Antibody dependent cellular cytotoxicity-inducing anti-EGFR antibodies as effective therapeutic option for cutaneous melanoma resistant to BRAF inhibitors.

Introduction: About 50% of cutaneous melanoma (CM) patients present activating BRAF mutations that can be effectively targeted by BRAF inhibitors (BRAFi). However, 20% of CM patients exhibit intrinsic drug resistance to BRAFi, while most of the others develop adaptive resistance over time. The mechanisms involved in BRAFi resistance are disparate and globally seem to rewire the cellular signaling profile by up-regulating different receptor tyrosine kinases (RTKs), such as the epidermal growth factor receptor (EGFR). RTKs inhibitors have not clearly demonstrated anti-tumor activity in BRAFi resistant models. To overcome this issue, we wondered whether the shared up-regulated RTK phenotype associated with BRAFi resistance could be exploited by using immune weapons as the antibody-dependent cell cytotoxicity (ADCC)-mediated effect of anti-RTKs antibodies, and kill tumor cells independently from the mechanistic roots. Methods and results: By using an in vitro model of BRAFi resistance, we detected increased membrane expression of EGFR, both at mRNA and protein level in 4 out of 9 BRAFi-resistant (VR) CM cultures as compared to their parental sensitive cells. Increased EGFR phosphorylation and AKT activation were observed in the VR CM cultures. EGFR signaling appeared dispensable for maintaining resistance, since small molecule-, antibody- and CRISPR-targeting of EGFR did not restore sensitivity of VR cells to BRAFi. Importantly, immune-targeting of EGFR by the anti-EGFR antibody cetuximab efficiently and specifically killed EGFR-expressing VR CM cells, both in vitro and in humanized mouse models in vivo, triggering ADCC by healthy donors' and patients' peripheral blood cells. Conclusion: Our data demonstrate the efficacy of immune targeting of RTKs expressed by CM relapsing on BRAFi, providing the proof-of-concept supporting the assessment of anti-RTK antibodies in combination therapies in this setting. This strategy might be expected to concomitantly trigger the crosstalk of adaptive immune response leading to a complementing T cell immune rejection of tumors.

Whole Genome Sequence Dataset of Mycobacterium tuberculosis Strains from Patients of Campania Region.

Tuberculosis (TB) is one of the deadliest infectious disorders in the world. To effectively TB manage, an essential step is to gain insight into the lineage of Mycobacterium tuberculosis (MTB) and the distribution of drug resistance. Although the Campania region is declared a cluster area for the infection, to contribute to the effort to understand TB evolution and transmission, still poorly known, we have generated a dataset of 159 genomes of MTB strains, from Campania region collected during 2018-2021, obtained from the analysis of whole genome sequence. The results show that the most frequent MTB lineage is the 4 according for 129 strains (81.11%). Regarding drug resistance, 139 strains (87.4%) were classified as multi susceptible, while the remaining 20 (12.58%) showed drug resistance. Among the drug-resistance strains, 8 were isoniazid-resistant MTB, 4 multidrug-resistant MTB, while only one was classified as pre-extensively drug-resistant MTB. This dataset expands the existing available knowledge on drug resistance and evolution of MTB, contributing to further TB-related genomics studies to improve the management of this disease.

Glucose Deprivation Promotes Pseudohypoxia and Dedifferentiation in Lung Adenocarcinoma.

