Caveolin 1 is overexpressed and amplified in a subset of basal-like and metaplastic breast carcinomas: a morphologic, ultrastructural, immunohistochemical, and in situ hybridization analysis

The distribution and significance of caveolin 1 (CAV1) expression in different breast cell types and role in breast carcinogenesis remain poorly understood. Both tumor-suppressive and oncogenic roles have been proposed for this protein. The aims of this study were to characterize the distribution of CAV1 in normal breast, benign breast lesions, breast cancer precursors, and metaplastic breast carcinomas; to assess the prognostic significance of CAV1 expression in invasive breast carcinomas; and to define whether CAV1 gene amplification is the underlying genetic mechanism driving CAV1 overexpression in breast carcinomas. Purpose: The distribution and significance of caveolin 1 (CAV1) expression in different breast cell types and role in breast carcinogenesis remain poorly understood. Both tumor-suppressive and oncogenic roles have been proposed for this protein. The aims of this study were to characterize the distribution of CAV1 in normal breast, benign breast lesions, breast cancer precursors, and metaplastic breast carcinomas; to assess the prognostic significance of CAV1 expression in invasive breast carcinomas; and to define whether CAV1 gene amplification is the underlying genetic mechanism driving CAV1 overexpression in breast carcinomas. Experimental Design: CAV1 distribution in frozen and paraffin-embedded whole tissue sections of normal breast was evaluated using immunohistochemistry, immunofluorescence, and immunoelectron microscopy. CAV1 expression was immunohistochemically analyzed in benign lesions, breast cancer precursors, and metaplastic breast carcinomas and in a cohort of 245 invasive breast carcinomas from patients treated with surgery followed by anthracycline-based chemotherapy. In 25 cases, CAV1 gene amplification was assessed by chromogenic in situ hybridization. Results: In normal breast, CAV1 was expressed in myoepithelial cells, endothelial cells, and a subset of fibroblasts. Luminal epithelial cells showed negligible staining. CAV1 was expressed in 90% of 39 metaplastic breast carcinomas and in 9.4% of 245 invasive breast cancers. In the later cohort, CAV1 expression was significantly associated with ‘basal-like’ immunophenotype and with shorter disease-free and overall survival on univariate analysis. CAV1 gene amplification was found in 13% of cases with strong CAV1 expression. Conclusions: The concurrent CAV1 amplification and overexpression call into question its tumor-suppressive effects in basal-like breast carcinomas

The landscape of RNA polymerase II-associated chromatin interactions in prostate cancer

Transcriptional dysregulation is a hallmark of prostate cancer (PCa). We mapped the RNA polymerase II-associated (RNA Pol II-associated) chromatin interactions in normal prostate cells and PCa cells. We discovered thousands of enhancer-promoter, enhancer-enhancer, as well as promoter-promoter chromatin interactions. These transcriptional hubs operate within the framework set by structural proteins - CTCF and cohesins - and are regulated by the cooperative action of master transcription factors, such as the androgen receptor (AR) and FOXA1. By combining analyses from metastatic castration-resistant PCa (mCRPC) specimens, we show that AR locus amplification contributes to the transcriptional upregulation of the AR gene by increasing the total number of chromatin interaction modules comprising the AR gene and its distal enhancer. We deconvoluted the transcription control modules of several PCa genes, notably the biomarker KLK3, lineage-restricted genes (KRT8, KRT18, HOXB13, FOXA1, ZBTB16), the drug target EZH2, and the oncogene MYC. By integrating clinical PCa data, we defined a germline-somatic interplay between the PCa risk allele rs684232 and the somatically acquired TMPRSS2-ERG gene fusion in the transcriptional regulation of multiple target genes - VPS53, FAM57A, and GEMIN4. Our studies implicate changes in genome organization as a critical determinant of aberrant transcriptional regulation in PCa

Characterization of the genomic features and expressed fusion genes in micropapillary carcinomas of the breast

