Gene amplification of ERBB2 and EGFR in adenocarcinoma in situ and intramucosal adenocarcinoma of Barrett\u27s esophagus

金沢大学医薬保健研究域医学系We examined 11 cases of carcinoma arising from Barrett\u27s esophagus consisting of two adenocarcinomas in situ (ACIS), two intramucosal adenocarcinomas, and seven overt invasive adenocarcinomas. Overexpression of p53 (implying a mutation of the p53 gene), ERBB2, and EGFR was measured by immunohistochemistry, and gene amplification of ERBB2 and EGFR was measured by fluorescence in situ hybridization (FISH). In all cases of ACIS and the intramucosal adenocarcinomas, almost all cancer cells overexpressed p53, however the populations overexpressing ERBB2 and EGFR varied in different cases: in one ACIS, ERBB2 was coexpressed in all the cancer cells, in the other ACIS and one intramucosal adenocarcinoma, ERBB2 was overexpressed in about 50% and only 10% of the p53-positive cells respectively. EGFR was co-expressed in 20% in the other intramucosal adenocarcinoma. Protein overexpression of ERBB2 or EGFR corresponded to the amplification of their respective genes on a cell by cell basis. These gene amplifications, however, were not found in the seven invasive adenocarcinomas. Thus we speculate that the gene amplification occurred late in the dysplasia-carcinoma sequence probably after the mutation of p53. Furthermore, new clonal expansion accompanied by tumor invasion might have extinguished the originally amplified genes in these tumors. © 2010 Japanese Society of Pathology and Blackwell Publishing Asia Pty Ltd

Non-incidental coamplification of Myc and ERBB2, and Myc and EGFR, in gastric adenocarcinomas

金沢大学大学院医学系研究科がん細胞学This study was conducted to assess the frequencies of protein overexpression and gene amplification of Myc and to identify the mechanisms of Myc gene amplification, especially with regards to its possible coamplification with ERBB2 or EGFR in gastric adenocarcinomas. By immunohistochemical analysis of a total of 300 formalin-fixed and paraffin-embedded gastric adenocarcinomas, the nuclear overexpression of MYC was found in 47 tumors (16%). A fluorescence in situ hybridization (FISH) analysis revealed that nine (19%) of the 47 tumors with protein overexpression had cancer cells with high levels of Myc amplification, whereas only seven (6%) of the 122 tumors without protein overexpression showed high-level Myc gene amplification. Such Myc amplification was significantly correlated with positive nuclear protein overexpression. The coamplification of ERBB2 or EGFR with Myc that was found in six and four cases, respectively, is believed to be non-incidental because those frequencies were significantly higher than the individual frequencies observed for the total examined cases (ERBB2: 7%; EGFR: 4%). The high levels of gene amplification of these three genes, as visualized by FISH, could be broadly classified into two typical types, namely, \u27multiple scattered signals\u27 and \u27large clustered signals\u27. Using two-color FISH, the coexistence of coamplified Myc and ERBB2, or Myc and EGFR, within single nuclei in various combinations of amplification types and copy numbers, could be ascertained in all nine cases, including one in which the synchronous \u27multiple scattered type\u27 coamplification of Myc and ERBB2 was observed. In three tumors, coamplification of ERBB2 and EGFR was found; however, ERBB2- and EGFR-amplified cell populations were separate and mutually exclusive. We propose that the non-incidental coamplification of Myc and either ERBB2 or EGFR occurred through translocation and subsequent rearrangement. © 2007 USCAP, Inc All rights reserved

Gene amplification of CCNE1, CCND1, and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

New and effective treatments for advanced gastric cancer are urgently needed. Cyclins E and D1 form a complex with cyclin-dependent kinase 2, 4, or 6 to regulate G1-S transition. The G1-S regulatory genes encoding cyclin E (CCNE1), cyclin D1 (CCND1), and CDK6 (CDK6) are frequently amplified in gastric cancer and may therefore influence molecularly targeted therapies against ERBB2 or EGFR when coamplified. A total of 179 formalin-fixed and paraffin-embedded gastric cancer specimens were examined for these gene amplifications by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization. Amplification of at least 1 G1-S regulatory gene was found in 35 tumors (CCNE1 amplification, 15% of samples; CCND1, 6%; CDK6, 1%). In 13 of the 35 tumors, dual-color fluorescence in situ hybridization identified coamplification of the G1-S regulatory genes with ERBB2, EGFR, and/or KRAS in single cancer nuclei. The observation that cells with G1-S regulatory gene amplification contained clonal subpopulations with coamplification of ERBB2, EGFR, or KRAS in 5 early and 3 advanced cancers suggests that amplification of the G1-S regulatory genes represents an early event, which precedes ERBB2, EGFR, or KRAS amplification. Amplified CCNE1, CCND1, and CDK6 in advanced gastric cancer may be potentially useful as direct targets for molecular therapy or for combination therapy with ERBB2 or EGFR inhibitors. Multiplex ligation-dependent probe amplification could be a useful tool for identification of patients who would benefit from such therapies. © 2016 Elsevier Inc.Embargo Period 12 month

