Epigenetic remodeling to improve the efficacy of immunotherapy in human glioblastoma: pre-clinical evidence for development of new immunotherapy approaches.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive primary brain tumor, that is refractory to standard treatment and to immunotherapy with immune-checkpoint inhibitors (ICI). Noteworthy, melanoma brain metastases (MM-BM), that share the same niche as GBM, frequently respond to current ICI therapies. Epigenetic modifications regulate GBM cellular proliferation, invasion, and prognosis and may negatively regulate the cross-talk between malignant cells and immune cells in the tumor milieu, likely contributing to limit the efficacy of ICI therapy of GBM. Thus, manipulating the tumor epigenome can be considered a therapeutic opportunity in GBM. METHODS: Microarray transcriptional and methylation profiles, followed by gene set enrichment and IPA analyses, were performed to study the differences in the constitutive expression profiles of GBM vs MM-BM cells, compared to the extracranial MM cells and to investigate the modulatory effects of the DNA hypomethylating agent (DHA) guadecitabine among the different tumor cells. The prognostic relevance of DHA-modulated genes was tested by Cox analysis in a TCGA GBM patients' cohort. RESULTS: The most striking differences between GBM and MM-BM cells were found to be the enrichment of biological processes associated with tumor growth, invasion, and extravasation with the inhibition of MHC class II antigen processing/presentation in GBM cells. Treatment with guadecitabine reduced these biological differences, shaping GBM cells towards a more immunogenic phenotype. Indeed, in GBM cells, promoter hypomethylation by guadecitabine led to the up-regulation of genes mainly associated with activation, proliferation, and migration of T and B cells and with MHC class II antigen processing/presentation. Among DHA-modulated genes in GBM, 7.6% showed a significant prognostic relevance. Moreover, a large set of immune-related upstream-regulators (URs) were commonly modulated by DHA in GBM, MM-BM, and MM cells: DHA-activated URs enriched for biological processes mainly involved in the regulation of cytokines and chemokines production, inflammatory response, and in Type I/II/III IFN-mediated signaling; conversely, DHA-inhibited URs were involved in metabolic and proliferative pathways. CONCLUSIONS: Epigenetic remodeling by guadecitabine represents a promising strategy to increase the efficacy of cancer immunotherapy of GBM, supporting the rationale to develop new epigenetic-based immunotherapeutic approaches for the treatment of this still highly deadly disease.

A novel microRNA signature for the detection of melanoma by liquid biopsy.

BACKGROUND: Melanoma is the deadliest form of skin cancer and metastatic disease is associated with a significant survival rate drop. There is an urgent need for consistent tumor biomarkers to scale precision medicine and reduce cancer mortality. Here, we aimed to identify a melanoma-specific circulating microRNA signature and assess its value as a diagnostic tool. METHODS: The study consisted of a discovery phase and two validation phases. Circulating plasma extracellular vesicles (pEV) associated microRNA profiles were obtained from a discovery cohort of metastatic melanoma patients and normal subjects as controls. A pEV-microRNA signature was obtained using a LASSO penalized logistic regression model. The pEV-microRNA signature was subsequently validated both in a publicly available dataset and in an independent internal cohort. RESULTS: We identified and validated in three independent cohorts a panel of melanoma-specific circulating microRNAs that showed high accuracy in differentiating melanoma patients from healthy subjects with an area under the curve (AUC) of 1.00, 0.94 and 0.75 respectively. Investigation of the function of the pEV-microRNA signature evidenced their possible immune suppressive role in melanoma patients. CONCLUSIONS: We demonstrate that a blood test based on circulating microRNAs can non-invasively detect melanoma, offering a novel diagnostic tool for improving standard care. Moreover, we revealed an immune suppressive role for melanoma pEV-microRNAs.

A "Dirty" Footprint: Macroinvertebrate diversity in Amazonian Anthropic Soils.

Amazonian rainforests, once thought to be pristine wilderness, are increasingly known to have been widely inhabited, modified, and managed prior to European arrival, by human populations with diverse cultural backgrounds. Amazonian Dark Earths (ADEs) are fertile soils found throughout the Amazon Basin, created by pre-Columbian societies with sedentary habits. Much is known about the chemistry of these soils, yet their zoology has been neglected. Hence, we characterized soil fertility, macroinvertebrate communities, and their activity at nine archeological sites in three Amazonian regions in ADEs and adjacent reference soils under native forest (young and old) and agricultural systems. We found 673 morphospecies and, despite similar richness in ADEs (385 spp.) and reference soils (399 spp.), we identified a tenacious pre-Columbian footprint, with 49% of morphospecies found exclusively in ADEs. Termite and total macroinvertebrate abundance were higher in reference soils, while soil fertility and macroinvertebrate activity were higher in the ADEs, and associated with larger earthworm quantities and biomass. We show that ADE habitats have a unique pool of species, but that modern land use of ADEs decreases their populations, diversity, and contributions to soil functioning. These findings support the idea that humans created and sustained high-fertility ecosystems that persist today, altering biodiversity patterns in Amazonia.

