Working together for the family: determination of HER oncogene co-amplifications in breast cancer.

HER2 is a well-studied tyrosine kinase (TK) membrane receptor which functions as a therapeutic target in invasive ductal breast carcinomas (IDC). The standard of care for the treatment of HER2-positive breast is the antibody trastuzumab. Despite specific treatment unfortunately, 20% of primary and 70% of metastatic HER2 tumors develop resistance. HER2 belongs to a gene family, with four members (HER1-4) and these members could be involved in resistance to anti-HER2 therapies. In this study we designed a probemix to detect the amplification of the four HER oncogenes in a single reaction. In addition, we developed a protocol based on the combination of MLPA with ddPCR to detect the tumor proportion of co-amplified HERs. On 111 IDC, the HER2 MLPA results were validated by FISH (Adjusted r 2 = 0,91, p < 0,0001), CISH (Adjusted r 2 = 0,938, p < 0,0001) and IHC (Adjusted r 2 = 0,31, p < 0,0001). HER1-4 MLPA results were validated by RT-qPCR assays (Spearman Rank test p < 0,05). Of the 111 samples, 26% presented at least one HER amplified, of which 23% showed co-amplifications with other HERs. The percentage of cells with HER2 co-amplified varied among the tumors (from 2-72,6%). Independent in-silico findings show that the outcome of HER2+ patients is conditioned by the status of HER3 and HER4. Our results encourage further studies to investigate the relationship with patient's response to single or combined treatment. The approach could serve as proof of principle for other tumors in which the HER oncogenes are involved.

TP73 DNA methylation and upregulation of ΔNp73 are associated with an adverse prognosis in breast cancer.

AIM: Accumulated evidence suggests that aberrant methylation of the TP73 gene and increased levels of ΔNp73 in primary tumours correlate with poor prognosis. However, little is known regarding the transcriptional and functional regulation of the TP73 gene in breast cancer. The aim of the present study was to determine the expression of the ΔNp73 isoform, its relationship with DNA methylation of TP73 and their clinical prognostic significance in breast cancer patients. METHODS: TP73 gene methylation was studied in TCGA datasets and in 70 invasive ductal breast carcinomas (IDCs). The expression of p73 isoforms was evaluated by immunohistochemistry (IHC) and Western blot and correlated with clinicopathological variables and clinical outcome. RESULTS: We observed that the methylation of diverse CpG islands of TP73 differed significantly between molecular subtypes. An inverse correlation was found between p73 protein expression and the methylation status of the TP73 gene. The expression of exon 3' of p73 (only expressed in ΔNp73) was significantly higher in patients with wild-type p53. Immunohistochemical analysis revealed that all p73 isoforms were localised in both the nuclear and cytoplasmic compartments. We confirmed a positive association between the expression of ∆Np73 and high histological grade. CONCLUSIONS: Our findings suggest that high expression of ΔNp73 could be used to determine the aggressiveness of IDCs and could be incorporated in the pathologist's report.

Epigenetic variations in breast cancer progression to lymph node metastasis.

Breast cancer is a heterogeneous disease characterized by the accumulation of genetic and epigenetic alterations that contribute to the development of regional and distant metastases. Lymph node metastasis (LNM) status is the single most important prognostic factor. Metastatic cancer cells share common molecular alterations with those of the primary tumor, but in addition, they develop distinct changes that allow the cancer to progress. There is an urgent need for molecular studies which focus on identifying genomic and epigenomic markers that can predict the progression to metastasis. The objective of this study was to identify epigenetic similarities and differences between paired primary breast tumor (PBT) and LNM. We employed Methylation-Specific-MLPA (Multiplex ligation-dependent probe amplification) to assess the methylation status of 33 cancer-related genes in a cohort of 50 paired PBT and LNM specimens. We found that the methylation index, which represents the degree of aberrantly methylated genes in a specimen, was maintained during the progression to LNM. However, some genes presented differential methylation profiles. Interestingly, PAX6 presented a significant negative correlation between paired PBT and LNM (p = 0.03), which indicated a switch from methylated to unmethylated status in the progression from PBT to LNM. We further identified that the methylation status of PAX6 on the identified CpG site functionally affected the expression of PAX6 at the mRNA level. Our study unraveled significant epigenetic changes during the progression from PBT to LNM, which may contribute to improved prognosis, prediction and therapeutic management of metastatic breast cancer patients.

DNA methylation index and methylation profile of invasive ductal breast tumors.

