ERBB2 amplifications in esophageal adenocarcinoma.
BACKGROUND
ERBB2 (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, Her-2-neu) gene amplification and overexpression has been reported in several types of cancer. The purpose of this study was to (1) determine the frequency of ERBB2 amplification (in comparison to other proto-oncogenes) in tumors from patients with esophageal adenocarcinoma, (2) characterize structural details of an ERBB2 amplicon in the esophageal adenocarcinoma cell line OE19 (contains a 100-fold ERBB2 amplification), and (3) test whether growth of the OE19 cell line is sensitive to the ERBB2 inhibitor trastuzumab (Herceptin; Genetech, Inc, San Francisco, CA).


METHODS
First, we determined the frequency, by Southern blotting techniques, of amplification of ERBB2 and 13 other proto-oncogenes in a panel of 25 esophageal adenocarcinoma tumors. Then, in a second panel of 10 tumor specimens, expression levels of the ERBB2 gene and of several other genes that flank ERBB2 on chromosome 17 were determined by microarray analysis. Next we characterized the ERBB2 amplicon in the esophageal adenocarcinoma cell line OE19 using cytogenetic methods and a Rec-A protein assisted restriction endonuclease mapping technique. Finally, an in vitro growth inhibition assay was used to measure the sensitivity of OE19 and OE33 cells to treatment with trastuzumab (humanized antibody to ERBB2).


RESULTS
ERBB2 was the most frequently amplified proto-oncogene among 25 esophageal adenocarcinoma tumors tested (greater than 10-fold amplification in 3 of 25 (12%) tumors tested). The OE19 cell line contains a 100-fold amplification of the ERBB2 gene, and highly expresses its messenger ribonucleic acid. Transcripts from genes that flank ERBB2 including GRB7, a protein linked to metastasis in esophageal cancer, also showed high levels of expression. In OE19 cells, the ERBB2 amplicon was localized to a homogeneously staining region of chromosome 14. Southern blots from the Rec-A protein assisted restriction endonuclease cleavage mapping experiments in OE19 showed a strong band of 210 kilobases in size, demonstrating that the main amplicon was a tandem repeat. In the in vitro growth inhibition assay, trastuzumab inhibited the OE19 and OE33 cells growth by 49% and 20%, respectively, at a saturating concentration of 20 microg/mL.


CONCLUSIONS
ERBB2 is the most frequently amplified proto-oncogene in esophageal adenocarcinoma among the genes that we tested. In the OE19 esophageal adenocarcinoma cell line, the ERBB2 amplicon is translocated onto chromosome 14, is amplified 100-fold at the deoxyribonucleic acid level, and is highly overexpressed at the messenger ribonucleic acid level. Finally, the growth of this cell line was inhibited by incubation with trastuzumab. These results demonstrate that a substantial number of esophageal adenocarcinomas have amplified copies of the ERBB2 gene, and that they may be responsive to ERBB2 targeted therapies such as trastuzumab.
Dahlberg
Peter S
PS
Department of Surgery, Division of Cardiovascular and Thoracic Surgery, Hennepin County Medical Center, Minneapolis, Minnesota 55455, USA. dahlb002@umn.edu
Jacobson
Blake A
BA
Dahal
Ganesh
G
Fink
James M
JM
Kratzke
Robert A
RA
Maddaus
Michael A
MA
Ferrin
Lance J
LJ
D000230:Adenocarcinoma / Q000235:genetics* / Q000473:pathology; D000911:Antibodies, Monoclonal / Q000494:pharmacology; D061067:Antibodies, Monoclonal, Humanized; D015139:Blotting, Southern; D045744:Cell Line, Tumor / Q000187:drug effects; D002886:Chromosomes, Human, Pair 17 / Q000235:genetics; D003199:Computer Systems; D004938:Esophageal Neoplasms / Q000235:genetics* / Q000473:pathology; D005784:Gene Amplification; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D018734:Genes, erbB-2; D006801:Humans; D016133:Polymerase Chain Reaction; D012150:Polymorphism, Restriction Fragment Length; D000090063:Proto-Oncogene Mas; D011519:Proto-Oncogenes; D012333:RNA, Messenger / Q000235:genetics; D012334:RNA, Neoplasm / Q000235:genetics; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors; D013274:Stomach Neoplasms / Q000473:pathology; D000068878:Trastuzumab
D000911:Antibodies, Monoclonal; D061067:Antibodies, Monoclonal, Humanized; D000090063:Proto-Oncogene Mas; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D018719:Erb-b2 Receptor Tyrosine Kinases; D000068878:Trastuzumab; C000715470:MAS1 protein, human