Micropapillary carcinoma ( MPC ) is a rare histological special type of breast cancer, characterized by an aggressive clinical behaviour and a pattern of copy number aberrations ( CNAs ) distinct from that of grade‐ and oestrogen receptor ( ER )‐matched invasive carcinomas of no special type ( IC‐NSTs ). The aims of this study were to determine whether MPCs are underpinned by a recurrent fusion gene(s) or mutations in 273 genes recurrently mutated in breast cancer. Sixteen MPCs were subjected to microarray‐based comparative genomic hybridization ( aCGH ) analysis and Sequenom OncoCarta mutation analysis. Eight and five MPCs were subjected to targeted capture and RNA sequencing, respectively. aCGH analysis confirmed our previous observations about the repertoire of CNAs of MPCs . Sequencing analysis revealed a spectrum of mutations similar to those of luminal B IC‐NSTs , and recurrent mutations affecting mitogen‐activated protein kinase family genes and NBPF10 . RNA ‐sequencing analysis identified 17 high‐confidence fusion genes, eight of which were validated and two of which were in‐frame. No recurrent fusions were identified in an independent series of MPCs and IC‐NSTs . Forced expression of in‐frame fusion genes ( SLC2A1–FAF1 and BCAS4–AURKA ) resulted in increased viability of breast cancer cells. In addition, genomic disruption of CDK12 caused by out‐of‐frame rearrangements was found in one MPC and in 13% of HER2 ‐positive breast cancers, identified through a re‐analysis of publicly available massively parallel sequencing data. In vitro analyses revealed that CDK12 gene disruption results in sensitivity to PARP inhibition, and forced expression of wild‐type CDK12 in a CDK12 ‐null cell line model resulted in relative resistance to PARP inhibition. Our findings demonstrate that MPCs are neither defined by highly recurrent mutations in the 273 genes tested, nor underpinned by a recurrent fusion gene. Although seemingly private genetic events, some of the fusion transcripts found in MPCs may play a role in maintenance of a malignant phenotype and potentially offer therapeutic opportunities. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/106752/1/path4325.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/106752/2/path4325-sup-0001-AppendixS1.pd

Mapping genetic vulnerabilities reveals BTK as a novel therapeutic target in oesophageal cancer

Objective Oesophageal cancer is the seventh most common cause of cancer-related death worldwide. Disease relapse is frequent and treatment options are limited. Design To identify new biomarker-defined therapeutic approaches for patients with oesophageal cancer, we integrated the genomic profiles of 17 oesophageal tumour-derived cell lines with drug sensitivity data from small molecule inhibitor profiling, identifying drug sensitivity effects associated with cancer driver gene alterations. We also interrogated recently described RNA interference screen data for these tumour cell lines to identify candidate genetic dependencies or vulnerabilities that could be exploited as therapeutic targets. Results By integrating the genomic features of oesophageal tumour cell lines with siRNA and drug screening data, we identified a series of candidate targets in oesophageal cancer, including a sensitivity to inhibition of the kinase BTK in MYC amplified oesophageal tumour cell lines. We found that this genetic dependency could be elicited with the clinical BTK/ERBB2 kinase inhibitor, ibrutinib. In both MYC and ERBB2 amplified tumour cells, ibrutinib downregulated ERK-mediated signal transduction, cMYC Ser-62 phosphorylation and levels of MYC protein, and elicited G(1) cell cycle arrest and apoptosis, suggesting that this drug could be used to treat biomarker-selected groups of patients with oesophageal cancer. Conclusions BTK represents a novel candidate therapeutic target in oesophageal cancer that can be targeted with ibrutinib. On the basis of this work, a proof-of-concept phase II clinical trial evaluating the efficacy of ibrutinib in patients with MYC and/or ERBB2 amplified advanced oesophageal cancer is currently underway (NCT02884453). Trial registration number NCT02884453; Pre-result

Clinical Utility of Circulating Tumour Cell Androgen Receptor Splice Variant-7 Status in Metastatic Castration-resistant Prostate Cancer.

