Gene copy mapping of the ERBB2/TOP2A region in breast cancer

ERBB2 is one of the most important oncogenes in breast cancer, and its disordered expression is commonly associated with gene amplification. Amplification of at least one gene near ERBB2, topoisomerase IIalpha (TOP2A), has been shown to be clinically significant, but the prevailing patterns of gene amplification in this region of chromosome arm 17q have not been studied systematically in clinical cases of breast cancer. For characterizing this region, a commercial ERBB2-containing contig probe and 7 probes prepared from single overlapping BAC and P1 clones lying telomeric to ERBB2 and including TOP2A were hybridized to 77 ERBB2-amplified archival breast tumor specimens from 75 patients. The 7 single-clone probes covered a region of approximately 650 kb starting 114 kb telomeric to ERBB2. Amplification of the ERBB2 contig target alone was found in 32% of the tumors, whereas all 8 probe targets were amplified in 12% of the tumors, based on an amplification criterion of there being more than or equal to 2 targets per chromosome 17 centromere. When one of the 7 overlapping probes encompassing TOP2A indicated amplification within a specimen, all probes telomeric to that probe usually showed amplification. Only 5 specimens had regions of normal or deleted targets separating 2 amplified targets. Also, tumors that showed deletion of TOP2A usually showed deletion of one or more contiguous targets. The observed patterns of amplification and deletion are consistent with the break-fusion-bridge model for gene amplification. TOP2A was amplified in 25% of all tumor specimens and was deleted in 24%, based on a deletion criterion of there being fewer than or equal to 0.75 targets per chromosome 17 centromere. Considering the relevance of the TOP2A gene product to anthracycline therapy and the wealth of other cancer-associated genes within the ERBB2/TOP2A region, the pattern of amplification and deletion near ERBB2 and TOP2A may have a dramatic effect on the malignant potential of breast carcinomas and their response to therapy.     

Gene amplification of ERBB2 and EGFR in adenocarcinoma in situ and intramucosal adenocarcinoma of Barrett's esophagus

We examined 11 cases of carcinoma arising from Barrett's 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.     

RNA interference-based functional dissection of the 17q12 amplicon in breast cancer reveals contribution of coamplified genes

DNA amplification is a frequent occurrence in cancer genomes. While tumor amplicons may harbor known oncogenes "driving" amplification, amplicons rarely comprise only single genes. The potential functional contribution of coamplified genes remains largely unexplored. In breast cancer, 20-30% of tumors exhibit amplification within chromosome band 17q12, containing the ERBB2 oncogene. Analysis of array-based comparative genomic hybridization and expression profiling data indicate that the minimum region of recurrent amplification (i.e., the amplicon "core") at 17q12 includes two other genes, GRB7 and STARD3, which exhibit elevated expression when amplified. Western blot analysis confirms overexpression of each at the protein level in breast cancer cell lines SKBR3 and BT474 harboring amplification. In these cell lines (but not in control MCF7 breast cancer cells lacking 17q12 amplification), targeted knockdown of ERBB2 expression using RNA interference (RNAi) methods results in decreased cell proliferation, decreased cell-cycle progression, and increased apoptosis. Notably, targeted knockdown of either GRB7 or STARD3 also leads to decreased cell proliferation and cell-cycle progression, albeit to a lesser extent compared with ERBB2 knockdown. We conclude that the amplification and resultant overexpression of genes coamplified with ERBB2 at 17q12 can contribute to proliferation levels of breast cancer cells. Our findings validate the utility of RNAi in the functional interrogation of tumor amplicons, and provide evidence for a contribution of coamplified genes to tumor phenotypes.     

