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.

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.

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%.

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.