Breast carcinogenesis is a multistep process that involves both genetic and epigenetic alterations. Identification of aberrantly methylated genes in breast tumors and their relation to clinical parameters can contribute to improved diagnostic, prognostic, and therapeutic decision making. Our objective in the present study was to identify the methylation status of 34 cancer-involved genes in invasive ductal carcinomas (IDC). Each of the 70 IDC cases analyzed had a unique methylation profile. The highest methylation frequency was detected in the WT1 (95.7%) and RASSF1 (71.4%) genes. Hierarchical cluster analysis revealed three clusters with different distribution of the prognostic factors tumor grade, lymph node metastasis, and proliferation rate. Methylation of TP73 was associated with high histological grade and high proliferation rate; methylation of RARB was associated with lymph node metastasis. Concurrent methylation of TP73 and RARB was associated with high histological grade, high proliferation rate, increased tumor size, and lymph node metastasis. Patients with more than six methylated genes had higher rates of relapse events and cancer deaths. In multivariate analysis, TP73 methylation and the methylation index were associated with disease outcome. Our results indicate that methylation index and methylation of TP73 and/or RARB are related to unfavorable prognostic factors in patients with IDC. These epigenetic markers should be validated in further studies to improve breast cancer management.

Aberrant DNA methylation of cancer-related genes in giant breast fibroadenoma: a case report.

INTRODUCTION: Giant fibroadenoma is an uncommon variant of benign breast lesions. Aberrant methylation of CpG islands in promoter regions is known to be involved in the silencing of genes (for example, tumor-suppressor genes) and appears to be an early event in the etiology of breast carcinogenesis. Only hypermethylation of p16INK4a has been reported in non-giant breast fibroadenoma. In this particular case, there are no previously published data on epigenetic alterations in giant fibroadenomas. Our previous results, based on the analysis of 49 cancer-related CpG islands have confirmed that the aberrant methylation is specific to malignant breast tumors and that it is completely absent in normal breast tissue and breast fibroadenomas. CASE PRESENTATION: A 13-year-old Hispanic girl was referred after she had noted a progressive development of a mass in her left breast. On physical examination, a 10 × 10 cm lump was detected and axillary lymph nodes were not enlarged. After surgical removal the lump was diagnosed as a giant fibroadenoma. Because of the high growth rate of this benign tumor, we decided to analyze the methylation status of 49 CpG islands related to cell growth control. We have identified the methylation of five cancer-related CpG islands in the giant fibroadenoma tissue: ESR1, MGMT, WT-1, BRCA2 and CD44. CONCLUSION: In this case report we show for the first time the methylation analysis of a giant fibroadenoma. The detection of methylation of these five cancer-related regions indicates substantial epigenomic differences with non-giant fibroadenomas. Epigenetic alterations could explain the higher growth rate of this tumor. Our data contribute to the growing knowledge of aberrant methylation in breast diseases. In this particular case, there exist no previous data regarding the role of methylation in giant fibroadenomas, considered by definition as a benign breast lesion.

P-cadherin and beta-catenin are useful prognostic markers in breast cancer patients; beta-catenin interacts with heat shock protein Hsp27.

The cadherin-catenin proteins have in common with heat shock proteins (HSP) the capacity to bind/interact proteins of other classes. Moreover, there are common molecular pathways that connect the HSP response and the cadherin-catenin protein system. In the present study, we have explored whether in breast cancer the HSP might interact functionally with the cadherin-catenin cell adhesion system. Beta-catenin was immunoprecipitated from breast cancer biopsy samples, and the protein complexes isolated in this way were probed with antibodies against HSP family members. We are thus the first to demonstrate a specific interaction between beta-catenin and Hsp27. However, beta-catenin did not bind Hsp60, Hsp70, Hsp90, gp96, or the endoplasmic reticulum stress response protein CHOP. To confirm the finding of Hsp27-beta-catenin interaction, the 27-kDa immunoprecipitated band was excised from one-dimensional polyacrylamide gel electrophoresis gels and submitted to liquid chromatography-tandem mass spectrometry with electrospray ionization, confirming a role for Hsp27. In addition, beta-catenin interacted with other proteins including heat shock transcription factor 1, P-cadherin, and caveolin-1. In human breast cancer biopsy samples, beta-catenin was coexpressed in the same tumor areas and in the same tumor cells that expressed Hsp27. However, this coexpression was strong when beta-catenin was present in the cytoplasm of the tumor cells and not when beta-catenin was expressed at the cell surface only. Furthermore, murine breast cancer cells transfected with hsp25 showed a redistribution of beta-catenin from the cell membrane to the cytoplasm. When the prognostic significance of cadherin-catenin expression was examined by immunohistochemistry in breast cancer patients (n = 215, follow-up = >10 years), we found that the disease-free survival and overall survival were significantly shorter for patients expressing P-cadherin and for patients showing expression of beta-catenin in the cytoplasm only (not at the cell surface). The interactions of beta-catenin with Hsp27 and with HSF1 may explain some of the molecular pathways that influence tumor cell survival and the clinical significance in the prognosis of the breast cancer patients.