High-resolution genomic profiles of breast cancer cell lines assessed by tiling BAC array comparative genomic hybridization

A BAC-array platform for comparative genomic hybridization was constructed from a library of 32,433 clones providing complete genome coverage, and evaluated by screening for DNA copy number changes in 10 breast cancer cell lines (BT474, MCF7, HCC1937, SK-BR-3, L56Br-C1, ZR-75-1, JIMT1, MDA-MB-231, MDA-MB-361, and HCC2218) and one cell line derived from fibrocystic disease of the breast (MCF10A). These were also characterized by gene expression analysis and found to represent all five recently described breast cancer subtypes using the "intrinsic gene set" and centroid correlation. Three cell lines, HCC1937 and L56BrC1 derived from BRCA1 mutation carriers and MDA-MB-231, were of basal-like subtype and characterized by a high frequency of low-level gains and losses of typical pattern, including limited deletions on 5q. Four estrogen receptor positive cell lines were of luminal A subtype and characterized by a different pattern of aberrations and high-level amplifications, including ERBB2 and other 17q amplicons in BT474 and MDA-MB-361. SK-BR-3 cells, characterized by a complex genome including ERBB2 amplification, massive high-level amplifications on 8q and a homozygous deletion of CDH1 at 16q22, had an expression signature closest to luminal B subtype. The effects of gene amplifications were verified by gene expression analysis to distinguish targeted genes from silent amplicon passengers. JIMT1, derived from an ERBB2 amplified trastuzumab resistant tumor, was of the ERBB2 subtype. Homozygous deletions included other known targets such as PTEN (HCC1937) and CDKN2A (MDA-MB-231, MCF10A), but also new candidate suppressor genes such as FUSSEL18 (HCC1937) and WDR11 (L56Br-C1) as well as regions without known genes. The tiling BAC-arrays constitute a powerful tool for high-resolution genomic profiling suitable for cancer research and clinical diagnostics.     

Concomitant gastrin and ERBB2 gene amplifications at 17q12–q21 in the intestinal type of gastric cancer

Our recent studies using comparative genomic hybridization showed that gain or amplification at the 17q12–q21 region is very common in the intestinal type of gastric cancer. Here, we describe a fluorescence in situ hybridization study with gastrin (GAS)‐specific and ERBB2‐specific probes on ten specimens of gastric carcinoma that, by using comparative genomic hybridization, showed 1) DNA copy number gain or amplification at 17q12–q21, a region known to harbor the GAS and ERBB2 genes (four cases); 2) gain of the entire chromosome 17 (three cases); or 3) normal copy number of chromosome 17 (three cases). GAS and ERBB2 protein expression was studied by Western immunoblotting from gastric cancer cell lines with or without gain at 17q12–q21 as well as a breast cancer cell line with ERBB2 amplification. Our results showed that simultaneous amplification of both GAS and ERBB2 was four‐ to ninefold in the tumors with the 17q12–q21 amplification. Both genes were amplified in the same nuclei, and the hybridization signals were localized to the same region of the nucleus. Overexpression of GAS and ERBB2 was observed by Western immunoblotting only in the gastric cancer cell line with gain at 17q12–q21. The ERBB2 amplification is also a recurrent change in breast cancer. To investigate whether the GAS amplification is unique in gastric cancer, fluorescence in situ hybridization analysis was performed on 40 breast cancer cell lines. The ERBB2 amplification was observed in 11 cell lines, but none of the lines showed the GAS amplification. This indicates that the formation of an amplicon, in which both the GAS and the ERBB2 genes are amplified, might be unique in gastric cancer, especially in its intestinal type, and that simultaneous amplification of both genes is important to the tumorigenesis of intestinal gastric cancer. We demonstrate here for the first time that a gene of a physiological hormone is amplified in tumors that originate from cells that normally secrete the hormone. Genes Chromosomes Cancer 24:24–29, 1999. © 1999 Wiley‐Liss, Inc.     