Increased utilization of glucose is a hallmark of cancer. Sodium-glucose transporter 2 (SGLT2) is a critical player in glucose uptake in early-stage and well-differentiated lung adenocarcinoma (LUAD). SGLT2 inhibitors, which are FDA approved for diabetes, heart failure, and kidney disease, have been shown to significantly delay LUAD development and prolong survival in murine models and in retrospective studies in diabetic patients, suggesting that they may be repurposed for lung cancer. Despite the antitumor effects of SGLT2 inhibition, tumors eventually escape treatment. Here, we studied the mechanisms of resistance to glucose metabolism-targeting treatments. Glucose restriction in LUAD and other tumors induced cancer cell dedifferentiation, leading to a more aggressive phenotype. Glucose deprivation caused a reduction in alpha-ketoglutarate (αKG), leading to attenuated activity of αKG-dependent histone demethylases and histone hypermethylation. The dedifferentiated phenotype depended on unbalanced EZH2 activity that suppressed prolyl-hydroxylase PHD3 and increased expression of hypoxia-inducible factor 1α (HIF1α), triggering epithelial-to-mesenchymal transition. Finally, a HIF1α-dependent transcriptional signature of genes upregulated by low glucose correlated with prognosis in human LUAD. Overall, this study furthers current knowledge of the relationship between glucose metabolism and cell differentiation in cancer, characterizing the epigenetic adaptation of cancer cells to glucose deprivation and identifying targets to prevent the development of resistance to therapies targeting glucose metabolism. SIGNIFICANCE: Epigenetic adaptation allows cancer cells to overcome the tumor-suppressive effects of glucose restriction by inducing dedifferentiation and an aggressive phenotype, which could help design better metabolic treatments.

RIZ2 at the crossroad of the EGF/EGFR signaling in colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is the third most deadly and fourth most diagnosed cancer worldwide. Despite the progress in early diagnosis and advanced therapeutic options, CRC shows a poor prognosis with a 5 year survival rate of ~ 45%. PRDM2/RIZ, a member of PR/SET domain family (PRDM), expresses two main molecular variants, the PR-plus isoform (RIZ1) and the PR-minus (RIZ2). The imbalance in their expression levels in favor of RIZ2 is observed in many cancer types. The full length RIZ1 has been extensively investigated in several cancers where it acts as a tumor suppressor, whereas few studies have explored the RIZ2 oncogenic properties. PRDM2 is often target of frameshift mutations and aberrant DNA methylation in CRC. However, little is known about its role in CRC. METHODS: We combined in-silico investigation of The Cancer Genome Atlas (TCGA) CRC datasets, cellular and molecular assays, transcriptome sequencing and functional annotation analysis to assess the role of RIZ2 in human CRC. RESULTS: Our in-silico analysis on TCGA datasets confirmed that PRDM2 gene is frequently mutated and transcriptionally deregulated in CRC and revealed that a RIZ2 increase is highly correlated with a significant RIZ1 downregulation. Then, we assayed several CRC cell lines by qRT-PCR analysis for the main PRDM2 transcripts and selected DLD1 cell line, which showed the lowest RIZ2 levels. Therefore, we overexpressed RIZ2 in these cells to mimic TCGA datasets analysis results and consequently to assess the PRDM2/RIZ2 role in CRC. Data from RNA-seq disclosed that RIZ2 overexpression induced profound changes in CRC cell transcriptome via EGF pathway deregulation, suggesting that RIZ2 is involved in the EGF autocrine regulation of DLD1 cell behavior. Noteworthy, the forced RIZ2 expression increased cell viability, growth, colony formation, migration and organoid formation. These effects could be mediated by the release of high EGF levels by RIZ2 overexpressing DLD1 cells. CONCLUSIONS: Our findings add novel insights on the putative RIZ2 tumor-promoting functions in CRC, although additional efforts are warranted to define the underlying molecular mechanism.

Host nasopharyngeal transcriptome dataset of a SARS-CoV-2 positive Italian cohort.

The ongoing COVID-19 pandemic caused by SARS-CoV-2 has affected millions of people worldwide and has significant implications for public health. Host transcriptomics profiling provides comprehensive understanding of how the virus interacts with host cells and how the host responds to the virus. COVID-19 disease alters the host transcriptome, affecting cellular pathways and key molecular functions. To contribute to the global effort to understand the virus's effect on host cell transcriptome, we have generated a dataset from nasopharyngeal swabs of 35 individuals infected with SARS-CoV-2 from the Campania region in Italy during the three outbreaks, with different clinical conditions. This dataset will help to elucidate the complex interactions among genes and can be useful in the development of effective therapeutic pathways.

