Copyright © Royal College of pathologists of Australasia. Unauthorized reproduction of this article is prohibited. HER2 status in gastric/gastro-oesophageal junctional cancers: should determination of gene amplification by SISH use HER2 copy number or HER2:CEP17 ratio? MARIAN PRIYANTHI KUMARASINGHE 1,2 ,WILLEM BASTIAAN DE BOER 1,2 ,TZE SHENG KHOR 1 , ESTHER M. OOI 3 ,NIC JENE 4 ,SURESHINI JAYASINGHE 4 AND STEPHEN B. FOX 4,5 1 PathWest Laboratory Medicine, Perth, 2 School of Pathology and Laboratory Medicine, University of Western Australia, Perth, 3 School of Medicine and Pharmacology, Royal Perth Hospital Unit, University of Western Australia, Perth, WA, 4 Department of Pathology, Peter MacCallum Cancer Centre, East Melbourne, and 5 Department of Pathology and Sir Peter MacCallum Department of Oncology, University of Melbourne, Melbourne, Vic, Australia Summary The aim of this study was to compare HER2 amplification, as determined by the HER2 copy number (CN) and the HER2/ CEP17 ratio, with protein expression in gastric and gastro- oesophageal junction (G/GOJ) adenocarcinoma. HER2 immunohistochemistry (IHC) and silver in situ hybridisation (SISH) were performed in 185 cases. Modified gastric criteria were used for IHC scoring. HER2 and CEP17 CNs were counted in at least 20 cancer cells and the ratio calculated as per previously defined protocols. These two SISH methods were statistically compared against the different IHC scores. Thirty-four cases showed amplification, by both methods in 29, and either method in five. IHC score was 3þ in 29 cases; 26 showed amplification by both methods, one by ratio only and two were not amplified. IHC score was 2þ in 24 cases; three showed amplification by both methods and two by either. One each of IHC 1þ and 0 showed an increased ratio but not CN. The HER2 CN and ratio for IHC score 3þ compared to scores 2þ,1þ and 0 were significantly different (all p < 0.01). The CN for IHC 2þ vs IHC 1þ and IHC 0 was significantly different (both p < 0.01) but the ratio was not ( p ¼ 0.5711 and p ¼ 0.2857, respectively). The CN and the ratio for scores 1þ and 0 were not significantly different ( p ¼ 0.9823 and p ¼ 0.9910, respectively). The HER2 CN differentiates between the different IHC scores better than the HER2:CEP17 ratio. Cases that show IHC3þ and high CN may not require calculation of the ratio. Furthermore, con- sideration should be given to the CN when IHC negative cases appear amplified by the ratio only. Key words: Amplification, copy number, double probe method, gastric/gastro- esophageal junctional carcinoma, HER2 status, ratio, single probe method. Received 17 June, revised 29 October, accepted 7 November 2013 INTRODUCTION HER2 positive status determines the eligibility for HER2 targeted therapy of advanced and metastatic gastric and gastro-oesophageal junction (G/GOJ) adenocarcinomas. The HER2 status can be determined by estimation of protein expression by immunohistochemistry (IHC) and/or assessment of HER2 gene copy number and centromeric probe 17 (CEP17) ratio by in situ hybridisation (ISH). ISH can be performed by bright field techniques such as chromogenic ISH (CISH) or silver ISH (SISH) or by dark field methods using fluorescence (FISH). Although there is evidence that bright field methods are superior to FISH in determining the gene amplification for G/GOJ cancers, 1–3 there is no universal acceptance of a method for testing at this point. 4,5–7 The Trastuzumab for Gastric Cancer (ToGA) study defined ISH positive status for G/GOJ cancer as HER2:CEP17 ratio 2.0 irrespective of the copy number. 4 This criterion has been used for clinical testing and in most studies related to the HER2 status of gastric/ GOJ cancer. The majority of studies show a perfect or near perfect correlation between IHC3þ expression and gene amplification. 4,8–10 However, they also report that a proportion of equivocal and negative IHC cases are ISH positive when the ratio is used. IHC is a semi-quantitative test that is subject to a variety of pre analytical and analytical issues involving tissue sampling, scoring criteria and inherent subjectivity of IHC interpretation. In contrast, ISH technique is considered superior, being more quantitative and reproducible. There are specific issues confounding HER2 testing in G/GOJ cancers by either method, such as the type of specimen used (i.e., endoscopic biopsies, resections or metastatic material) and a greater degree of heterogeneity reported in these cancers. 2,8 Considering the special issues, a modified IHC scoring system was established for HER2 testing of G/GOJ cancers. 9 Superiority of the modified/gastric cancer scoring system has been validated by others subsequently. 4,10 A positive IHC reaction in G/GOJ cancers includes basolateral/lateral membrane staining as opposed to the requirement of complete membrane staining in breast carcinomas. Additionally the cut-off for a positive test is only 10% positive tumour cells for resections and a cluster of five positive tumour cells for endoscopic biopsies. 9,10 There are two methods to establish gene amplification. One is the single probe method by counting the actual HER2 copy number (CN) per nucleus, the cut-off for ampli- fication being 6.0. The other is using dual probes to calculate the ratio of HER2 genes to centromere 17 (HER2/CEP17), the cut-off for amplification being 2.0. The ratio distinguishes increased HER2 gene copy number secondary to extra copies of CEP17 that may occur due to true polysomy or Pathology (April 2014) 46(3), pp. 184–187 ANATOMICAL PATHOLOGY Print ISSN 0031-3025/Online ISSN 1465-3931 # 2014 Royal College of Pathologists of Australasia DOI: 10.1097/PAT.0000000000000075