Epigenomic Profiling Advises Therapeutic Potential of Leukotriene Receptor Inhibitors for a Subset of Triple-Negative Breast Tumors.

Triple-negative breast cancer (TNBC) is the most aggressive molecular subtype, with a poor survival rate compared to others subtypes. For a long time, chemotherapy was the only systemic treatment for TNBC, and the identification of actionable molecular targets might ultimately improve the prognosis for TNBC patients. We performed a genome-wide analysis of DNA methylation at CpG islands on a collection of one hundred ten breast carcinoma samples and six normal breast tissue samples using reduced representation bisulfite sequencing with the XmaI restriction enzyme (XmaI-RRBS) and identified a subset of TNBC samples with significant hypomethylation at the LTB4R/LTB4R2 genes' CpG islands, including CpG dinucleotides covered with cg12853742 and cg21886367 HumanMethylation 450K microarray probes. Abnormal DNA hypomethylation of this region in TNBC compared to normal samples was confirmed by bisulfite Sanger sequencing. Gene expression generally anticorrelates with promoter methylation, and thus, the promoter hypomethylation detected and confirmed in our study might be revealed as an indirect marker of high LTB4R/LTB4R2 expression using a simple methylation-sensitive PCR test. Analysis of RNA-seq expression and DNA methylation data from the TCGA dataset demonstrates that the expression of the LTB4R and LTB4R2 genes significantly negatively correlates with DNA methylation at both CpG sites cg12853742 (R = -0.4, p = 2.6 × 10-6; R = -0.21, p = 0.015) and cg21886367 (R = -0.45, p = 7.3 × 10-8; R = -0.24, p = 0.005), suggesting the upregulation of these genes in tumors with abnormal hypomethylation of their CpG island. Kaplan-Meier analysis using the TCGA-BRCA gene expression and clinical data revealed poorer overall survival for TNBC patients with an upregulated LTB4R. To this day, only the leukotriene inhibitor LY255283 has been tested on an MCF-7/DOX cell line, which is a luminal A breast cancer molecular subtype. Other studies compare the effects of Montelukast and Zafirlukast (inhibitors of the cysteinyl leukotriene receptor, which is different from LTB4R/LTB4R2) on the MDA-MB-231 (TNBC) cell line, with high methylation and low expression levels of LTB4R. In our study, we assess the therapeutic effects of various drugs (including leukotriene receptor inhibitors) with the DepMap gene effect and drug sensitivity data for TNBC cell lines with hypomethylated and upregulated LTB4R/LTB4R2 genes. LY255283, Minocycline, Silibinin, Piceatannol, Mitiglinide, 1-Azakenpaullone, Carbetocin, and Pim-1-inhibitor-2 can be considered as candidates for the additional treatment of TNBC patients with tumors demonstrating LTB4R/LTB4R2 hypomethylation/upregulation. Finally, our results suggest that the epigenetic status of leukotriene B4 receptors is a novel, potential, predictive, and prognostic biomarker for TNBC. These findings might improve individualized therapy for TNBC patients by introducing new therapeutic adjuncts as anticancer agents.

DNA Methylation and Prospects for Predicting the Therapeutic Effect of Neoadjuvant Chemotherapy for Triple-Negative and Luminal B Breast Cancer.