Cardiopulmonary Exercise Testing in Children and Young Adolescents after a Multisystem Inflammatory Syndrome: Physical Deconditioning or Residual Pathology?

Multisystem inflammatory syndrome in children (MIS-C) is a serious health condition that imposes a long-term follow-up. The purpose of our pilot study is to evaluate the usefulness of the cardiopulmonary stress test (CPET) in the follow-up after MIS-C. All patients admitted for MIS-C in our hospital in the 12 months preceding the date of observation were considered for inclusion in the study. Pre-existing cardio-respiratory diseases and/or the lack of collaboration were the exclusion criteria. At enrolment, each subject passed a cardiological examination, rest ECG, echocardiogram, 24 h Holter-ECG, blood tests, and a CPET complete of spirometry. A total of 20 patients met the inclusion criteria (11.76 ± 3.29 years, 13 male). In contrast to the normality of all second-level investigations, CPET showed lower-than-expected peakVO2 and peak-oxygen-pulse values (50% of cases) and higher-than-expected VE/VCO2-slope values (95% of cases). A statistically significant inverse correlation was observed between P-reactive-protein values at admission and peakVO2/kg values (p = 0.034), uric acid values at admission, and peakVO2 (p = 0.011) or peak-oxygen-pulse expressed as a percentage of predicted (p = 0.021), NT-proBNP values at admission and peakVO2 expressed as a percentage of predicted (p = 0.046). After MIS-C (4-12 months) relevant anomalies can be observed at CPET, which can be a valuable tool in the follow-up after this condition.

Glucose deprivation promotes pseudo-hypoxia and de-differentiation in lung adenocarcinoma.

Increased utilization of glucose is a hallmark of cancer. Several studies are investigating the efficacy of glucose restriction by glucose transporter blockade or glycolysis inhibition. However, the adaptations of cancer cells to glucose restriction are unknown. Here, we report the discovery that glucose restriction in lung adenocarcinoma (LUAD) induces cancer cell de-differentiation, leading to a more aggressive phenotype. Glucose deprivation causes a reduction in alpha-ketoglutarate (αKG), leading to attenuated activity of αKG-dependent histone demethylases and histone hypermethylation. We further show that this de-differentiated phenotype depends on unbalanced EZH2 activity, causing inhibition of prolyl-hydroxylase PHD3 and increased expression of hypoxia inducible factor 1α (HIF1α), triggering epithelial to mesenchymal transition. Finally, we identified an HIF1α-dependent transcriptional signature with prognostic significance in human LUAD. Our studies further current knowledge of the relationship between glucose metabolism and cell differentiation in cancer, characterizing the epigenetic adaptation of cancer cells to glucose deprivation and identifying novel targets to prevent the development of resistance to therapies targeting glucose metabolism.

Functional Relationships between Long Non-Coding RNAs and Estrogen Receptor Alpha: A New Frontier in Hormone-Responsive Breast Cancer Management.

In the complex and articulated machinery of the human genome, less than 2% of the transcriptome encodes for proteins, while at least 75% is actively transcribed into non-coding RNAs (ncRNAs). Among the non-coding transcripts, those ≥200 nucleotides long (lncRNAs) are receiving growing attention for their involvement in human diseases, particularly cancer. Genomic studies have revealed the multiplicity of processes, including neoplastic transformation and tumor progression, in which lncRNAs are involved by regulating gene expression at epigenetic, transcriptional, and post-transcriptional levels by mechanism(s) that still need to be clarified. In breast cancer, several lncRNAs were identified and demonstrated to have either oncogenic or tumor-suppressive roles. The functional understanding of the mechanisms of lncRNA action in this disease could represent a potential for translational applications, as these molecules may serve as novel biomarkers of clinical use and potential therapeutic targets. This review highlights the relationship between lncRNAs and the principal hallmark of the luminal breast cancer phenotype, estrogen receptor α (ERα), providing an overview of new potential ways to inhibit estrogenic signaling via this nuclear receptor toward escaping resistance to endocrine therapy.

