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

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

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

Molecular Alterations of KIT Oncogene in Gliomas

Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK), is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117) immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17) and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH) and quantitative real-time PCR (qRT-PCR) were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179) of cases, namely in 25% (1/4) of pilocytic astrocytomas, 25% (5/20) of diffuse astrocytomas, 20% (1/5) of anaplastic astrocytomas, 19.5% (15/77) of glioblastomas and one third (3/9) of anaplastic oligoastrocytomas. Only 5.7% (2/35) of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0–22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24) of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK inhibitors