FGFR1 emerges as a potential therapeutic target for lobular breast carcinomas.

PURPOSE: Classic lobular carcinomas (CLC) account for 10% to 15% of all breast cancers. At the genetic level, CLCs show recurrent physical loss of chromosome16q coupled with the lack of E-cadherin (CDH1 gene) expression. However, little is known about the putative therapeutic targets for these tumors. The aim of this study was to characterize CLCs at the molecular genetic level and identify putative therapeutic targets. EXPERIMENTAL DESIGN: We subjected 13 cases of CLC to a comprehensive molecular analysis including immunohistochemistry for E-cadherin, estrogen and progesterone receptors, HER2/neu and p53; high-resolution comparative genomic hybridization (HR-CGH); microarray-based CGH (aCGH); and fluorescent and chromogenic in situ hybridization for CCND1 and FGFR1. RESULTS: All cases lacked the expression of E-cadherin, p53, and HER2, and all but one case was positive for estrogen receptors. HR-CGH revealed recurrent gains on 1q and losses on 16q (both, 85%). aCGH showed a good agreement with but higher resolution and sensitivity than HR-CGH. Recurrent, high level gains at 11q13 (CCND1) and 8p12-p11.2 were identified in seven and six cases, respectively, and were validated with in situ hybridization. Examination of aCGH and the gene expression profile data of the cell lines, MDA-MB-134 and ZR-75-1, which harbor distinct gains of 8p12-p11.2, identified FGFR1 as a putative amplicon driver of 8p12-p11.2 amplification in MDA-MB-134. Inhibition of FGFR1 expression using small interfering RNA or a small-molecule chemical inhibitor showed that FGFR1 signaling contributes to the survival of MDA-MB-134 cells. CONCLUSIONS: Our findings suggest that receptor FGFR1 inhibitors may be useful as therapeutics in a subset of CLCs.

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 40x and 63x 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.

Distribution of p63, cytokeratins 5/6 and cytokeratin 14 in 51 normal and 400 neoplastic human tissue samples using TARP-4 multi-tumor tissue microarray.

p63, cytokeratin (CK) 5/6 and CK 14 have been employed in diagnostic pathology as markers of basal, squamous and myoepithelial differentiation in several types of human neoplasms; however, there is scant data on the concurrent expression of these markers in large series of human neoplasms. We analyzed the distribution of these three immunohistochemical markers in 51 normal human tissue samples, 350 carcinomas, 25 malignant melanomas (MMs), and 25 glioblastomas using three serial sections of tissue array research program (TARP)-4 multi-tumor tissue microarray. Also, we performed double immunostainings to characterize the differential distribution of p63/CK 5/6 and p63/CK 14 in normal breast, salivary gland and skin. p63, CK 5/6 and CK 14 were expressed in basal cells of the prostate and respiratory epithelia and in breast and bronchial myoepithelial cells. p63 was also expressed in cytotrophoblast cells of human placenta and in scattered cells of lymph node germinal center. CK 5/6 and CK 14 also stained the cytoplasm of basal cells of esophageal stratified squamous epithelium and transitional epithelial cells of the bladder. No mesenchymal, neural, endothelial, smooth muscle or adipose cells were stained by any of the markers. p63, CK 5/6, and CK 14 were respectively expressed in 92.6%, 75.0%, and 52.9% of the squamous cell carcinomas of the lung, 10.2%, 20.0%, and 7.4% of the ductal carcinomas of the breast, 12.9%, 34.4%, and 11.8% of the serous and 25.0%, 0%, and 0% of the endometrioid carcinomas of the ovary. Lung, prostate and colonic adenocarcinomas, as well as MMs and glioblastomas were only rarely decorated by one of the markers. Only matched samples of 16 squamous cell carcinomas and two ductal carcinomas of the breast co-expressed these three markers. In double immunostainings, p63-CK 5/6, as well as p63-CK 14 were co-expressed by basal/myoepithelial cells of the salivary glands and basal cells of the epidermis. Our results demonstrate that p63, CK 5/6 and CK 14 may be used together in immunohistochemical panels to characterize squamous differentiation in poorly differentiated carcinomas or carcinomas of unknown origin.