LINE-1 hypomethylation is associated with poor outcomes in locoregionally advanced oropharyngeal cancer.

BACKGROUND AND PURPOSE: Currently, human papillomavirus (HPV) positivity represents a strong prognostic factor for both reduced risk of relapse and improved survival in patients with oropharyngeal squamous cell carcinoma (OPSCC). However, a subset of HPV-positive OPSCC patients still experience poor outcomes. Furthermore, HPV-negative OPSCC patients, who have an even higher risk of relapse, are still lacking suitable prognostic biomarkers for clinical outcome. Here, we evaluated the prognostic value of LINE-1 methylation level in OPSCC patients and further addressed the relationship between LINE-1 methylation status and p53 protein expression as well as genome-wide/gene-specific DNA methylation. RESULTS: In this study, DNA was extracted from 163 formalin-fixed paraffin-embedded tissue samples retrospectively collected from stage III-IVB OPSCC patients managed with curative intent with up-front treatment. Quantitative methylation-specific PCR revealed that LINE-1 hypomethylation was directly associated with poor prognosis (5-year overall survival-OS: 28.1% for LINE-1 methylation < 35% vs. 69.1% for ≥ 55%; p < 0.0001). When LINE-1 methylation was dichotomized as < 55% versus ≥ 55%, interaction with HPV16 emerged: compared with hypermethylated HPV16-positive patients, subjects with hypomethylated HPV16-negative OPSCC reported an adjusted higher risk of death (HR 4.83, 95% CI 2.24-10.38) and progression (HR 4.54, 95% CI 2.18-9.48). Tumor protein p53 (TP53) gene is often mutated and overexpressed in HPV-negative OPSCC. Since p53 has been reported to repress LINE-1 promoter, we then analyzed the association between p53 protein expression and LINE-1 methylation levels. Following p53 immunohistochemistry, results indicated that among HPV16-negative patients with p53 ≥ 50%, LINE-1 methylation levels declined and remained stable at approximately 43%; any HPV16-positive patient reported p53 ≥ 50%. Finally, DNA methylation analysis demonstrated that genome-wide average methylation level at cytosine-phosphate-guanine sites was significantly lower in HPV16-negative OPSCC patients who relapsed within two years. The subsequent integrative analysis of gene expression and DNA methylation identified 20 up-regulated/hypomethylated genes in relapsed patients, and most of them contained LINE-1 elements in their promoter sequences. CONCLUSIONS: Evaluation of the methylation level of LINE-1 may help in identifying the subset of OPSCC patients with bad prognosis regardless of their HPV status. Aberrant LINE-1 hypomethylation might occur along with TP53 mutations and lead to altered gene expression in OPSCC.

Whole-Exome Sequencing Revealed New Candidate Genes for Human Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is a complex disease affecting young adults. It is a pathological condition impairing myocardium activity that leads to heart failure and, in the most severe cases, transplantation, which is currently the only possible therapy for the disease. DCM can be attributed to many genetic determinants interacting with environmental factors, resulting in a highly variable phenotype. Due to this complexity, the early identification of causative gene mutations is an important goal to provide a genetic diagnosis, implement pre-symptomatic interventions, and predict prognosis. The advent of next-generation sequencing (NGS) has opened a new path for mutation screening, and exome sequencing provides a promising approach for identifying causal variants in known genes and novel disease-associated candidates. We analyzed the whole-exome sequencing (WES) of 15 patients affected by DCM without overloading (hypertension, valvular, or congenital heart disease) or chronic ischemic conditions. We identified 70 pathogenic or likely pathogenic variants and 1240 variants of uncertain clinical significance. Gene ontology enrichment analysis was performed to assess the potential connections between affected genes and biological or molecular function, identifying genes directly related to extracellular matrix organization, transcellular movement through the solute carrier and ATP-binding cassette transporter, and vitamin B12 metabolism. We found variants in genes implicated to a different extent in cardiac function that may represent new players in the complex genetic scenario of DCM.

Targeted molecular profiling of epithelial ovarian cancer from Italian BRCA wild-type patients with a BRCA and PARP pathways gene panel.

