Gene expression analysis of invasive breast carcinoma yields differential patterns in luminal subtypes of breast cancer

Breast cancer is a heterogeneous disease, and new biomarkers are needed for more accurate classification and prediction of prognosis. The goal of this study is to assess the expression of breast cancer classification genes, to identify new molecular signatures in different intrinsic subtypes of breast cancer and to correlate their expression with different clinical variables. The study included 84 female patients newly diagnosed with non-metastatic breast cancer at the outpatient clinic at the National Cancer Institute, Cairo University, Egypt. Detection of 17 breast cancer classification genes was done using RT-PCR in tumor and normal tissues. Estrogen receptor (ER), progesterone receptor (PR), HER2, and Ki67 expression were assessed using IHC assay for intrinsic subtyping. Combined expression of FOXA1 and GATA3 was statistically higher in luminal subtypes in comparison to non-luminal subtypes. In Luminal A subtype; GRB7, EGFR, PTGS2, ID1, and KRT5 were significantly downregulated. FOXA1 and GATA3 were significantly upregulated in luminal B subtype, where EGFR and PTGS2 were significantly downregulated. While ESR1, EGFR, KRT5 and PTGS2 showed significantly low expression in tumor tissue in Her2 enriched subtype, TFF3 was significantly downregulated in triple negative subtype. GATA3 and FOXA1 expression exhibited significant correlation with tumor grade. Furthermore, GATA3, FOXA1, ESR1, and ID1 were also correlated significantly with staging of the tumor. Combined expression of ESR1, FOXA1 and GATA3 represents a molecular signature of luminal subtypes. Long term follow-up is needed to investigate the prognostic effect of breast cancer classification genes found in this study.     

Mutation screening of 10 cancer susceptibility genes in unselected breast cancer patients

Variants of cancer susceptibility genes other than BRCA1/2 have been proved to be associated with increased risks of breast cancer. This study was performed to investigate the spectrum and prevalence of mutations in 10 cancer susceptibility genes in paired tumor/normal tissues of 292 unselected Chinese breast cancer patients. We performed an analysis of germline and somatic variants in ATM, CDH1, CHEK2, ESR1, GATA3, MAP3K1, MSH2, PALB2, RB1 and STK11 genes by integrating microfluidic PCR‐based target enrichment and next‐generation sequencing technologies. In total, 3 germline and 25 somatic deleterious mutations were found among 27 patients (9.25%), and 17 of them were novel mutations. Most deleterious mutations were prevalent in luminal A invasive breast cancer (P = .014). We also observed 83 variants of uncertain significance (VUS) in 100 patients (34.25%), 23 of which were predicted to be deleterious by in silico prediction programs (MetaSVM and MetaLR). VUS carriers had higher positive rate of lymph node metastasis than non‐carriers (P = .008) and were predominantly present in ER+ tumors (P = .018). Our findings would enhance the understanding of the molecular mechanisms of breast cancer in Chinese population.     

A family with a complex clinical presentation characterized by arrhythmogenic right ventricular dysplasia/cardiomyopathy and features of branchio‐oculo‐facial syndrome

Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) is a familial form of cardiomyopathy typically caused by mutations in genes that encode an element of the cardiac desmosome. Branchio‐oculo‐facial syndrome (BOFS) is a craniofacial disorder caused by TFAP2A mutations. In a family segregating ARVD/C, some members also had features of BOFS. Genetic testing for ARVD/C identified a mutation in PKP2, encoding plakophilin‐2, a component of the cardiac desmosome. Evaluation of dysmorphology by chromosome microarray (CMA) identified a 4.4 Mb deletion at chromosome 6p24 that included both TFAP2A and DSP, encoding desmoplakin, an additional component of the cardiac desmosome implicated in ARVD/C. A family member with both the 6p24 deletion and PKP2 mutation had more severe cardiac dysfunction. These findings suggest that this contiguous gene deletion contributes to both ARVD/C and BOFS, and that DSP haploinsufficiency may contribute to cardiomyopathy. This family provides a clinical example that underscores the need for careful evaluation in clinical scenarios where genetic heterogeneity is known to exist. Finally, it suggests that individuals with unexplained cardiomyopathy and dysmorphic facial features may benefit from CMA analysis. © 2013 Wiley Periodicals, Inc.     