Clinicopathological significance of platelet-derived growth factor (PDGF)-B and vascular endothelial growth factor-A expression, PDGF receptor-β phosphorylation, and microvessel density in gastric cancer

Background: Angiogenesis is important in the growth and metastasis of various kinds of solid tumors, including gastric cancers. The angiogenic process is triggered by several key growth factors, including vascular endothelial growth factor (VEGF)-A and platelet-derived growth factor (PDGF)-B, that are secreted by tumors. Our aim was to define: i) the expression pattern of VEGF-A and PDGF-B in tumor cells and the activation of PDGF receptor (PDGFR)-β tyrosine kinase in stromal cells of human gastric adenocarcinomas; and ii) the relationship between VEGF-A and PDGF-B expression and microvessel density (MVD), to determine if there is a rationale for a new therapeutic strategy.Methods: A series of 109 gastric adenocarcinoma cases that had undergone surgical resection was examined immunohistochemically using antibodies against VEGF-A, PDGF-B, and CD34, followed by further examination of PDGFR-β phosphorylation by immunoblotting analysis.Results: MVD was higher in diffuse-type than intestinal-type cancers (p < 0.001). VEGF-A overexpression correlated to PDGF-B overexpression in both the intestinal-type (p < 0.005) and diffuse-type (p < 0.0001) groups, indicating that VEGF-A and PDGF-B are secreted simultaneously in the same tumor, and may thus play important roles together in angiogenesis. However, several differences between intestinal-type and diffuse-type cancers were observed. In the diffuse-type cancer group, higher MVD was related to the PDGF-B proportion (p < 0.05) and VEGF-A overexpression (p < 0.05), but not to PDGF-B overexpression or the VEGF-A proportion. On the other hand, in the intestinal-type cancer group, higher MVD was correlated to overexpression (p < 0.005), intensity (p < 0.05), and proportion (p < 0.05) of PDGF-B, but not of VEGF-A. In addition, phosphorylation of PDGFR-β was correlated with depth of cancer invasion at statistically significant level.Conclusions: Our results indicate that PDGF-B, which is involved in the maintenance of microvessels, plays a more important role in angiogenesis in intestinal-type gastric carcinomas than VEGF-A, which plays a key role mainly in the initiation of new blood vessel formation. In contrast, VEGF-A has a critical role for angiogenesis more in diffuse-type cancers, but less in those of intestinal type. Thus, a therapy targeting the PDGF-B signaling pathway could be effective for intestinal-type gastric carcinoma, whereas targeting VEGF-A or both VEGF-A and PDGF-B signaling pathways could be effective for diffuse-type gastric carcinomas. © 2010 Suzuki et al; licensee BioMed Central Ltd

Semi-comprehensive analysis of gene amplification in gastric cancers using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

The prognosis of patients with gastric carcinomas at an advanced stage still remains dismal, and therefore novel therapeutic modalities are urgently needed. Since the successful targeting of amplified ERBB2 with a humanized monoclonal antibody, the amplified genes of other receptor tyrosine kinases such as EGFR, FGFR2, and MET, as well as those of other cell regulator genes, are being considered as candidate targets of molecular therapy. The aim of the present study was to determine the amplification status of 26 genes, which are frequently amplified in solid cancers, in advanced gastric cancers. A total of 93 formalin-fixed and paraffin-embedded advanced gastric cancer tissues were examined by multiple ligation-dependent probe amplification, and 32 cases with ‘gain’ or ‘amplified’ status of 16 genes were further examined for the respective gene amplification by fluorescence in situ hybridization (FISH) and for the respective protein overexpression by immunohistochemistry. The frequencies of gene amplifications in advanced gastric cancers were as follows: ERBB2 (13 cases, 14%), FGFR2 (7 cases, 8%), MYC (7 cases, 8%), TOP2A (7 cases, 8%), MET (4 cases, 4%), MDM2 (4 cases, 4%), CCND1 (3 cases, 3%), FGF10 (2 cases, 3%), and EGFR (1 case, 1%). Amplification of the receptor tyrosine kinases genes occurred in a mutually exclusive manner except for one tumor in which ERBB2 and FGFR2 were both amplified but in different cancer cells. Co-amplification of ERBB2 and MYC, and EGFR and CCND1, in single nuclei but on different amplicons, was confirmed in one case each. Attempts at correlating the FISH status with the immunohistochemical staining pattern showed variable results from complete concordance to no correlation. In conclusion, combination of multiple ligation-dependent probe amplification and FISH analysis is a feasible approach for obtaining the semi-comprehensive genetic information that is necessary for personalized molecular targeted therapy.Modern Pathology advance online publication, 6 March 2015; doi:10.1038/modpathol.2015.33