Ovarian cancer (OC) is the fifth most common type of cancer in women and the fourth most common cause of cancer death in women. Identification of pathogenic variants in OC tissues has an important clinical significance for therapeutic and prevention purposes. This study aims to evaluate the mutational profile of a patient cohort, negative for BRCA1/2 germinal variants and Mismatch Repair defects, using next-generation sequencing (NGS) approach on DNA from formalin-fixed paraffin-embedded samples. We used a custom NGS panel, targeting 34 cancer-related genes, mainly of the BRCA and PARP pathways, and analyzed NGS data to identify somatic and germline variants in Italian patients affected by primary epithelial ovarian cancer. We analyzed 75 epithelial ovarian cancer tissues and identified 54 pathogenic variants and 56 variants of unknown significance. TP53 was characterized by the highest mutational rate, occurring in 55% of tested epithelial ovarian cancers (EOCs). Interestingly, a subset of 8 EOCs showed pathogenic variants of homologous recombination pathway, which could be sensitive to PARP-inhibitor therapies. Germline analysis of actionable genes revealed 4 patients carrier of pathogenic germline variants respectively of RAD51C (2 patients), RAD51D, and PALB2. Molecular profiling of EOCs using our custom NGS panel has enabled the detection of both somatic and germline variants, allowing the selection of patients suitable for targeted therapies, and the identification of high-risk OC families that can benefit from genetic counseling and testing.

Identification of functional pathways and molecular signatures in neuroendocrine neoplasms by multi-omics analysis.

BACKGROUND: Neuroendocrine neoplasms (NENs) represent a heterogeneous class of rare tumors with increasing incidence. They are characterized by the ability to secrete peptide hormones and biogenic amines but other reliable biomarkers are lacking, making diagnosis and identification of the primary site very challenging. While in some NENs, such as the pancreatic ones, next generation sequencing technologies allowed the identification of new molecular hallmarks, our knowledge of the molecular profile of NENs from other anatomical sites is still poor. METHODS: Starting from the concept that NENs from different organs may be clinically and genetically correlated, we applied a multi-omics approach by combining multigene panel testing, CGH-array, transcriptome and miRNome profiling and computational analyses, with the aim to highlight common molecular and functional signatures of gastroenteropancreatic (GEP)-NENs and medullary thyroid carcinomas (MTCs) that could aid diagnosis, prognosis and therapy. RESULTS: By comparing genomic and transcriptional profiles, ATM-dependent signaling emerged among the most significant pathways at multiple levels, involving gene variations and miRNA-mediated regulation, thus representing a novel putative druggable pathway in these cancer types. Moreover, a set of circulating miRNAs was also selected as possible diagnostic/prognostic biomarkers useful for clinical management of NENs. CONCLUSIONS: These findings depict a complex molecular and functional landscape of NENs, shedding light on novel therapeutic targets and disease biomarkers to be exploited.

Combinatorial targeting of a chromatin complex comprising Dot1L, menin and the tyrosine kinase BAZ1B reveals a new therapeutic vulnerability of endocrine therapy-resistant breast cancer.