High-resolution genomic and expression analyses of copy number alterations in breast tumors

Analysis of recurrent DNA amplification can lead to the identification of cancer driver genes, but this process is often hampered by the low resolution of existing copy number analysis platforms. Fifty-one breast tumors were profiled for copy number alterations (CNAs) with the high-resolution Affymetrix 500K SNP array. These tumors were also expression-profiled and surveyed for mutations in selected genes commonly mutated in breast cancer (TP53, CDKN2A, ERBB2, KRAS, PIK3CA, PTEN). Combined analysis of common CNAs and mutations revealed putative associations between features. Analysis of both the prevalence and amplitude of CNAs defined regions of recurrent alteration. Compared with previous array comparative genomic hybridization studies, our analysis provided boundaries for frequently altered regions that were approximately one-fourth the size, greatly reducing the number of potential alteration-driving genes. Expression data from matched tumor samples were used to further interrogate the functional relevance of genes located in recurrent amplicons. Although our data support the importance of some known driver genes such as ERBB2, refined amplicon boundaries at other locations, such as 8p11-12 and 11q13.5-q14.2, greatly reduce the number of potential driver genes and indicate alternatives to commonly suggested driver genes in some cases. For example, the previously reported recurrent amplification at 17q23.2 is reduced to a 249 kb minimal region containing the putative driver RPS6KB1 as well as the putative oncogenic microRNA mir-21. High-resolution copy number analysis provides refined insight into many breast cancer amplicons and their relationships to gene expression, point mutations and breast cancer subtype classifications. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Oncogene amplification in breast cancer.

To refine the analysis of gene amplification in breast cancer, the authors have developed sensitive methods that can be used to screen nucleic acid prepared from a variety of sources. In their analysis, Southern hybridization and DNA dot-blot analysis were used to screen 49 breast cancer DNAs for Myc, Neu, and Int-2 gene amplification. The analysis detected minimal one extra gene copy) as well as expanded (two or more extra gene copies) gene amplifications, and in addition, distinguished between gene amplification and aneuploidy as the cause of extra gene copies. These quantitative methods were adapted to patient specimens routinely available in the anatomic pathology laboratory, including fresh tumor tissue, tumor nuclei discarded during estrogen receptor analysis, and paraffin blocks. One minimal gene amplification was found in three cases of intraductal cancer. Of 25 cases of nonmetastatic invasive cancer, 28% had at least one extra Myc gene, whereas 24% had Neu, and 21% had Int-2 gene amplification. Of 21 cases of metastatic invasive cancer, 43% had Myc, 43% had Neu, and 40% had Int-2 gene amplification. Among the nonmetastatic cancers, 47% had one, 12% had two, and 4% had three amplified genes. Within the metastatic cancers, 48% had one, 28% had two, and 5% had three amplified genes. Our data suggest relationships between tumor progression and both incidence and size of Myc, Neu, and Int-2 gene amplification.     

A newly established metastatic breast tumor cell line with integrated amplified copies of ERBB2 and double minute chromosomes

A continuous line of human mammary tumor cells, called 21MT, has been established in culture from a pleural effusion of a 36-year-old woman with metastatic breast cancer. The cells are epithelial as shown by morphology and expression of keratins and are mammary tumor cells as shown by expression of the HMFG-2 antigenic determinant. The cells grow well both in DFCI-1, a partially defined medium containing pituitary extract and 1% fetal bovine serum, and in alpha-minimum essential medium (alpha-MEM) supplemented with 10% serum, epidermal growth factor (EGF), insulin, and hydrocortisone. Karyotypic analysis of cells at early passage has shown the presence of rearranged (marker) chromosomes as well as aneuploidy with a net DNA content in the tetraploid range, confirmed by DNA cytofluorography, as well as double minute chromosomes in about 5% of the cells. Southern blots have revealed a 40-fold amplification of the ERBB2 gene and a 50-fold overexpression of its mRNA. The amplification of ERBB2 DNA was localized by in situ hybridization to one of the marker chromosomes but not to the double minutes. It is inferred, therefore, that at least two genes have been amplified in these cells.     

High-level DNA amplifications are common genetic aberrations in B-cell neoplasms.

