ERBB2 Amplifications in Esophageal Adenocarcinoma Peter S. Dahlberg, MD, PhD, Blake A. Jacobson, PhD, Ganesh Dahal, PhD, James M. Fink, MD, PhD, Robert A. Kratzke, MD, PhD, Michael A. Maddaus, MD, Lance J. Ferrin, MD, PhD  The Annals of Thoracic Surgery  Volume 78, Issue 5, Pages 1790-1800 (November 2004) DOI: 10.1016/j.athoracsur.2004.05.037

Fig 1 ERBB2 signaling; normally ERBB2, pairs with other epidermal growth factor receptor family receptors that have bound their respective ligands. A phosphorylation cascade is activated and results in activation of multiple intracellular pathways including mitogen activated protein kinase and phosphoinositide 3-kinase, which promote cell cycle progression and inhibit apoptosis. When ERBB2 is overexpressed as the result of a gene amplification event, the receptor can homodimerize, and it is constitutively active. (EGF = epidermal growth factor; EGFR = epidermal growth factor receptor; MAPK = mitogen activated protein kinase; P = phosphate; PI3K = phosphoinositide 3-kinase.) The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)

Fig 2 ERBB2 amplicon mapping using Rec-A protein assisted restriction endonuclease (RARE) cleavage of genomic deoxyribonucleic acid (DNA). (A) Strategy. If multiple copies of an amplified gene are present as a tandem repeat within a contiguous stretch of DNA, cutting at a single site (indicated by the clear vertical line) will yield fragments with a molecular weight determined by the length of the repetitive unit. (B) The RARE cleavage. The technique takes advantage of the ability of Rec-A protein from E coli to pair an oligonucleotide at a unique site of homology to form a three-stranded structure called a synaptic complex (oligonucleotide and two strands of native DNA). This acts as a sequence-specific “masking tape” for DNA and protects the site from methylation during subsequent treatment with EcoR1 methylase. After removal of the synaptic complex, digestion with EcoR1 results in cutting of genomic DNA at a single site. (Modified from Ferrin LJ, et al, Science; 1991;254:1494–7 [12], with permission.) The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)

Fig 3 ERBB2 copy number in 25 esophageal cancers and 2 cell lines. Deoxyribonucleic acid, isolated from tumor specimens and cell lines, was electrophoresed through agarose, transferred onto a nylon membrane, and hybridized with an ERBB2 probe. Densitometry was performed to determine copy number (per haploid genome equivalent; ie, normal would be 1.0). Labels correspond to those in Table 1. High level amplification of ERBB2 was in samples 7, 11c, and 31 as well as in both esophageal adenocarcinoma cell lines, OE19 and OE33. The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)

Fig 4 ERBB2 microarray expression. Relative expression levels of ERBB2 were determined by microarray analysis using Affymetrix U95 chips. Each chip has 3 oligonucleotides representing ERBB2 transcripts. Sample groups from left to right are specimens from normal esophagus, esophageal adenocarcinoma, esophageal adenocarcinoma metastasis, normal stomach, and the 2 esophageal adenocarcinoma cell lines OE19 and OE33. Expression values identified by squares, circles, and triangles represent messenger RNA levels of ERBB2 measured by 3 different Affymetrix ERBB2 oligonucleotide probes. (EAC = esophageal adenocarcinoma; ESO = normal esophagus; MET = metastasis; STO = normal stomach.) The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)

Fig 5 Immunohistochemical staining of ERBB2 protein in cell line OE19. Cell line OE19 was stained with antibody to ERBB2 using a commercially available method (HercepTest). Cells stained 3+ indicating high-level protein expression. Cells from the KATO-III gastric adenocarcinoma cell line that does not have an ERBB2 amplification were scored as 0 (figure not shown). The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)

Fig 6 Karyotype analysis of esophageal cancer cell line OE19. The karyotype of OE19 was complex, which is typical of carcinoma cell lines. There were numerous deletions and additions. Of note, there were two relatively normal appearing chromosome 17 seconds. No double minute chromosomes were apparent, but there was an obvious homogeneous staining region found on chromosome 14 (circled). (mar = marker chromosomes.) The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)

Fig 7 Fluorescence in situ hybridization of ERBB2 deoxyribonucleic acid (DNA) in esophageal cancer cell lines OE19 (A) and OE33 (B). Fluorescence in situ hybridization was performed utilizing the Vysis PathVysion ERBB2 DNA Probe Kit. The ERBB2 DNA appears pink and a control probe to the chromosome 17 is green. Reverse banding techniques identified the site of amplification as the homogenously staining region seen in the OE19 karyotype on chromosome 14. The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)

Fig 8 Rec-A protein assisted restriction endonuclease (RARE) cleavage mapping of OE19 esophageal carcinoma cell genomic deoxyribonucleic acid (DNA). The RARE cleavage mapping was used to identify the size of the amplicon repeat in OE19 cells. An oligonucleotide corresponding to the 51 nucleic acids spanning the exon 10 EcoR1 site in the ERBB2 gene was synthesized. After protection of the site with Rec-A protein, the genomic DNA was treated with EcoR1 methylase to block all other EcoR1 sites. The synaptic complex was removed and the DNA digested with EcoR1. Cleavage occurs only at the targeted site that was protected from methylation. Genomic DNA was then subjected to pulsed field electrophoresis through agarose and probed with an ERBB2 probe from exon 10. The gel has a single prominent band at 210 kb and a faint band at 420 kb (probably represented a doublet of the main amplicon). These results are consistent with an ERBB2 amplicon of 210 kb configured as a tandem repeat. These results indicate that the amplicon contains sizable amounts of DNA from outside the 29 kb ERBB2 gene. The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)

Fig 9 Trastuzumab sensitivity of cancer cell lines. The dependence of cell growth on ERBB2 protein expression was tested by measuring the ability of trastuzumab (Herceptin) to inhibit cell growth in vitro. Cells were incubated in media with a saturating dose of 20 μg/mL of trastuzumab or with control antibody. Cells were counted after 96 hours. OE19 is an esophageal adenocarcinoma cell line with a 100-fold ERBB2 amplification, OE 33 is an esophageal adenocarcinoma cell line with a 14-fold ERBB2 amplification, SK-BR-3 and BT-474 are breast cancer cell lines with about a 10-fold ERBB2 amplification, and KATO-III is a gastric adenocarcinoma cell line that does not have an ERBB2 amplification. Mean ± standard error of 4 to 6 independent experiments are shown. Growth of all cell lines with ERBB2 amplifications were significantly inhibited by treatment with Herceptin (p < 0.05 Duncan multiple range test). The Annals of Thoracic Surgery 2004 78, 1790-1800DOI: (10.1016/j.athoracsur.2004.05.037)