Abstract Background Detection of androgen receptor splice variant-7 (AR-V7) mRNA in circulating tumour cells (CTCs) is associated with worse outcome in metastatic castration-resistant prostate cancer (mCRPC). However, studies rarely report comparisons with CTC counts and biopsy AR-V7 protein expression. Objective To determine the reproducibility of AdnaTest CTC AR-V7 testing, and associations with clinical characteristics, CellSearch CTC counts, tumour biopsy AR-V7 protein expression and overall survival (OS). Design, setting, and participants CTC AR-V7 status was determined for 227 peripheral blood samples, from 181 mCRPC patients with CTC counts (202 samples; 136 patients) and matched mCRPC biopsies (65 samples; 58 patients). Outcome measurements and statistical analysis CTC AR-V7 status was associated with clinical characteristics, CTC counts, and tissue biopsy AR-V7 protein expression. The association of CTC AR-V7 status and other baseline variables with OS was determined. Results and limitations Of the samples, 35% were CTC+/AR-V7+. CTC+/AR-V7+ samples had higher CellSearch CTC counts (median CTC; interquartile range [IQR]: 60, 19–184 vs 9, 2–64; Mann-Whitney test p Conclusions Studies reporting the prognostic relevance of CTC AR-V7 status must account for CTC counts. Discordant CTC AR-V7 results and AR-V7 protein expression in matched, same-patient biopsies are reported. Patient summary Liquid biopsies that determine circulating tumour cell androgen receptor splice variant-7 status have the potential to impact treatment decisions in metastatic castration-resistant prostate cancer patients. Robust clinical qualification of these assays is required before their routine use

Metaplastic breast carcinomas exhibit EGFR, but not HER2, gene amplification and overexpression: immunohistochemical and chromogenic in situ hybridization analysis

INTRODUCTION: Metaplastic breast carcinomas constitute a heterogeneous group of neoplasms, accounting for less than 1% of all invasive mammary carcinomas. Approximately 70–80% of metaplastic breast carcinomas overexpress the epidermal growth factor receptor (EGFR). Human epidermal growth factor receptor (HER)2 and EGFR have attracted much attention in the medical literature over the past few years owing to the fact that humanized monoclonal antibodies against HER2 and therapies directed against the extracellular ligand-binding domain or the intracellular tyrosine kinase domain of EGFR have proven successful in treating certain types of human cancer. We investigated whether HER2 and EGFR overexpression was present and evaluated gene amplification in a series of metaplastic breast carcinomas. METHOD: Twenty-five metaplastic breast carcinomas were immunohistochemically analyzed using a monoclonal antibody (31G7) for EGFR and two antibodies for HER2 (Herceptest and CB11) and scored using the Herceptest scoring system. Gene amplification was evaluated by chromogenic in situ hybridization using Zymed Spot-Light EGFR and HER2 amplification probe. The results were evaluated by bright field microscopy under 40× and 63× objective lenses. RESULTS: Nineteen (76%) metaplastic breast carcinomas exhibited EGFR ovexpression, and among these EGFR amplification (defined either by large gene clusters or >5 signals/nucleus in >50% of neoplastic cells) was detected in seven cases (37%): three carcinomas with squamous differentiation and four spindle cell carcinomas. One case exhibited HER2 overexpression of grade 2+ (>10% of cells with weak to moderate complete membrane staining), but HER2 gene amplification was not detected. CONCLUSION: Metaplastic breast carcinomas frequently overexpressed EGFR, which was associated with EGFR gene amplification in one-third of cases. Our findings suggest that some patients with metaplastic breast carcinomas might benefit from novel therapies targeting EGFR. Because most metaplastic breast carcinomas overexpress EGFR without gene amplification, further studies to evaluate EGFR activating mutations are warranted

Determination of HER2 amplification status on tumour DNA by digital PCR.

Determination of the presence of HER2 amplification by quantitative PCR has been challenging, in part due to chromosomal instability and identification of a robust a reference region. We assessed the potential of digital PCR for highly accurate assessment of DNA concentration with EFTUD2 as chromosome 17 reference probe. We assessed a HER2:EFTDU2 ratio by digital PCR assay in the microdissected DNA from 18 HER2 amplified and 58 HER2 non-amplified cancers. The HER2:EFTUD2 ratio had high concordance with conventionally defined HER2 status with a sensitivity of 100% (18/18) and a specificity of 98% (57/58). The HER2:EFTUD2 digital PCR assay has potential to accurately assess HER2 amplification status