bioterapia dei Tumori—Italian Network for Tumor Biotherapy) is a non-profit association created in 2004 to promote and foster scientific and operative interactions among Italian professionals involved in the field of cancer bioimmunotherapy. To date, more than 100 members representing over 40 national academic, regulatory, and industrial groups are part of the NIBIT. Aim of the annual meeting of the NIBIT is the discussion of recent preclinical and clinical results obtained by various Italian groups of the Network in the field of cancer immunology and biotherapy. Traditionally, the NIBIT meeting also hosts foreign speakers for keynote lectures on hot topics in the NIBIT field of interest. Like every year, the eighth annual meeting of the NIBIT took place in the Certosa of Pontignano, a Tuscan Carthusian monastery close to Siena. The 2010 main topics were as follows: (1) immunology of cancer stem cells; (2) prostate cancer immunotherapy; (3) immunomodulation therapy; (4) new aspects in cancer vaccines; (5) NIBIT and other Italian clinical trials.

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.

Cutaneous melanoma is a very aggressive neoplasia of melanocytic origin with constantly growing incidence and mortality rates world-wide. Epigenetic modifications (i.e., alterations of genomic DNA methylation patterns, of post-translational modifications of histones, and of microRNA profiles) have been recently identified as playing an important role in melanoma development and progression by affecting key cellular pathways such as cell cycle regulation, cell signalling, differentiation, DNA repair, apoptosis, invasion and immune recognition. In this scenario, pharmacologic inhibition of DNA methyltransferases and/or of histone deacetylases were demonstrated to efficiently restore the expression of aberrantly-silenced genes, thus re-establishing pathway functions. In light of the pleiotropic activities of epigenetic drugs, their use alone or in combination therapies is being strongly suggested, and a particular clinical benefit might be expected from their synergistic activities with chemo-, radio-, and immuno-therapeutic approaches in melanoma patients. On this path, an important improvement would possibly derive from the development of new generation epigenetic drugs characterized by much reduced systemic toxicities, higher bioavailability, and more specific epigenetic effects.

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.

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.

Cancer/testis antigens (CTA) are tumor-associated antigens expressed during ontogenesis, in a number of solid tumors but not in normal tissues except testis. Most of these CTA are highly immunogenic, eliciting a humoral and cellular response in the patients with advanced cancer, and are useful for tumor-specific immunotherapy. Medullary thyroid carcinoma (MTC) is a neoplasm derived from the parafollicular cells of the thyroid and occurs in either a sporadic or a familial form. In the present study, we examined by RT-PCR the expression of a number of genes encoding CTA in 23 surgical samples of sporadic MTC. Among the 11 cDNA antigens examined, RAGE, MAGE-4, and GAGE 1-2, were not expressed in any of the tissues. SSX 2 was present only in one tissue, whereas BAGE, GAGE 1-6, MAGE-1, MAGE-2, MAGE-3, and SSX 1-5 were detected in two to five samples. NY-ESO-1 cDNA was the most frequent, being detected in 15 of 23 examined samples (65.2%). Six (26.1%) tissues did not express any CTA-specific mRNA, whereas 10 tumors expressed only one gene (43.5%), 3 (21.4%) expressed 2 genes, and 4 displayed a broad CTA gene expression. NY-ESO-1 expression in primary MTC tissues significantly correlated with tumor recurrence. The presence of specific anti-NY-ESO-1 antibodies was searched in the sera of MTC-affected patients examined by ELISA using recombinant NY-ESO-1 protein. A humoral response against this CTA was detected in 6 of 11 NY-ESO-1 expressing patients (54.5%), and in 1 of 6 patients with NY-ESO-1-negative tumor. No anti-NY-ESO-1 antibodies were detected in healthy subjects (n = 17). The presence of anti-NY-ESO-1 antibodies was searched also in the sera of MTC affected patients whose tissues were not available for CTA analysis. Anti-NY-ESO-1 antibodies were present in 15 of 42 sera (35.7%), demonstrating that MTC is a neoplasm frequently associated with humoral immune response to NY-ESO-1. Serological survey may be useful as a way to identify patients with humoral immune response to NY-ESO-1 that provide a new attractive target for vaccine-based immunotherapy of MTC.