BACKGROUND: Targeting vulnerabilities of cancer cells by inhibiting key regulators of cell proliferation or survival represents a promising way to overcome resistance to current therapies. In breast cancer (BC), resistance to endocrine therapy results from constitutively active or aberrant estrogen receptor alpha (ERα) signaling to the genome. Targeting components of the ERα pathway in these tumors represents, therefore, a rational way toward effective new treatments. Interaction proteomics identified several proteins associated with ERα in BC cells, including epigenetic complexes controlling gene transcription comprising the scaffold protein menin and the histone methyltransferase Dot1L. METHODS: We combined chromatin immunoprecipitation, transcriptome sequencing, siRNA-mediated gene knockdown (kd), pharmacological inhibition coupled to cellular and functional assays and interaction proteomics in antiestrogen (AE)-sensitive and AE-resistant human BC cell models to: map menin and Dot1L chromatin localization, search for their common and specific target genes, measure the effects of single or combinatorial knockdown or pharmacological inhibition of these proteins on cell proliferation and survival, and characterize their nuclear interactomes. RESULTS: Dot1L and menin associate in MCF-7 cells chromatin, where they co-localize in a significant fraction of sites, resulting in co-regulation of genes involved, among others, in estrogen, p53, HIF1α and death receptor signaling, regulation of cell cycle and epithelial-to-mesenchymal transition. Specific inhibitors of the two factors synergize with each other for inhibition of cell proliferation of AE (tamoxifen or fulvestrant)-sensitive and AE-resistant BC cells. Menin and Dot1L interactomes share a sizeable fraction of their nuclear partners, the majority being known BC fitness genes. Interestingly, these include B-WICH and WINAC complexes that share BAZ1B, a bromodomain protein comprising a tyrosine-protein kinase domain playing a central role in chromatin remodeling and transcriptional regulation. BAZ1B kd caused significant inhibition of ERα expression, proliferation and transcriptome changes resulting in inhibition of estrogen, myc, mTOR, PI3K and AKT signaling and metabolic pathways in AE-sensitive and AE-resistant BC cells. CONCLUSIONS: Identification of a functional interplay between ERα, Dot1L, menin and BAZ1B and the significant effects of their co-inhibition on cell proliferation and survival in cell models of endocrine therapy-resistant BC reveal a new therapeutic vulnerability of these aggressive diseases.

Histone Methyltransferase DOT1L as a Promising Epigenetic Target for Treatment of Solid Tumors.

The histone lysine methyltransferase DOT1L (DOT1-like histone lysine methyltransferase) is responsible for the epigenetic regulation of gene expression through specific methylation of lysine79 residue of histone H3 (H3K79) in actively transcribed genes. Its normal activity is crucial for embryonic development and adult tissues functions, whereas its aberrant functioning is known to contribute to leukemogenesis. DOT1L is the only lysine methyltransferase that does not contain a SET domain, which is a feature that allowed the development of selective DOT1L inhibitors that are currently investigated in Phase I clinical trials for cancer treatment. Recently, abnormal expression of this enzyme has been associated with poor survival and increased aggressiveness of several solid tumors. In this review evidences of aberrant DOT1L expression and activity in breast, ovarian, prostate, colon, and other solid tumors, and its relationships with biological and clinical behavior of the disease and response to therapies, are summarized. Current knowledge of the structural basis of DOT1L ability to regulate cell proliferation, invasion, plasticity and stemness, cell cycle progression, cell-to-cell signaling, epithelial-to-mesenchymal transition, and chemoresistance, through cooperation with several molecular partners including noncoding RNAs, is also reviewed. Finally, available options for the treatment of therapeutically challenging solid tumors by targeting DOT1L are discussed.

The pleiotropic roles of circular and long noncoding RNAs in cutaneous melanoma.

Cutaneous melanoma (CM) is a very aggressive disease, often characterized by unresponsiveness to conventional therapies and high mortality rates worldwide. The identification of the activating BRAFV600 mutations in approximately 50% of CM patients has recently fueled the development of novel small-molecule inhibitors that specifically target BRAFV600 -mutant CM. In addition, a major progress in CM treatment has been made by monoclonal antibodies that regulate the immune checkpoint inhibitors. However, although target-based therapies and immunotherapeutic strategies have yielded promising results, CM treatment remains a major challenge. In the last decade, accumulating evidence points to the aberrant expression of different types of noncoding RNAs (ncRNAs) in CM. While studies on microRNAs have grown exponentially leading to significant insights on CM biology, the role of circular RNAs (circRNAs) and long noncoding RNAs (lncRNAs) in this tumor is less understood, and much remains to be discovered. Here, we summarize and critically review the available evidence on the molecular functions of circRNAs and lncRNAs in BRAFV600 -mutant CM and CM immunogenicity, providing recent updates on their functional role in targeted therapy and immunotherapy resistance. In addition, we also include an evaluation of several algorithms and databases for prediction and validation of circRNA and lncRNA functional interactions.

