High expression of the vacuole membrane protein 1 (VMP1) is a potential marker of poor prognosis in HER2 positive breast cancer.

BACKGROUND: Fusion genes result from genomic structural changes, which can lead to alterations in gene expression that supports tumor development. The aim of the study was to use fusion genes as a tool to identify new breast cancer (BC) genes with a role in BC progression. METHODS: Fusion genes from breast tumors and BC cell lines were collected from publications. RNA-Seq data from tumors and cell lines were retrieved from databanks and analyzed for fusions with SOAPfuse or the analysis was purchased. Fusion genes identified in both tumors (n = 1724) and cell lines (n = 45) were confirmed by qRT-PCR and sequencing. Their individual genes were ranked by selection criteria that included correlation of their mRNA level with copy number. The expression of the top ranked gene was measured by qRT-PCR in normal tissue and in breast tumors from an exploratory cohort (n = 141) and a validation cohort (n = 277). Expression levels were correlated with clinical and pathological factors as well as the patients' survival. The results were followed up in BC cohorts from TCGA (n = 818) and METABRIC (n = 2509). RESULTS: Vacuole membrane protein 1 (VMP1) was the most promising candidate based on specific selection criteria. Its expression was higher in breast tumor tissue than normal tissue (p = 1x10-4), and its expression was significantly higher in HER2 positive than HER2 negative breast tumors in all four cohorts analyzed. High expression of VMP1 associated with breast cancer specific survival (BCSS) in cohort 1 (hazard ratio (HR) = 2.31, CI 1.27-4.18) and METABRIC (HR = 1.26, CI 1.02-1.57), and also after adjusting for HER2 expression in cohort 1 (HR = 2.03, CI 1.10-3.72). BCSS was not significant in cohort 2 or TCGA cohort, which may be due to differences in treatment regimens. CONCLUSIONS: The results suggest that high VMP1 expression is a potential marker of poor prognosis in HER2 positive BC. Further studies are needed to elucidate how VMP1 could affect pathways supportive of tumorigenesis.

High expression of ZNF703 independent of amplification indicates worse prognosis in patients with luminal B breast cancer.

Amplification of 8p12-p11 is relatively common in breast cancer and several genes within the region have been suggested to affect breast tumor progression. The aim of the study was to map the amplified 8p12-p11 region in a large set of breast tumors in an effort to identify the genetic driver and to explore its impact on tumor progression and prognosis. Copy number alterations (CNAs) were mapped in 359 tumors, and gene expression data from 577 tumors (359 tumors included) were correlated with CNA, clinical-pathological factors, and protein expression (39 tumors). 8p12-p11 was amplified in 11.4% of tumors. The smallest region of amplification harbored one full-length gene, ZNF703. ZNF703 mRNA expression was significantly higher in estrogen receptor (ER)-positive than ER-negative tumors (P = 2 × 10(-16)), a reflection of high expression in luminal tumors. Forty-eight percent of tumors with ZNF703 amplification were luminal B tumors in which the best correlation between DNA copy number and mRNA was seen (P = 1.2 × 10(-7)) as well as correlation between mRNA and protein expression (P = 0.02). High ZNF703 mRNA correlated with poor survival in patients with ER-positive luminal B tumors (log rank P = 0.04). Furthermore, high ZNF703 mRNA expression correlated with poor outcome in patients with ZNF703 copy number neutral, ER-positive, luminal B tumors (log rank P = 0.004). The results support ZNF703 as the driver gene of the 8p12 amplification and suggest that independent of amplification, high expression of the gene affects prognosis in luminal B tumors.

CpG island hypermethylation of BRCA1 and loss of pRb as co-occurring events in basal/triple-negative breast cancer.

Triple-negative breast cancer (TNBC) occurs in approximately 15% of all breast cancer patients, and the incidence of TNBC is greatly increased in BRCA1 mutation carriers. This study aimed to assess the impact of BRCA1 promoter methylation with respect to breast cancer subtypes in sporadic disease. Tissue microarrays (TMAs) were constructed representing tumors from 303 patients previously screened for BRCA1 germline mutations, of which a subset of 111 sporadic tumors had previously been analyzed with respect to BRCA1 methylation. Additionally, a set of eight tumors from BRCA1 mutation carriers were included on the TMAs. Expression analysis was performed on TMAs by immunohistochemistry (IHC) for BRCA1, pRb, p16, p53, PTEN, ER, PR, HER2, CK5/6, EGFR, MUC1 and Ki-67. Data on BRCA1 aberrations and IHC expression was examined with respect to breast cancer-specific survival. The results demonstrate that CpG island hypermethylation of BRCA1 significantly associates with the basal/triple-negative subtype. Low expression of pRb, and high/intense p16, were associated with BRCA1 promoter hypermethylation, and the same effects were seen in BRCA1 mutated tumors. The expression patterns of BRCA1, pRb, p16 and PTEN were highly correlated, and define a subgroup of TNBCs characterized by BRCA1 aberrations, high Ki-67 (≥ 40%) and favorable disease outcome. In conclusion, our findings demonstrate that epigenetic inactivation of the BRCA1 gene associates with RB/p16 dysfunction in promoting TNBCs. The clinical implications relate to the potential use of targeted treatment based on PARP inhibitors in sporadic TNBCs, wherein CpG island hypermethylation of BRCA1 represents a potential marker of therapeutic response.