Gene amplification is one of the molecular mechanisms resulting in the up-regulation of gene expression. In non-Hodgkin's lymphomas, such gene amplifications have been identified rarely. Using comparative genomic hybridization, a technique that has proven to be very sensitive for the detection of high-level DNA amplifications, we analyzed 108 cases of B-cell neoplasms (42 chronic B-cell leukemias, 5 mantle cell lymphomas, and 61 aggressive B-cell lymphomas). Twenty-four high-level amplifications were identified in 13% of the patients and mapped to 15 different genomic regions. Regions most frequently amplified were bands Xq26-28, 2p23-24, and 2p14-16 as well as 18q21 (three times each). Amplification of several proto-oncogenes and a cell cycle control gene (N-MYC (two cases), BCL2, CCND2, and GLI) located within the amplified regions was demonstrated by Southern blot analysis or fluorescence in situ hybridization to interphase nuclei of tumor cells. These data demonstrate that gene amplifications in B-cell neoplasms are much more frequent than previously assumed. The identification of highly amplified DNA regions and genes included in the amplicons provides important information for further analyses of genetic events involved in lymphomagenesis.     

Gene amplification and gene dosage in cell lines derived from squamous cell carcinoma of the head and neck.

Gene amplification and related alterations in gene dosage were analyzed in a series of 34 cell lines derived from different human head and neck squamous cell carcinomas (SCCHN). INT2 gene amplification was observed in 62%, MYC gene amplification in 24%, and EGFR gene amplification in 21% of the cell lines. There was a strong correlation between EGFR gene amplification and increased copies of the ERBB2 gene on chromosome 17, suggesting a synergistic selection for these two genes either during cancer progression or in culture. Two abnormalities showed a significant correlation with clinical course: MYC gene amplification showed an inverse correlation with tumor recurrence (r = -0.44, p = 0.01), and a small increase in MYCL gene copies on chromosome I correlated with the presence of metastases (r = 0.61, p = 0.001). This altered MYCL gene dosage might represent a chromosome translocation rather than true gene amplification. In addition to gene amplification, 79% of the cell lines had increased copies of chromosome 8. Comparison of the cell lines with several of the corresponding primary tumors demonstrated that most gene amplifications were already present in the primary tumors, although some appeared de novo in cell culture. These studies indicate that gene amplification, especially of INT2, is a prominent abnormality in head and neck squamous cell cancer. Aneuploidy and chromosomal lesions other than gene amplification were also found to alter the dosage of several oncogenes specifically.     

Effects of ERBB2 amplicon size and genomic alterations of chromosomes 1, 3, and 10 on patient response to trastuzumab in metastatic breast cancer

Trastuzumab is widely used for advanced breast cancer patients with ERBB2-amplified tumors. Nevertheless, over half of these patients do not have an objective response. One reason may be altered expression of genes that might compensate for ERBB2 inhibition. We previously mapped the gene-rich region of chromosome 17 telomeric to ERBB2, and reported considerable variability in the telomeric extent of the ERBB2 amplicon. Here we examined whether the variable amplicon size may be associated with patient response to trastuzumab. In addition, we looked at associations between response and several signaling pathway-related genes unrelated to the ERBB2 amplicon, including AKT3, PTEN, PIK3CA, and PTGS2. In 35 patients with ERBB2-amplified metastatic breast cancer, with 40% overall response to trastuzumab, fluorescence in situ hybridization identified the telomeric extent of the ERBB2 amplicon and the status of the several pathway-related genes. Objective response strongly correlated with the telomeric amplicon size, with 62% of patients with shorter amplicons responding, compared with only 7% of patients with longer amplicons (P = 0.0015). Abnormal copy number of PTGS2 was marginally associated with objective response (P = 0.066), while abnormal copy numbers of two reference loci, 1q25 and the chromosome 10 centromere, were significantly associated with response. Pairwise combinations of copy number status of these loci and ERBB2 amplicon size provided stronger associations and identified a group of patients without responders. These results suggest that patient selection for trastuzumab may be improved by considering ERBB2 amplicon size and genomic status of the 1q25, PTGS2, and centromere 10 loci.     