Despite advances in the diagnosis and treatment of breast cancer (BC), the main cause of deaths is resistance to existing therapies. An approach to improve the effectiveness of therapy in patients with aggressive BC subtypes is neoadjuvant chemotherapy (NACT). Yet, the response to NACT for aggressive subtypes is less than 65% according to large clinical trials. An obvious fact is the lack of biomarkers predicting the therapeutic effect of NACT. In a search for epigenetic markers, we performed genome-wide differential methylation screening by XmaI-RRBS in cohorts of NACT responders and nonresponders, for triple-negative (TN) and luminal B tumors. The predictive potential of the most discriminative loci was further assessed in independent cohorts by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), a promising method for the implementation of DNA methylation markers in diagnostic laboratories. The selected most informative individual markers were combined into panels demonstrating cvAUC = 0.83 (TMEM132D and MYO15B markers panel) for TN tumors and cvAUC = 0.76 (TTC34, LTBR and CLEC14A) for luminal B tumors. The combination of methylation markers with clinical features that correlate with NACT effect (clinical stage for TN and lymph node status for luminal B tumors) produces better classifiers, with cvAUC = 0.87 for TN tumors and cvAUC = 0.83 for luminal B tumors. Thus, clinical characteristics predictive of NACT response are independently additive to the epigenetic classifier and in combination improve prediction.

Parental Origin of the RB1 Gene Mutations in Families with Low Penetrance Hereditary Retinoblastoma.

Our aim was to identify RB1 alterations causing hereditary low penetrance retinoblastoma and to evaluate how the parental origin of an RB1 mutation affects its phenotypic expression. By NGS and MLPA, RB1 mutations were found in 191 from 332 unrelated retinoblastoma patients. Among patients with identified RB1 mutations but without clinical family history of retinoblastoma, 7% (12/175) were found to have hereditary disease with one of the parents being an asymptomatic carrier of an RB1 mutation. Additionally, in two families with retinoblastoma history, mutations were inherited by probands from unaffected parents. Overall, nine probands inherited RB1 mutations from clinically unaffected fathers and five, from mothers. Yet, we gained explanations of maternal "unaffectedness" in most cases, either as somatic mosaicism or as clinical presentation of retinomas in involution, rendering the proportion of paternal to maternal truly asymptomatic mutation carriers as 9:1 (p = 0.005). This observation supports an assumption that parental origin of an RB1 mutation influences the likelihood of developing retinoblastoma. Additionally, our study revealed a relatively high frequency of asymptomatic carriage of the RB1 mutations among the parents of retinoblastoma patients, highlighting the utmost necessity of molecular analysis among the probands' relatives irrespective of their clinical status and family history of retinoblastoma.

Mutations in Epigenetic Regulation Genes in Gastric Cancer.

We have performed mutational profiling of 25 genes involved in epigenetic processes on 135 gastric cancer (GC) samples. In total, we identified 79 somatic mutations in 49/135 (36%) samples. The minority (n = 8) of mutations was identified in DNA methylation/demethylation genes, while the majority (n = 41), in histone modifier genes, among which mutations were most commonly found in KMT2D and KMT2C. Somatic mutations in KMT2D, KMT2C, ARID1A and CHD7 were mutually exclusive (p = 0.038). Mutations in ARID1A were associated with distant metastases (p = 0.03). The overall survival of patients in the group with metastases and in the group with tumors with signet ring cells was significantly reduced in the presence of mutations in epigenetic regulation genes (p = 0.036 and p = 0.041, respectively). Separately, somatic mutations in chromatin remodeling genes correlate with low survival rate of patients without distant metastasis (p = 0.045) and in the presence of signet ring cells (p = 0.0014). Our results suggest that mutations in epigenetic regulation genes may be valuable clinical markers and deserve further exploration in independent cohorts.

Abnormal promoter DNA hypermethylation of the integrin, nidogen, and dystroglycan genes in breast cancer.