Genomic profiling of breast tumours in relation to BRCA abnormalities and phenotypes.

INTRODUCTION: Germline mutations in the BRCA1 and BRCA2 genes account for a considerable fraction of familial predisposition to breast cancer. Somatic mutations in BRCA1 and BRCA2 have not been found and the involvement of these genes in sporadic tumour development therefore remains unclear. METHODS: The study group consisted of 67 primary breast tumours with and without BRCA1 or BRCA2 abnormalities. Genomic alterations were profiled by high-resolution (~7 kbp) comparative genome hybridisation (CGH) microarrays. Tumour phenotypes were analysed by immunohistochemistry on tissue microarrays using selected biomarkers (ER, PR, HER-2, EGFR, CK5/6, CK8, CK18). RESULTS: Classification of genomic profiles through cluster analysis revealed four subgroups, three of which displayed high genomic instability indices (GII). Two of these GII-high subgroups were enriched with either BRCA1- or BRCA2-related tumours whereas the third was not BRCA-related. The BRCA1-related subgroup mostly displayed non-luminal phenotypes, of which basal-like were most prominent, whereas the other two genomic instability subgroups BRCA2- and GII-high-III (non-BRCA), were almost entirely of luminal phenotype. Analysis of genome architecture patterns revealed similarities between the BRCA1- and BRCA2 subgroups, with long deletions being prominent. This contrasts with the third instability subgroup, not BRCA-related, where small gains were more prominent. CONCLUSIONS: The results suggest that BRCA1- and BRCA2-related tumours develop largely through distinct genetic pathways in terms of the regions altered while also displaying distinct phenotypes. Importantly, we show that the development of a subset of sporadic tumours is similar to that of either familial BRCA1- or BRCA2 tumours. Despite their differences, we observed clear similarities between the BRCA1- and BRCA2-related subgroups reflected in the type of genomic alterations acquired with deletions of long DNA segments being prominent. This suggests similarities in the mechanisms promoting genomic instability for BRCA1- and BRCA2-associated tumours, possibly relating to deficiency in DNA repair through homologous recombination. Indeed, this feature characterized both familial and sporadic tumours displaying BRCA1- or BRCA2-like spectrums of genomic alterations. The importance of these findings lies in the potential benefit from targeted therapy, through the use of agents leading to DNA double-strand breaks such as PARP inhibitors (olaparib) and cisplatin, for a much larger group of patients than the few BRCA1 and BRCA2 germline mutation carriers.

Establishment of three human breast epithelial cell lines derived from carriers of the 999del5 BRCA2 Icelandic founder mutation.

Germ line mutations in BRCA1 and BRCA2 account for a large proportion of inherited breast and ovarian cancer. Both genes are involved in DNA repair by homologous recombination and are thought to play a vital role in maintaining genomic stability. A major drawback for long-term functional studies of BRCA in general and BRCA2 in particular has been a lack of representative human breast epithelial cell lines. In the present study, we have established three cell lines from two patients harboring the 999del5 germ line founder mutation in the BRCA2 gene. Primary cultures were established from cellular outgrowth of explanted tissue and subsequently transfected with a retroviral construct containing the HPV-16 E6 and E7 oncogenes. Paired cancer-derived and normal-derived cell lines were established from one patient referred to as BRCA2-999del5-2T and BRCA2-999del5-2N, respectively. In addition, one cell line was derived from cancer-associated normal tissue from another patient referred to as BRCA2-999del5-1N. All three cell lines showed characteristics of breast epithelial cells as evidenced by expression of breast epithelial specific cytokeratins. Cytogenetic analysis showed marked chromosomal instability with tetraploidy and frequent telomeric associations. In conclusion, we have established three breast epithelial cell lines from two patients carrying the BRCA2 Icelandic 999del5 founder mutation. These cell lines form the basis for further studies on carcinogenesis and malignant progression of breast cancer on a defined genetic background.