Increased copy number at 17q22-q24 by CGH in breast cancer is due to high-level amplification of two separate regions

Studies by comparative genomic hybridization (CGH) have defined a chromosomal site at 17q22-q24 that is often overrepresented in breast cancer, neuroblastoma, and several other tumor types. Due to the limited resolution and dynamic range of CGH, it remains unclear whether this gain reflects high-level amplification of small subregion(s) or low-level gain of most of the distal 17q. We used 32 physically mapped 17q probes to construct more accurate copy number profiles for 14 breast cancer cell lines by interphase fluorescence in situ hybridization (FISH). Six cell lines (43%) showed an increased copy number of the 17q22-q24 region by CGH, and seven (50%) by FISH. FISH copy number profiles had a substantially higher dynamic range than did CGH profiles. FISH revealed two independent, highly amplified regions (A and B) at 17q23, separated by about 5 Mb of non-amplified DNA. These regions were distinctly telomeric from the ERBB2 gene locus. However, region A was often co-amplified with ERBB2, whereas B was amplified in cell lines that showed no ERBB2 amplification. We conclude that distal 17q gains recently discovered in breast cancer by CGH are due to high-level amplifications of two different regions at 17q23. This chromosomal region has previously been reported to undergo allelic loss and therefore was thought to harbor a tumor suppressor gene. The present FISH data provide support for the presence, and a starting point for the positional isolation, of 17q23 genes whose upregulation by amplification may play a role in the progression of breast cancer and many other tumor types. Genes Chromosomes Cancer 20:372–376, 1997. © 1997 Wiley-Liss, Inc.     

ERBB2 kinase domain mutation in a gastric cancer metastasis

ERBB2 is a member of the epidermal growth factor receptor (EGFR) family. Recent studies revealed that the kinase domain of the ERBB2 gene was mutated in human cancers, including gastric cancer. Despite the importance of cancer metastasis in the pathogenesis of cancers, data on the ERBB2 kinase domain mutation in cancer metastasis are lacking. In this study, to explore the possibility that ERBB2 mutation is involved in the metastasis mechanism, we analyzed the kinase domain of ERBB2 for the detection of somatic mutations in 58 gastric adenocarcinomas with lymph node metastasis. We found one ERBB2 mutation, which was detected in the lymph node metastasis, but not in the primary tumor of the same patient. The ERBB2 mutation was a missense mutation which substituted an amino acid in exon 21 (V832I). We simultaneously analyzed the somatic mutations of EGFR, K-RAS, PIK3CA and BRAF genes in the sample with the ERBB2 mutation, and found that this metastatic carcinoma did not harbor any of the mutations. Our data suggest that ERBB2 kinase domain mutation occasionally occurs in metastatic gastric carcinoma and might play a role in the metastatic process of some gastric carcinomas.     

Co-amplification of 8p12 and 11q13 in breast cancers is not the result of a single genomic event

Epithelial cancers frequently have multiple amplifications, and particular amplicons tend to occur together. These co-amplifications have been suggested to result from amplification of pre-existing junctions between two chromosomes, that is, translocation junctions. We investigated this hypothesis for two amplifications frequent in breast cancer, at 8p12 and 11q13, which had been reported to be associated in Southern blot studies. We confirmed that both genomic amplification and expression of genes was correlated between the frequently-amplified regions of 8p and 11q, in array CGH and microarray expression data, supporting the importance of co-amplification. We examined by FISH the physical structure of co-amplifications that we had identified by array CGH, in five breast cancer cell lines (HCC1500, MDA-MB-134, MDA-MB-175, SUM44, and ZR-75-1), four breast tumors, and a pancreatic cancer cell line (SUIT2). We found a variety of arrangements: amplification of translocation junctions; entirely independent amplification of the two regions on separate chromosomes; and separate amplification of 8p and 11q sequences in distinct sites on the same rearranged chromosome. In this last arrangement, interphase nuclei often showed intermingling of FISH signals from 8p12 and 11q13, giving a false impression that the sequences were interdigitated. We conclude that co-amplification of the main 8p and 11q amplicons in breast tumors is not usually the result of a preceding translocation event but most likely reflects selection of clones that have amplified both loci. This article contains supplementary material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.     