DNA methylation analysis of the HIF‐1α prolyl hydroxylase domain genes PHD1, PHD2, PHD3 and the factor inhibiting HIF gene FIH in invasive breast carcinomas

Huang K T, Mikeska T, Dobrovic A & Fox S B
(2010) Histopathology 57, 451–460
DNA methylation analysis of the HIF‐1α prolyl hydroxylase domain genes PHD1, PHD2, PHD3 and the factor inhibiting HIF gene FIH in invasive breast carcinomas Aims: Hypoxia‐inducible factor‐1 (HIF‐1) activity is regulated by prolyl hydroxylase (PHD1, PHD2, PHD3) and factor inhibiting HIF‐1 (FIH) that target the α subunit of HIF‐1 (HIF‐1α) for proteosomal degradation. We hypothesised that the elevated HIF‐1α level is due in some tumours to epigenetic silencing by DNA hypermethylation of the promoter region of one or more of the PHDs and FIH genes. The aims were to define the presence or absence of promoter methylation of PHDs and FIH in cell lines of various sources and breast carcinomas and, if present, determine its effect on mRNA and protein expression. Methods and results: Tumour cell lines (n = 20) and primary invasive breast carcinomas (n = 168) were examined for promoter region DNA methylation using methylation‐sensitive high‐resolution melting. There was evidence of PHD3 but not of PHD1, PHD2 or FIH DNA methylation in breast cancer (SkBr3) and leukaemic (HL60 and CCRF‐CEM) cell lines, but there was no evidence of methylation in any of 168 breast cancers. Only the high‐level PHD3 methylation seen in leukaemic cell lines correlated with absent mRNA and protein expression. Conclusions: Methylation‐induced epigenetic silencing of PHD1, PHD2, PHD3 and FIH is unlikely to underlie up‐regulated HIF‐1α expression in human breast cancer but may play a role in other tumour types.     

Proteasome ubiquitin receptor PSMD4 is an amplification target in breast cancer and may predict sensitivity to PARPi

Poly (ADP‐ribose) polymerase 1 (PARP1) is an enzyme involved in DNA repair under investigation as a chemotherapeutic target. Current randomized phase three trials of PARPi in metastatic breast cancer are limited to patients with documented BRCA1/2 mutations and no biomarker of PARPi beyond BRCA status is available. In an effort to identify novel biomarkers for PARP inhibition, we created a cell line (HCC1187/TALRES) resistant to the PARP1 inhibitor talazoparib. Herein we show by array‐CGH that HCC1187/TALRES has a selective loss of the proteasome ubiquitin receptor PSMD4 amplicon resulting in significant down‐regulation of PSMD4. Conversely, we find that breast cancer cell lines that have copy number gain or amplification for PSMD4 are significantly more sensitive to talazoparib. Functional studies reveal that knock‐down of PSMD4 in amplified breast cancer cells and loss of the PSMD4 amplicon result in knock‐down of PARP1 protein. We show that PSMD4 is amplified and overexpressed in breast cancer and its overexpression correlates with poor survival. Knock‐down of PSMD4 results in a significant decrease in cell growth. We provide evidence that PSMD4 is a proteasomal amplification target in breast cancer that PSMD4 amplification confers sensitivity to PARP inhibition, and that PSMD4 amplification is lost in the process of acquiring resistance to PARPi. Finally, this study shows not only that PSMD4 copy number correlates with PARPi sensitivity, but also, that it may be a better predictor of sensitivity to PARPi than BRCA1/2 mutation.     

Detection of novel germline mutations in six breast cancer predisposition genes by targeted next‐generation sequencing

In this study, a customized amplicon‐based target sequencing panel was designed to enrich the whole exon regions of six genes associated with the risk of breast cancer. Targeted next‐generation sequencing (NGS) was performed for 146 breast cancer patients (BC), 71 healthy women with a family history of breast cancer (high risk), and 55 healthy women without a family history of cancer (control). Sixteen possible disease‐causing mutations on four genes were identified in 20 samples. The percentages of possible disease‐causing mutation carriers in the BC group (8.9%) and in the high‐risk group (8.5%) were higher than that in the control group (1.8%). The BRCA1 possible disease‐causing mutation group had a higher prevalence in family history and triple‐negative breast cancer, while the BRCA2 possible disease‐causing mutation group was younger and more likely to develop axillary lymph node metastasis (P < 0.05). Among the 146 patients, 47 with a family history of breast cancer were also sequenced with another 14 moderate‐risk genes. Three additional possible disease‐causing mutations were found on PALB2, CHEK2, and PMS2 genes, respectively. The results demonstrate that the six‐gene targeted NGS panel may provide an approach to assess the genetic risk of breast cancer and predict the clinical prognosis of breast cancer patients.