The Effects of Physical Inactivity and Exercise at Home in Young Patients with Congenital Heart Disease during the COVID-19 Pandemic.

Background: The COVID-19 pandemic had a significant impact on the population's ability to be physically active. Purpose: Evaluate the effect of the COVID-19 mitigation measures on exercise tolerance in patients with congenital heart disease (CHD). Materials and methods: All subjects (880, 6-18 years old) who performed a stress test at our hospital from October 2020 to February 2021 and had a similar test one year earlier were enrolled. A questionnaire on the degree of physical activity carried out in 2020 concerning the period prior to the pandemic was compiled. Exercise tolerance and the main anthropometric parameters between the first and second tests were compared. Results: 110 subjects (11.9 ± 4.1 years) were included in the study. The percentage of patients engaged in regular physical activity (RPA) decreased significantly during the pandemic (p < 0.001), and BMI increased significantly (p < 0.001), except among the subjects who began RPA during the lockdown, whereas test duration did not decrease significantly overall but increased in this last subgroup (p < 0.05) Conclusions: The COVID-19 lockdown led to a less active lifestyle with a significant increase in BMI in our group of CHD. These data could have negative effects on the risk profile of this population. RPA practiced at home seems to be effective in counteracting such effects.

BRCA Methylation Testing Identifies a Subset of Ovarian Carcinomas without Germline Variants That Can Benefit from PARP Inhibitor.

Homologous Recombination Deficiency (HRD) is a frequent feature of high-grade epithelial ovarian carcinoma (EOC), associated with sensitivity to PARP-inhibitors (PARPi). The best characterized causes of HRD in EOCs are germline or somatic mutations in BRCA1 and BRCA2 genes. Although promoter methylation is a well-known mechanism of gene transcriptional repression, few data have been published about BRCA gene methylation in EOCs. In this retrospective study, we quantitatively analyzed by pyrosequencing a selected series of 90 formalin-fixed (FFPE) primary EOCs without BRCA germline mutations. We identified 20/88 (22.7%) EOCs showing BRCA promoter methylation, including 17/88 (19.3%) in BRCA1 and 4/86 (4.6%) in BRCA2 promoters, one of which showing concomitant BRCA1 methylation. Mean methylation levels were 49.6% and 45.8% for BRCA1 and BRCA2, respectively, with methylation levels ≥50% in 10/20 methylated EOCs. Constitutive BRCA methylation was excluded by testing blood-derived DNA. In conclusion, pyrosequencing methylation analysis of BRCA genes is a robust, quantitative and sensitive assay applicable to FFPE samples. Remarkably, a considerable subset of germline BRCA-negative EOCs showed somatic methylation and, likely, HRD. A subpopulation of women with BRCA methylation, even without BRCA mutations, could potentially benefit from PARP-inhibitors; further clinical studies are needed to clarify the predictive role of somatic BRCA methylation of PARP-therapy response.

Identification of Antiestrogen-Bound Estrogen Receptor α Interactomes in Hormone-Responsive Human Breast Cancer Cell Nuclei.

Estrogen receptor alpha (ERα) is a ligand-inducible transcription factor which mediates estrogen actions in hormone-responsive tumors and is targeted by effective anticancer therapies based on the ERα antagonist ligands, selective estrogen receptor modulators (such as Tamoxifen/TAM) or disruptors (such as Fulvestrant/ICI). Despite its importance for cancer therapy, including acquired resistance to endocrine therapy, the molecular basis of ERα response to different ligands is not fully known to date. Interaction proteomics shows great potential to identify and characterize molecular mechanisms of disease based on physical and functional protein-protein interaction networks. Tandem affinity purification coupled to mass spectrometry is applied here for mapping in hormone-responsive breast cancer cells nuclei, the ERα interactomes, induced by each of the two classes of antiestrogens. The results provide new insights on the molecular bases for antiestrogen-mediated control of ERα function and reveal new potential ways to overcome endocrine therapy resistance in cancer.