Amplification of ERBB2 (HER2) in embryonal rhabdomyosarcoma: A potential treatment target in rare cases?

The ERBB2 gene encodes a receptor tyrosine kinase also known as HER2. The gene is amplified and overexpressed in one-fifth of breast carcinomas; patients with such tumors benefit from targeted treatment with trastuzumab or other drugs blocking the receptor. In addition, ERBB2 has been shown to be amplified and/or overexpressed in a variety of other malignancies. Notably, both alveolar and embryonal rhabdomyosarcoma (RMS), especially in children, often show increased expression of ERBB2. Although high-level amplification of the gene has not been described in RMS, its frequent expression at the cell surface of RMS cells has been exploited for chimeric antigen receptor T-cell (CAR T)-based treatment strategies. We here describe two cases of pediatric, fusion-negative embryonal RMS with high-level amplification of the ERBB2 gene. One patient is currently treated with conventional chemotherapy for a recently detected standard risk RMS, whereas the other patient died from metastatic disease. Both tumors displayed focal amplicons (210 and 274 Kb, respectively) in chromosome band 17q12, with proximal and distal borders corresponding to those typically seen in breast cancer. In both tumors, the ERBB2 amplicon correlated with high expression at the RNA and protein levels. Thus, breast cancer-like ERBB2 amplification is a very rare, but recurrent feature of pediatric RMS, and should be exploited as an alternative treatment target.     

FISH analysis of gene aberrations (MYC, CCND1, ERBB2, RB, and AR) in advanced prostatic carcinomas before and after androgen deprivation therapy

Genetic mechanisms leading to androgen-independent growth in advanced prostatic carcinomas (PC) are still poorly understood. Analysis of genes potentially involved in the regulation of tumor cell proliferation and apoptosis might confer better insight into this process and might lead to improved therapeutic strategies. Fluorescence in situ hybridization (FISH) analysis of dissociated nuclei with DNA probes for MYC (8q24)/#8, cyclin D1 gene (CCND1; 11q13)/#11, ERBB2 (17q13)/#17, the androgen receptor gene (AR; Xq12)/#X, and the retinoblastoma gene (RB; 13q14) was applied to formalin-fixed tissue from 63 patients with advanced PC after androgen deprivation therapy (ADT); matched tumor tissue before ADT was also available in 22 of these cases. The cut-points used were: "increased copy number," > or = 30% of all nuclei with increased FISH signals (centromere and/or gene); "amplification," > or = 15% of nuclei with "increased gene copy number." CCND1 and MYC gene "amplifications" were present before ADT in 25% and 33% of the cases, respectively; the frequency of these "amplifications" increased to 37% and 57% after ADT. Loss of the RB gene was nearly four times more frequent after ADT than before therapy (22% versus 6%). AR and ERBB2 gene "amplifications" occurred only after ADT in 36% and 30% of cases, respectively. With the exception of the AR gene, the copy number increase was low. After treatment, MYC and AR gene "amplifications" correlated with the proliferation rate (Ki-67/MIB1 index; p = 0.01 and p = 0.04), whereas ERBB2 "amplifications" were associated with increased apoptotic index (PCD/TUNEL; p = 0.016). However, no correlation between FISH results and clinical follow-up could be established. FISH analysis of genes putatively involved in PC progression revealed characteristic patterns of aberrations in advanced PC before and after ADT. Distinct changes in gene copy number before and after therapy suggests possible involvement of these genes in the escape from androgen control.