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

PURPOSE: Pharmacologic 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 DESIGN: Cutaneous 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. RESULTS: SGI-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. CONCLUSIONS: Our 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.

Guadecitabine plus ipilimumab in unresectable melanoma: five-year follow-up and integrated multi-omic analysis in the phase 1b NIBIT-M4 trial.

Association with hypomethylating agents is a promising strategy to improve the efficacy of immune checkpoint inhibitors-based therapy. The NIBIT-M4 was a phase Ib, dose-escalation trial in patients with advanced melanoma of the hypomethylating agent guadecitabine combined with the anti-CTLA-4 antibody ipilimumab that followed a traditional 3 + 3 design (NCT02608437). Patients received guadecitabine 30, 45 or 60 mg/m2/day subcutaneously on days 1 to 5 every 3 weeks starting on week 0 for a total of four cycles, and ipilimumab 3 mg/kg intravenously starting on day 1 of week 1 every 3 weeks for a total of four cycles. Primary outcomes of safety, tolerability, and maximum tolerated dose of treatment were previously reported. Here we report the 5-year clinical outcome for the secondary endpoints of overall survival, progression free survival, and duration of response, and an exploratory integrated multi-omics analysis on pre- and on-treatment tumor biopsies. With a minimum follow-up of 45 months, the 5-year overall survival rate was 28.9% and the median duration of response was 20.6 months. Re-expression of immuno-modulatory endogenous retroviruses and of other repetitive elements, and a mechanistic signature of guadecitabine are associated with response. Integration of a genetic immunoediting index with an adaptive immunity signature stratifies patients/lesions into four distinct subsets and discriminates 5-year overall survival and progression free survival. These results suggest that coupling genetic immunoediting with activation of adaptive immunity is a relevant requisite for achieving long term clinical benefit by epigenetic immunomodulation in advanced melanoma patients.

Amazonian earthworm biodiversity is heavily impacted by ancient and recent human disturbance.

Despite the importance of earthworms for soil formation, more is needed to know about how Pre-Columbian modifications to soils and the landscape. Gaining a deeper understanding is essential for comprehending the historical drivers of earthworm communities and the development of effective conservation strategies in the Amazon rainforest. Human disturbance can significantly impact earthworm diversity, especially in rainforest soils, and in the particular case of the Amazonian rainforest, both recent and ancient anthropic practices may be important. Amazonian Dark Earths (ADEs) are fertile soils found throughout the Amazon Basin, created by sedentary habits and intensification patterns of pre-Colombian societies primarily developed in the second part of the Holocene period. We have sampled earthworm communities in three Brazilian Amazonian (ADEs) and adjacent reference soils (REF) under old and young forests and monocultures. To better assess taxonomic richness, we used morphology and the barcode region of the COI gene to identify juveniles and cocoons and delimit Molecular Operational Taxonomic Units (MOTUs). Here we suggest using Integrated Operational Taxonomical units (IOTUs) which combine both morphological and molecular data and provide a more comprehensive assessment of diversity, while MOTUs only rely on molecular data. A total of 970 individuals were collected, resulting in 51 taxonomic units (IOTUs, MOTUs, and morphospecies combined). From this total, 24 taxonomic units were unique to REF soils, 17 to ADEs, and ten were shared between both soils. The highest richness was found in old forest sites for ADEs (12 taxonomic units) and REFs (21 taxonomic units). The beta-diversity calculations reveal a high species turnover between ADEs and REF soils, providing evidence that ADEs and REFs possess distinct soil biota. Furthermore, results suggest that ADE sites, formed by Pre-Columbian human activities, conserve a high number of native species in the landscape and maintain a high abundance, despite their long-term nature.

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

BACKGROUND: The 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. METHODS: Genome-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. RESULTS: Unsupervised 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. CONCLUSIONS: A 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.

Landscape of immune-related signatures induced by targeting of different epigenetic regulators in melanoma: implications for immunotherapy.