Clonal profiling of mixed lobular and ductal carcinoma revealed by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

A needle biopsy of a mass in the right breast of a 36-year-old woman revealed invasive ductal carcinoma (IDC), and approximately 20% of cancer cells showed unequivocal membranous staining with the HercepTest. After systemic therapy with trastuzumab and paclitaxel followed by FEC (fluorouracil + epirubicin + cyclophosphamide), a right mastectomy was performed. By histological and immunohistochemical examinations, the resected tumor consisted mainly of E-cadherin-negative invasive lobular carcinoma (ILC), and the rest was ERBB2-positive IDC; thus, the diagnosis was mixed ductal and lobular carcinoma. Multiplex ligation-dependent probe amplification and fluorescence in situ hybridization (FISH) analyses revealed that ILC and IDC shared high-level amplification of CCND1 in homogeneously staining regions (HSR) and that IDC had an additional HSR-type amplicon of ERBB2. These findings strongly indicate that IDC and ILC had a common precursor cell with CCND1 amplification. Review of the biopsy specimen with FISH showed IDC with gene amplifications of CCND1 and ERBB2 as a minor component, IDC without amplification of CCND1 or ERBB2 as a major component, and a minute portion of ILC with CCND1 amplification. We speculate that chemotherapy and trastuzumab caused a marked reduction in IDC; however, ILC with CCND1 amplification was resistant to chemotherapy and grew. © 2014 Japanese Society of Pathology and Wiley Publishing Asia Pty Ltd

Gene amplification of ESR1 in breast cancers-fact or fiction? A fluorescence in situ hybridization and multiplex ligation-dependent probe amplification study

Oestrogen receptor-alpha (ERα), encoded by the ESR1 gene located on 6q25, is a nuclear transcription factor. Since it was reported in 2007 that more than 20% of breast cancers show ESR1 gene amplification, there has been considerable controversy about its frequency and clinical significance. We set out to assess the frequency and levels of ESR1 amplification in breast cancers. In a total of 106 breast needle biopsy specimens examined by immunohistochemistry, 78 tumours contained more than 10% ERα-positive cancer cells. In fluorescence in situ hybridization (FISH) analysis with an ESR1-specific probe, variously extended ESR1 signals were found in ERα-expressing cells. Some of these were indistinguishable from large clustered signals generally accepted to mean high-level gene amplification in homogeneously staining regions (HSRs), and could be considered to represent gene amplification. However, with RNase treatment, the \u27HSR-like\u27 signals changed to small compact signals, and are thus thought to represent concentrated RNA. FISH using two differently labelled probes corresponding to the non-overlapping 5\u27- and 3\u27-end portions of the ESR1 gene on touch smears showed a preserved spatial relationship of the 3\u27 to 5\u27 sequence of ESR1, therefore strongly suggesting that the RNA consisted of primary transcripts. Using touch smears obtained from 51 fresh tumours, precise enumeration of ESR1 signals with a correction by the number of centromere 6 on FISH after RNase A treatment revealed that three tumours (5.9%) had tumour cells with one to three additional copies of ESR1 as predominant subpopulations. This infrequent and low level of gene amplification of ESR1 was also detected as a \u27gain\u27 of the gene by analysis with multiplex ligation-dependent probe amplification (MLPA). The consistent results from immunohistochemistry, FISH, and MLPA in the present study settle the long-standing debate concerning gene amplification of ESR1 in breast carcinoma. © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Lt

Genetic aberrations as the targets of oncology research: Involvement of paraffin-embedded tissues

Cancer is a complex and heterogeneous group of diseases which have been generally classified by their clinical and histopathological features. The genomes of cancer cells are altered by diverse mechanisms and these genetic aberrations lead to a variety of pathological changes. A number of technological advances have allowed us to analyze the cancer genome by various ‘-omics’ techniques, and have accelerated the exploration for the primary genetic aberrations that drive cancer. The state-of-the-art technologies that have developed over the past few decades have enabled researchers to catalogue these genetic aberrations in detail. These aberrations include changes in gene structure and the copy number, mutation, and modification of DNA. Simultaneously, there have been significant achievements in the translation of the genomic discoveries “from the bench to the bed”, which have provided valuable contributions to the progress in cancer therapy. One technology that has been central to these research efforts has been the histopathology of cancer specimens, particularly the use of formalin-fixed, paraffin-embedded tissues. In this overview, we consider the development of oncology research from the past to current efforts, and highlight the roles of histopathology and paraffin-embedded tissues in these efforts