Cell transmembrane receptors and extracellular matrix components play a pivotal role in regulating cell activity and providing for the concerted integration of cells in the tissue structures. We have assessed DNA methylation in the promoter regions of eight integrin genes, two nidogen genes, and the dystroglycan gene in normal breast tissues and breast carcinomas (BC). The protein products of these genes interact with the basement membrane proteins LAMA1, LAMA2, and LAMB1; abnormal hypermethylation of the LAMA1, LAMA2, and LAMB1 promoters in BC has been described in our previous publications. In the present study, the frequencies of abnormal promoter hypermethylation in BC were 13% for ITGA1, 31% for ITGA4, 4% for ITGA7, 39% for ITGA9, 38% for NID1, and 41% for NID2. ITGA2, ITGA3, ITGA6, ITGB1, and DAG1 promoters were nonmethylated in normal and BC samples. ITGA4, ITGA9, and NID1 promoter hypermethylation was associated with the HER2 positive tumors, and promoter hypermethylation of ITGA1, ITGA9, NID1 and NID2 was associated with a genome-wide CpG island hypermethylated BC subtype. Given that ITGA4 is not expressed in normal breast, one might suggest that its abnormal promoter hypermethylation in cancer is non-functional and is thus merely a passenger epimutation. Yet, this assumption is not supported by our finding that it is not associated with a hypermethylated BC subtype. ITGA4 acquires expression in a subset of breast carcinomas, and methylation of its promoter may be preventive against expression in some tumors. Strong association of abnormal ITGA4 hypermethylation with the HER2 positive tumors (p = 0.0025) suggests that simultaneous presence of both HER2 and integrin α4 receptors is not beneficial for tumor cells. This may imply HER2 and integrin α4 signaling pathways interactions that are yet to be discovered.

Analysis of miRNA Expression in Patients with Rheumatoid Arthritis during Olokizumab Treatment.

Rheumatoid arthritis (RA) is the most common autoimmune disease worldwide. Epigenetic alternations of microRNAs (miRNAs) can contribute to its pathogenesis and progression. As the first line therapy with DMARDs is not always successful, other drugs and therapeutic targets should be applied. This study aims to measure the expression level of plasma miRNAs in RA patients treated with olokizumab and to evaluate their potential as prognostic biomarkers. The expression of 9 miRNAs was quantified in 103 RA patients before treatment and at weeks 12 and 24 of olokizumab therapy by reverse transcription-polymerase chain reaction (RT-PCR) assay and analyzed in groups of responders and non-responders. Almost all miRNAs changed their expression during therapy. The ROC curve analysis of the most prominent of them together with consequent univariate and multivariate regression analysis revealed statistically significant associations with the olokizumab therapy efficiency scores for miR-26b, miR-29, miR-451, and miR-522. Therefore, these miRNAs might be a potential therapeutic response biomarker.

DNA methylation markers panel can improve prediction of response to neoadjuvant chemotherapy in luminal B breast cancer.

Despite the advantages of neoadjuvant chemotherapy (NACT), associated toxicity is a serious complication that renders monitoring of the patients' response to NACT highly important. Thus, prediction of tumor response to treatment is imperative to avoid exposure of potential non-responders to deleterious complications. We have performed genome-wide analysis of DNA methylation by XmaI-RRBS and selected CpG dinucleotides differential methylation of which discriminates luminal B breast cancer samples with different sensitivity to NACT. With this data, we have developed multiplex methylation sensitive restriction enzyme PCR (MSRE-PCR) protocol for determining the methylation status of 10 genes (SLC9A3, C1QL2, DPYS, IRF4, ADCY8, KCNQ2, TERT, SYNDIG1, SKOR2 and GRIK1) that distinguish BC samples with different NACT response. Analysis of these 10 markers by MSRE-PCR in biopsy samples allowed us to reveal three top informative combinations of markers, (1) IRF4 and C1QL2; (2) IRF4, C1QL2, and ADCY8; (3) IRF4, C1QL2, and DPYS, with the areas under ROC curves (AUCs) of 0.75, 0.78 and 0.74, respectively. A classifier based on IRF4 and C1QL2 better meets the diagnostic panel simplicity requirements, as it consists of only two markers. Diagnostic accuracy of the panel of these two markers is 0.75, with the sensitivity of 75% and specificity of 75%.

Clinical relevance of somatic mutations in main driver genes detected in gastric cancer patients by next-generation DNA sequencing.