BACKGROUND: Improvement of efficacy of immune checkpoint blockade (ICB) remains a major clinical goal. Association of ICB with immunomodulatory epigenetic drugs is an option. However, epigenetic inhibitors show a heterogeneous landscape of activities. Analysis of transcriptional programs induced in neoplastic cells by distinct classes of epigenetic drugs may foster identification of the most promising agents. METHODS: Melanoma cell lines, characterized for mutational and differentiation profile, were treated with inhibitors of DNA methyltransferases (guadecitabine), histone deacetylases (givinostat), BET proteins (JQ1 and OTX-015), and enhancer of zeste homolog 2 (GSK126). Modulatory effects of epigenetic drugs were evaluated at the gene and protein levels. Master molecules explaining changes in gene expression were identified by Upstream Regulator (UR) analysis. Gene set enrichment and IPA were used respectively to test modulation of guadecitabine-specific gene and UR signatures in baseline and on-treatment tumor biopsies from melanoma patients in the Phase Ib NIBIT-M4 Guadecitabine + Ipilimumab Trial. Prognostic significance of drug-specific immune-related genes was tested with Timer 2.0 in TCGA tumor datasets. RESULTS: Epigenetic drugs induced different profiles of gene expression in melanoma cell lines. Immune-related genes were frequently upregulated by guadecitabine, irrespective of the mutational and differentiation profiles of the melanoma cell lines, to a lesser extent by givinostat, but mostly downregulated by JQ1 and OTX-015. GSK126 was the least active drug. Quantitative western blot analysis confirmed drug-specific modulatory profiles. Most of the guadecitabine-specific signature genes were upregulated in on-treatment NIBIT-M4 tumor biopsies, but not in on-treatment lesions of patients treated only with ipilimumab. A guadecitabine-specific UR signature, containing activated molecules of the TLR, NF-kB, and IFN innate immunity pathways, was induced in drug-treated melanoma, mesothelioma and hepatocarcinoma cell lines and in a human melanoma xenograft model. Activation of guadecitabine-specific UR signature molecules in on-treatment tumor biopsies discriminated responding from non-responding NIBIT-M4 patients. Sixty-five % of the immune-related genes upregulated by guadecitabine were prognostically significant and conferred a reduced risk in the TCGA cutaneous melanoma dataset. CONCLUSIONS: The DNMT inhibitor guadecitabine emerged as the most promising immunomodulatory agent among those tested, supporting the rationale for usage of this class of epigenetic drugs in combinatorial immunotherapy approaches.

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

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.

Epigenetic Immune Remodeling of Mesothelioma Cells: A New Strategy to Improve the Efficacy of Immunotherapy.

Malignant pleural mesothelioma (MPM) is an aggressive malignancy with a severe prognosis, and with a long-standing need for more effective therapeutic approaches. However, treatment with immune checkpoint inhibitors is becoming an increasingly effective strategy for MPM patients. In this scenario, epigenetic modifications may negatively regulate the interplay between immune and malignant cells within the tumor microenvironment, thus contributing to the highly immunosuppressive contexture of MPM that may limit the efficacy of immunotherapy. Aiming to further improve prospectively the clinical efficacy of immunotherapeutic approaches in MPM, we investigated the immunomodulatory potential of different classes of epigenetic drugs (i.e., DNA hypomethylating agent (DHA) guadecitabine, histone deacetylase inhibitors VPA and SAHA, or EZH2 inhibitors EPZ-6438) in epithelioid, biphasic, and sarcomatoid MPM cell lines, by cytofluorimetric and real-time PCR analyses. We also characterized the effects of the DHA, guadecitabine, on the gene expression profiles (GEP) of the investigated MPM cell lines by the nCounter platform. Among investigated drugs, exposure of MPM cells to guadecitabine, either alone or in combination with VPA, SAHA and EPZ-6438 demonstrated to be the main driver of the induction/upregulation of immune molecules functionally crucial in host-tumor interaction (i.e., HLA class I, ICAM-1 and cancer testis antigens) in all three MPM subtypes investigated. Additionally, GEP demonstrated that treatment with guadecitabine led to the activation of genes involved in several immune-related functional classes mainly in the sarcomatoid subtype. Furthermore, among investigated MPM subtypes, DHA-induced CDH1 expression that contributes to restoring the epithelial phenotype was highest in sarcomatoid cells. Altogether, our results contribute to providing the rationale to develop new epigenetically-based immunotherapeutic approaches for MPM patients, potentially tailored to the specific histologic subtypes.

