All study TMAs were based on a series of 547 gastric cancer core biopsy specimens assembled and provided by Prof. Kreipe (Hannover). All individual patients gave written informed consent for biological studies at their initial presentation. All samples were obtained from surgery performed for diagnostic and/or therapeutic purposes and were used according to German ethical regulations. The study followed the guidelines of the Declaration of Helsinki and patient identity of the pathological specimens remained anonymous in the context of this study.
In a first step, a 30-core TMA set was pre-selected that provided a representative of all tumor types (intestinal, mixed, and diffuse) according to Lauren classification as well as different HER2 expression and amplification levels. Core size was 0.4 cm and each represented a different tumor sample. HER2 assessment was performed using different commercial assays according to the manufacturers’ instructions at the different participating sites (n = 8). IHC immunostaining was conducted using HercepTest™ (Dako Denmark A/S, Glostrup, Denmark) and/or the PATHWAY® HER2/neu (4B5) antibody (Ventana Medical Systems SA, Illkirch, France). HER2 amplification was determined by FISH assays, using either HER2 FISH pharmDX™ (Dako Denmark A/S) or PathVysion® (Abbott Laboratories, Des Plaines, IL, USA). Automated bright-field dual-color silver in situ hybridization (SISH) assay (BDISH; Inform™, Ventana Medical Systems SA) was used to determine gene amplification at three of the participating sites [26 (link)]. Evaluation was performed according to the modified gastric cancer testing protocol [11 (link)] taking incomplete basolateral or only lateral staining into account. As TMA cores were tested analogous to biopsies the 10% cut-off was recorded but not regarded for the final scoring (i.e., 1+, 2+, and 3+). FISH and BDISH were performed according to the manufacturers’ recommendations with ratios above 2.0 being considered amplified.
In a second step, the complete TMA sample series of 547 tumor cores was used to determine inter-observer variation of HER2 expression (staining intensity and area stained) scoring independent of inter-laboratory staining variation. Thus, TMAs used for evaluation were already IHC stained using the 4B5 antibody (Ventana Medical Systems SA) at the Hannover laboratory that supplied samples.
Data for the first 30-core TMA set were presented and discussed at a 2-day consensus meeting conducted at the Institute of Pathology, Charite, Berlin (27/28 March 2009). After a consensus was reached about specific issues concerning HER2 scoring by IHC in gastric cancer, the second full set of 547 cores were evaluated independently by six German pathologists (GB, MD, HH, JR, SA, AW). The complete 547 TMA set was scanned (Provito GmbH, Berlin, Germany) and provided as virtual slides to the panelists. By use of this data set, all cases that resulted in discordant IHC scores between observers were then individually discussed at a separate meeting in Düsseldorf (10 June 2009) to determine the most reproducible practical guideline for HER2 testing in gastric cancer. Statistical analyses were performed using the statistical program R version 1.9.1 and Microsoft® Excel®. Kappa statistics was calculated according to the method of Conger (1980) by package “irr” of program R [33 (link)]. In order validate these guidelines in routine practice a series of n = 447 prospective diagnostic gastric cancer samples have been tested at five different participating sites throughout Germany which comprised either a biopsy tissue block or one representative tissue block of resection specimen. Thereby, four sites followed the algorithm as proposed by EMEA with IHC (4B5, Ventana) being used first and one center applied both IHC and ISH (BDISH, Ventana) to all n = 152 specimens at their site.
In a first step, a 30-core TMA set was pre-selected that provided a representative of all tumor types (intestinal, mixed, and diffuse) according to Lauren classification as well as different HER2 expression and amplification levels. Core size was 0.4 cm and each represented a different tumor sample. HER2 assessment was performed using different commercial assays according to the manufacturers’ instructions at the different participating sites (n = 8). IHC immunostaining was conducted using HercepTest™ (Dako Denmark A/S, Glostrup, Denmark) and/or the PATHWAY® HER2/neu (4B5) antibody (Ventana Medical Systems SA, Illkirch, France). HER2 amplification was determined by FISH assays, using either HER2 FISH pharmDX™ (Dako Denmark A/S) or PathVysion® (Abbott Laboratories, Des Plaines, IL, USA). Automated bright-field dual-color silver in situ hybridization (SISH) assay (BDISH; Inform™, Ventana Medical Systems SA) was used to determine gene amplification at three of the participating sites [26 (link)]. Evaluation was performed according to the modified gastric cancer testing protocol [11 (link)] taking incomplete basolateral or only lateral staining into account. As TMA cores were tested analogous to biopsies the 10% cut-off was recorded but not regarded for the final scoring (i.e., 1+, 2+, and 3+). FISH and BDISH were performed according to the manufacturers’ recommendations with ratios above 2.0 being considered amplified.
In a second step, the complete TMA sample series of 547 tumor cores was used to determine inter-observer variation of HER2 expression (staining intensity and area stained) scoring independent of inter-laboratory staining variation. Thus, TMAs used for evaluation were already IHC stained using the 4B5 antibody (Ventana Medical Systems SA) at the Hannover laboratory that supplied samples.
Data for the first 30-core TMA set were presented and discussed at a 2-day consensus meeting conducted at the Institute of Pathology, Charite, Berlin (27/28 March 2009). After a consensus was reached about specific issues concerning HER2 scoring by IHC in gastric cancer, the second full set of 547 cores were evaluated independently by six German pathologists (GB, MD, HH, JR, SA, AW). The complete 547 TMA set was scanned (Provito GmbH, Berlin, Germany) and provided as virtual slides to the panelists. By use of this data set, all cases that resulted in discordant IHC scores between observers were then individually discussed at a separate meeting in Düsseldorf (10 June 2009) to determine the most reproducible practical guideline for HER2 testing in gastric cancer. Statistical analyses were performed using the statistical program R version 1.9.1 and Microsoft® Excel®. Kappa statistics was calculated according to the method of Conger (1980) by package “irr” of program R [33 (link)]. In order validate these guidelines in routine practice a series of n = 447 prospective diagnostic gastric cancer samples have been tested at five different participating sites throughout Germany which comprised either a biopsy tissue block or one representative tissue block of resection specimen. Thereby, four sites followed the algorithm as proposed by EMEA with IHC (4B5, Ventana) being used first and one center applied both IHC and ISH (BDISH, Ventana) to all n = 152 specimens at their site.