Somatic mutation profiling in gastric cancer (GC) enables main driver mutations to be identified and their clinical and prognostic value to be evaluated. We investigated 77 tumour samples of GC by next-generation sequencing (NGS) with the Ion AmpliSeq Hotspot Panel v2 and a custom panel covering six hereditary gastric cancer predisposition genes (BMPR1A, SMAD4, CDH1, TP53, STK11 and PTEN). Overall, 47 somatic mutations in 14 genes were detected; 22 of these mutations were novel. Mutations were detected most frequently in the CDH1 (13/47) and TP53 (12/47) genes. As expected, somatic CDH1 mutations were positively correlated with distant metastases (p = 0.019) and tumours with signet ring cells (p = 0.043). These findings confirm the association of the CDH1 mutations with diffuse GC type. TP53 mutations were found to be significantly associated with a decrease in overall survival in patients with Lauren diffuse-type tumours (p = 0.0085), T3-T4 tumours (p = 0.037), and stage III-IV tumours (p = 0.013). Our results confirm that the detection of mutations in the main driver genes may have a significant prognostic value for GC patients and provide an independent GC-related set of clinical and molecular genetic data.

Genome-wide methylotyping resolves breast cancer epigenetic heterogeneity and suggests novel therapeutic perspectives.

Aim: To provide a breast cancer (BC) methylotype classification by genome-wide CpG islands bisulfite DNA sequencing. Materials & methods: XmaI-reduced representation bisulfite sequencing DNA methylation sequencing method was used to profile DNA methylation of 110 BC samples and 6 normal breast samples. Intrinsic DNA methylation BC subtypes were elicited by unsupervised hierarchical cluster analysis, and cluster-specific differentially methylated genes were identified. Results & conclusion: Overall, six distinct BC methylotypes were identified. BC cell lines constitute a separate group extremely highly methylated at the CpG islands. In turn, primary BC samples segregate into two major subtypes, highly and moderately methylated. Highly and moderately methylated superclusters, each incorporate three distinct epigenomic BC clusters with specific features, suggesting novel perspectives for personalized therapy.

Genetic screening of Russian Usher syndrome patients toward selection for gene therapy.

BACKGROUND: Usher syndrome (USH) is heterogeneous in nature and requires genetic test for diagnosis and management. Mutations in USH associated genes are reported in some populations except Russians. Here, we first time represented the mutation spectrum of a Russian USH cohort. METHODS: Twenty-eight patients with USH were selected from 3214 patients from Deaf-Blind Support Foundation "Con-nection" during 2014-2016 following the observational study NCT03319524. Complete ophthalmologic, ENT, and vestibular medical tests were done for clinical characterization. NGS, MLPA, and Sanger sequencing were considered for genetic analysis. RESULTS: Around 53.57% and 39.28% patients had USH1 and USH2, respectively; 17.85% cases (n = 5/28) had no known mutation. Eleven (73.33%) subjects showed variations in USH1 associated genes MYO7A (72.72%), CDH23 (9.09%), PCDH15 (9.09%), and USH1C (9.09%). Eleven mutations are detected in MYO7A where 54.54% are novel. MYO7A: p.Q18* was most frequent (27.27%) mutation and is associated with early manifestation and most severe clinical picture. Two novel mutations (p.E1301* and c.158-?_318+?del) are detected in PCDH15 gene. Around 90.90% patients suspected to be USH2 are confirmed by genetic testing. Eleven mutations detected in the USH2A gene, where 27.27% were novel. Most common USH2A mutation is p.W3955* (50%) followed by p.E767fs, p.R1653*, and c.8682-9A> G (20% each). CONCLUSION: The Russian USH cohort shows both novel and known USH mutations. Clinically the prevalence of USH2 is low (39.28%) and the frequency of MYO7A mutations responsible for USH1B is very high (63.63%, N = 7/11) compared to other cohorts. These seven patients carrying MYO7A mutations are preliminarily eligible for the UshStat® gene therapy.