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

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

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.

[Social considerations affecting acceptance of HPV vaccination in Colombia. A systematic review].

OBJECTIVE: To identify social factors influencing the acceptance of human papilloma virus (HPV) vaccination in the Colombian population before and after the unexpected and poorly defined event of unknown etiology which occurred in 2014. METHODS: A systematic review of the literature was conducted in the following databases: Scopus, Web of Science, Medline via PubMed, Embase, Online Health Library (Biblioteca Virtual en Salud) and Ovid, and also in Google Scholar, academic repositories and in Colombian health institutions, using the terms "recombinant tetravalent vaccine against Human Papilloma Virus types 6, 11, 16, 18", "Colombia", "Papilloma" in order to primarily identify systematic reviews, quantitative and qualitative studies, narrative reviews, focusing on social aspects such as education, access, relationship with healthcare staff and role of the media which may have acted as barriers or facilitators for the acceptance of HPV vaccination in Colombia between 2006-2018. A narrative synthesis of the data was made. RESULTS: Twenty-four documents were included. The importance attached by parents, adolescents, providers and the media to having greater knowledge about HPV and its association with cervical cancer was identified. The relevance of good communication among healthcare professions and the community to enable adequate sharing of information regarding the risks and benefits of the vaccines was recognized. The inclusion of the vaccine in health insurance plans made access easier. The media must be involved as facilitators in vaccination programs. CONCLUSIONS: Education regarding HPV, patient-centered healthcare and adequate media coverage influence the acceptance of HPV vaccination in the Colombian population. Close follow-up of any vaccine-related adverse events is required.

TITULO: ASPECTOS SOCIALES QUE HAN AFECTADO LA ACEPTACIÓN DE LA VACUNACIÓN CONTRA EL VIRUS DEL PAPILOMA HUMANO EN COLOMBIA. UNA REVISIÓN SISTEMÁTICA. OBJETIVO: Establecer los aspectos sociales que afectaron la aceptación de la vacuna del virus del papi- loma humano (VPH) en la población colombiana antes y después del evento mal definido e inusitado de etiología desconocida sucedido en 2014. METODOS: Se realizó una búsqueda sistemática de literatura en las bases de datos: Scopus, Web of Science, Medline vía PubMed, Embase, Biblioteca Virtual en Salud y Ovid; además, en Google Académico y en repositorios de universidades y en instituciones de salud en Colombia, con los términos: "Vacuna Tetravalente Recombinante contra el Virus del Papiloma Humano Tipos 6, 11, 16, 18", "Colombia", "Papiloma" y sus correspondientes términos en inglés, para identificar principalmente revisiones sistemáticas, estudios cuantitativos y cualitativos, y revisiones narrativas que se enfocaran en aspectos sociales como: educación, acceso, relación con el personal de salud, papel de los medios de comunicación, que pudieran haber actuado como barreras o facilitadores para la aceptación de vacunación para VPH en Colombia en el periodo 2006-2018. Se hace una síntesis narrativa de la información. RESULTADOS: Se incluyeron 24 documentos. Se identificó la importancia, para los padres, adolescentes, proveedores y los medios, de tener un mejor conocimiento del VPH y su relación con el cáncer de cuello uterino (CCU). Se reconoce la relevancia de una buena comunicación entre las profesiones de la salud y la comunidad para informar adecuadamente tanto los beneficios como los riesgos de la vacuna. La inclusión en los planes de aseguramiento facilitó el acceso a esta por parte de la población. Los medios de comunicación deben ser considerados para que actúen como facilitadores de los programas de vacunación. CONCLUSIONES: Educar en el conocimiento del VPH, una atención en salud centrada en el paciente y una adecuada cobertura de los medios de comunicación influencian la aceptación del programa de vacuna- ción contra VPH en la población colombiana. Se requiere seguir haciendo seguimiento estricto de los efectos adversos asociados a la vacuna.

Circulating Levels of PD-L1 in Mesothelioma Patients from the NIBIT-MESO-1 Study: Correlation with Survival.

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 < 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 < 0.01)) or FC values above (p < 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 < 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.

Guadecitabine Plus Ipilimumab in Unresectable Melanoma: The NIBIT-M4 Clinical Trial.

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.

Immunomodulatory Properties of DNA Hypomethylating Agents: Selecting the Optimal Epigenetic Partner for 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.

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

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

Epigenetic targets for immune intervention in human malignancies.

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

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

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