P63-driven nuclear accumulation of beta-catenin is not a frequent event in human neoplasms.

deltaN-p63 isoforms may act as oncogenes owing to their ability to bind to p53-reporter genes without inciting their transcription, thus blocking the p53-driven cell cycle arrest and apoptosis. A novel mechanism linking p63 and Wnt pathways has recently been proposed. Briefly, in vitro studies using squamous cell carcinoma cell lines have suggested that deltaN-p63 may block the phosphorylation of beta-catenin, leading to its nuclear accumulation and triggering beta-catenin-responsive transcription of genes related to proliferation and oncogenic biological behavior. To test this new mechanism, the coexpression of deltaN-p63 and beta-catenin was evaluated in a large cohort of human neoplasms. Two serial sections of TARP-4 multi-tumor tissue microarray, composed of 51 normal tissue cores and 400 human neoplasms [breast (n = 75), colon (n = 75), lung (n = 75), prostate (n = 75) and ovary (n = 50) neoplasms, melanoma (n = 25), and glioblastoma (n = 25)] were subjected to immunohistochemistry with deltaN-p63 and beta-catenin monoclonal antibodies. p63 nuclear expression and beta-catenin membranous, cytoplasmic, membranous + cytoplasmic, and nuclear localization were evaluated. deltaN-p63 expression and beta-catenin nuclear localization were found in 92.6% and 0% of squamous cell carcinomas, 8.9% and 0% of breast carcinomas, 13.8% and 0% of lung adenocarcinomas, 1.4% and 23.2% of colon adenocarcinomas, 0% and 4.8% of prostate adenocarcinomas, 11.1% and 5% of ovary carcinomas, 9.0% and 9.1% of malignant melanomas, and 12.5% and 40.0% of glioblastomas, respectively. No statistically significant association between deltaN-p63 and nuclear beta-catenin expression was found for all tumors. At variance with squamous cell carcinoma cell lines, p63-driven nuclear accumulation of beta-catenin is an unusual phenomenon in human neoplasms. Caution should be exercised when translating the results of studies performed on cell lines to human neoplasms.

Unlocking pathology archives for molecular genetic studies: a reliable method to generate probes for chromogenic and fluorescent in situ hybridization.

Chromogenic (CISH) and fluorescent (FISH) in situ hybridization have emerged as reliable techniques to identify amplifications and chromosomal translocations. CISH provides a spatial distribution of gene copy number changes in tumour tissue and allows a direct correlation between copy number changes and the morphological features of neoplastic cells. However, the limited number of commercially available gene probes has hindered the use of this technique. We have devised a protocol to generate probes for CISH that can be applied to formalin-fixed, paraffin-embedded tissue sections (FFPETS). Bacterial artificial chromosomes (BACs) containing fragments of human DNA which map to specific genomic regions of interest are amplified with phi29 polymerase and random primer labelled with biotin. The genomic location of these can be readily confirmed by BAC end pair sequencing and FISH mapping on normal lymphocyte metaphase spreads. To demonstrate the reliability of the probes generated with this protocol, four strategies were employed: (i) probes mapping to cyclin D1 (CCND1) were generated and their performance was compared with that of a commercially available probe for the same gene in a series of 10 FFPETS of breast cancer samples of which five harboured CCND1 amplification; (ii) probes targeting cyclin-dependent kinase 4 were used to validate an amplification identified by microarray-based comparative genomic hybridization (aCGH) in a pleomorphic adenoma; (iii) probes targeting fibroblast growth factor receptor 1 and CCND1 were used to validate amplifications mapping to these regions, as defined by aCGH, in an invasive lobular breast carcinoma with FISH and CISH; and (iv) gene-specific probes for ETV6 and NTRK3 were used to demonstrate the presence of t(12;15)(p12;q25) translocation in a case of breast secretory carcinoma with dual colour FISH. In summary, this protocol enables the generation of probes mapping to any gene of interest that can be applied to FFPETS, allowing correlation of morphological features with gene copy number.

Pleomorphic lobular carcinoma of the breast: role of comprehensive molecular pathology in characterization of an entity.

Immunohistochemical analysis of E-cadherin has changed the way lobular neoplasia is perceived. It has helped to classify difficult cases of carcinoma in situ with indeterminate features and led to the identification of new variants of lobular carcinoma. Pleomorphic lobular carcinoma (PLC) and pleomorphic lobular carcinoma in situ (PLCIS), recently described variants of invasive and in situ classic lobular carcinoma, are reported to be associated with more aggressive clinical behaviour. Although PLC/PLCIS show morphological features of classic lobular neoplasia and lack E-cadherin expression, it is still unclear whether these lesions evolve through the same genetic pathway as lobular carcinomas or are high-grade ductal neoplasms that have lost E-cadherin. Here we have analysed a case of extensive PLCIS and invasive PLC associated with areas of E-cadherin-negative carcinoma in situ with indeterminate features, using immunohistochemistry, chromogenic in situ hybridization, high-resolution comparative genomic hybridization (CGH) and array-based CGH. We observed that all lesions lacked E-cadherin and beta-catenin and showed gain of 1q and loss of 16q, features that are typical of lobular carcinomas but are not seen in high-grade ductal lesions. In addition, amplifications of c-myc and HER2 were detected in the pleomorphic components, which may account for the high-grade features in this case and the reported aggressive clinical behaviour of these lesions. Taken together, these data suggest that at least some PLCs may evolve from the same precursor or through the same genetic pathway as classic lobular carcinomas.