Giulia Parisi

The biology of cancer testis antigens: Putative function, regulation and therapeutic potential

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.

Epigenetics of human cutaneous melanoma: setting the stage for new therapeutic strategies

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.

Methylation levels of the "long interspersed nucleotide element-1" repetitive sequences predict survival of melanoma patients

BackgroundThe 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.MethodsOverall 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.ResultsHypomethylation (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).ConclusionLINE-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.

Immunomodulatory activity of SGI-110, a 5-aza-2′-deoxycytidine-containing demethylating dinucleotide

PurposePharmacologic DNA hypomethylation holds strong promises in cancer immunotherapy due to its immunomodulatory activity on neoplastic cells. Searching for more efficient DNA hypomethylating agents to be utilized to design novel immunotherapeutic strategies in cancer, we investigated the immunomodulatory properties of the new DNA hypomethylating agent SGI-110, that is resistant to in vivo inactivation by cytidine deaminase.Experimental designCutaneous melanoma, mesothelioma, renal cell carcinoma, and sarcoma cells were treated in vitro with SGI-110. RT-PCR, quantitative RT-PCR, quantitative methylation-specific PCR, and flow cytometric analyses were performed to investigate changes induced by SGI-110 in the constitutive immune profile of cancer cells. The recognition by gp100-specific CTL of gp100-positive melanoma cells, treated or not with SGI-110, was tested by LDH release assays.ResultsSGI-110 induced/up-regulated the expression of investigated cancer/testis antigens (CTA) (i.e., MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A10, GAGE 1-2, GAGE 1-6, NY-ESO-1, and SSX 1-5) in all cancer cell lines studied, both at mRNA and at protein levels. Quantitative methylation-specific PCR analyses identified a hypomethylation of MAGE-A1 and NY-ESO-1 promoters in SGI-110-treated neoplastic cells, demonstrating a direct role of pharmacologic DNA demethylation in CTA induction. SGI-110 also up-regulated the expression of HLA class I antigens and of ICAM-1, resulting in an improved recognition of cancer cells by gp100-specific CTL.ConclusionsOur findings show that SGI-110 is a highly attractive therapeutic agent to comprehensively increase immunogenicity and immune recognition of neoplastic cells, and provide the scientific rationale for its clinical development to design novel chemo-immunotherapeutic approaches in cancer patients.

Whole genome methylation profiles as independent markers of survival in stage IIIC melanoma patients

BackgroundThe clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis. This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM.MethodsGenome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients. Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles. Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm. The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses.ResultsUnsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients. Patients with a “favorable” methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis. Median OS of stage IIIC patients with a “favorable” vs. “unfavorable” methylation profile were 31.5 and 10.4 months, respectively. The 5 year OS for stage IIIC patients with a “favorable” methylation profile was 41.2% as compared to 0% for patients with an “unfavorable” methylation profile. Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for “unfavorable” methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045). A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified.ConclusionsA discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients. Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.

Antitumor activity of epigenetic immunomodulation combined with CTLA-4 blockade in syngeneic mouse models

The multifaceted immunomodulatory activity of DNA hypomethylating agents improves immunogenicity and immune recognition of neoplastic cells; thus, we predicted they could be utilized to design new immunotherapeutic combinations in cancer. Testing this hypothesis, the antitumor efficacy of the DNA hypomethylating agent 5-aza-2′-deoxycytidine (5-AZA-CdR) combined with the anti-CTLA-4 monoclonal antibody (mAb) 9H10 in syngeneic transplantable murine models was investigated. Murine mammary carcinoma TS/A or mesothelioma AB1 cells were injected in BALB/c, athymic nude, and SCID/Beige mice that were treated with 5-AZA-CdR, mAb 9H10, or their combination. Tumor volumes were captured at different time-points; molecular and immunohistochemical assays investigated changes in neoplastic and normal tissues. A significant antitumor effect of 5-AZA-CdR combined with mAb 9H10 was found: compared to controls, a 77% (p < 0.01), 54% (p < 0.01) and 33% (p = 0.2) decrease in TS/A tumor growth was induced by 5-AZA-CdR combined with mAb 9H10, 5-AZA-CdR or mAb 9H10, respectively. These antitumor activities were confirmed utilizing the AB1 model. 5-AZA-CdR-based regimens induced a promoter-demethylation-sustained tumor expression of cancer testis antigens. MHC class I expression was up-regulated by 5-AZA-CdR. Antitumor efficacy of 5-AZA-CdR in athymic nude and SCID/Beige mice was not increased by mAb 9H10. In BALB/c mice, combined treatment induced the highest tumor infiltration by CD3+ lymphocytes, which included both CD8+ and CD4+ T cells; no such infiltrates were observed in normal tissues. This significant immune-related antitumor activity of 5-AZA-CdR combined with CTLA-4 blockade, demonstrated in highly aggressive mouse tumor models, provides a strong scientific rationale to implement epigenetically-based immunotherapies in cancer patients.

Epigenetic remodelling of gene expression profiles of neoplastic and normal tissues: immunotherapeutic implications

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.

Stability of BRAF V600E mutation in metastatic melanoma: new insights for therapeutic success?

Stability of BRAF V600E mutation in metastatic melanoma: new insights for therapeutic success?

Epigenetics of melanoma: implications for immune-based therapies

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.

Epigenetic markers of prognosis in 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.