Amplification involving the CEP17 region may lead to false negative results of HER2 gene amplification by FISH.
Authors: Tse CH , Gown AM , Goldstein LC , Kandalaft PL , Baird G , Kussick SL , Barry TS 
Published: December 2010

Abstract #1079

Background: Detection of HER2 gene amplification by fluorescence in-situ hybridization (FISH) is one of the key tests predicting clinical response of breast cancer to trastuzumab. According to the recent CAP/ASCO guidelines for HER2 testing, the tumor is considered positive for HER2 gene amplification when the HER2/CEP17 ratio is greater than 2.2, negative if less than 1.8, and equivocal if the ratio is between 1.8 and 2.2. The need to use CEP17 as reference for HER2 evaluation is supported by the observation that an increased HER2 gene copy number as a result of chromosome 17 polysomy may not bear the same clinical significance as HER2 gene amplification. However, response to trastuzumab has been observed in some cases with polysomy 17. This study aims to examine the reliability of using CEP17 for chromosome 17 enumeration.&#x2028; Material and methods:&#x2028; 40 cases of HER2 FISH performed at the PhenoPath Laboratories between October 2007 and May 2008 having a mean CEP17/cell of over 2.5 were selected. In order to confirm the elevated chromosome 17 copy number, FISH mapping studies were performed using probes for the Smith-Magenis syndrome (SMS) and retinoic acid receptor alpha (RARA) loci which reside on the short arm and long arm of chromosome 17 respectively and are telomeric to the centromere/HER2 loci. Chromosome 17 status was determined by correlating the mean copy numbers of HER2, CEP17, SMS and RARA. A gene locus with signal number/cell between 1.4 and 2.4 was considered consistent with eusomy. Additional studies of the TP53 locus which is telomeric to SMS on 17p was performed on a subset of cases having only one gene locus in the eusomic range.&#x2028; Results:&#x2028; The CEP17/cell of the 40 cases ranges from 2.61 to &gt;10 with a median of 3.92. 32 cases (80%) show at least one gene locus with copy numbers consistent with eusomy (17 [42.5%] have one gene locus and 15 [37.5%] have two gene loci showing eusomy). The TP53 locus was studied in 7 cases having only one gene locus in the eusomic range. Six of these cases also showed gene copy numbers consistent with eusomy. Overall, 9 cases (22.5%) would have the HER2 amplification status upgraded (negative to positive, negative to equivocal or equivocal to positive) if a gene locus showing eusomy was used as reference for HER2 evaluation.&#x2028; Discussion:&#x2028; The result of the present study shows that CEP17 may not be a reliable internal reference standard in HER2 FISH studies in a subset of cases. A majority of the apparent polysomy cases based on CEP17 copy number likely represent low-level amplification of the chromosome 17 centromere and would have been shown to be eusomic if another gene locus were used as a reference. The HER2/CEP17 ratio could be falsely low in as high as 22% of the cases with apparent polysomy 17. Additional FISH studies of other gene loci on the chromosome 17 are recommended in all HER2 FISH negative/equivocal cases with apparent polysomy 17.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1079.