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

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

MYC amplification in breast cancer: a chromogenic in situ hybridisation study

Aims: To analyse the correlation between MYC amplification and various clinicopathological features and outcome in a cohort of 245 patients with invasive breast carcinoma treated with surgery followed by anthracycline-based chemotherapy. Given the high prevalence of MYC amplification in tumours of BRCA1 mutation carriers and the similarities between these and sporadic "basal-like" carcinomas, the prevalence of MYC amplification in "basal-like" breast carcinomas was investigated. Methods: MYC gene copy number was assessed on tissue microarrays containing duplicate cores of 245 invasive breast carcinomas by means of chromogenic in situ hybridisation using SpotLight C-MYC amplification probe and chromosome 8 centromeric probe (CEP8). Signals were evaluated at 4006 magnification; 30 morphologically unequivocal neoplastic cells in each core were counted for the presence of the gene and CEP8 probes. Results: Amplification was defined as a MYC: CEP8 ratio > 2. Signals for both MYC and CEP8 were assessable in 196/245 (80%) tumours. MYC amplification was found in 19/196 cases (9.7%) and was not associated with tumour size, histological grade, positivity for oestrogen receptor, progesterone receptor, HER2, epidermal growth factor, cytokeratins 14, 5/6 and 17, MIB1 or p53. Only 4% of basal-like carcinomas showed MYC amplification, compared to 8.75% and 10.7% of luminal and HER2 tumours respectively. On univariate analysis, MYC amplification displayed a significant association with shorter metastasis-free and overall survival and proved to be an independent prognostic factor on multivariate survival analysis. Conclusion: MYC amplification is not associated with "basal-like" phenotype and proved to be an independent prognostic factor for breast cancer patients treated with anthracycline-based chemotherapy

Molecular alterations of kit oncogene in gliomas, Cell Oncol 29

Abstract. 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

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.FCT(SFRH/BI/15257/2004). This work was partially supported by NOVARTIS Oncology, Portugal, and Breakthrough Breast Cancer, UK

Genomic profiling of histological special types of breast cancer

Histological special types of breast cancer have distinctive morphological features and account for up to 25 % of all invasive breast cancers. We sought to determine whether at the genomic level, histological special types of breast cancer are distinct from grade- and estrogen receptor (ER)-matched invasive carcinomas of no special type (IC-NSTs), and to define genes whose expression correlates with gene copy number in histological special types of breast cancer. We characterized 59 breast cancers of ten histological special types using array-based comparative genomic hybridization (aCGH). Hierarchical clustering revealed that the patterns of gene copy number aberrations segregated with ER-status and histological grade, and that samples from each of the breast cancer histological special types preferentially clustered together. We confirmed the patterns of gene copy number aberrations previously reported for lobular, micropapillary, metaplastic, and mucinous carcinomas. On the other hand, metaplastic and medullary carcinomas were found to have genomic profiles similar to those of grade- and ER-matched IC-NSTs. The genomic aberrations observed in invasive carcinomas with osteoclast-like stromal giant cells support its classification as IC-NST variant. Integrative aCGH and gene expression analysis led to the identification of 145 transcripts that were significantly overexpressed when amplified in histological special types of breast cancer. Our results illustrate that together with histological grade and ER-status, histological type is also associated with the patterns and complexity of gene copy number aberrations in breast cancer, with adenoid cystic and mucinous carcinomas being examples of ER-negative and ER-positive breast cancers with distinctive repertoires of gene copy number aberration

High-throughput detection of fusion genes in cancer using the Sequenom MassARRAY platform

Fusion genes have pivotal roles in the development and progression of human cancer and offer potential for rational drug design. Massively parallel sequencing has identified a panoply of in-frame expressed fusion genes, but early reports suggest that the majority of these are present at very low prevalence or are private events. Conventional methods for the identification of recurrent expressed fusion genes in large cohorts of cancers (eg fluorescence in situ hybridization (FISH) and reverse transcriptase PCR (RT-PCR)) are time consuming and prone to artifacts. Here, we describe a novel high-throughput strategy for the detection of recurrent fusion genes in cancer based on the Sequenom MassARRAY platform. Fusion genes were initially identified by massively parallel sequencing of breast cancer cell lines. For each fusion gene, two Sequenom probes were designed. Primary human breast cancers and cancer cell lines were interrogated for 10 fusion genes. Sensitivity, specificity, and predictive values of the MassARRAY method were then determined using FISH and qRT-PCR as the 'gold standard.' By combining two probes per fusion gene, the negative and positive predictive values were 100 and 71.4%, respectively. All fusion genes identified by massively parallel sequencing were accurately detected. No recurrent fusion genes were found. The MassARRAY-based approach described here may, therefore, be employed as a high-throughput screening tool for known fusion genes in human cancer. In keeping with other highly sensitive assays, further refinement of this technique is necessary to reduce the number of false-positive results. Laboratory Investigation (2011) 91, 1491-1501; doi:10.1038/labinvest.2011.110; published online 1 August 201