© 2013 APMIS. Published by John Wiley & Sons Ltd. DOI 10.1111/apm.12215
APMIS 122: 755–760
Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH NARA YOON, IN-GU DO and EUN YOON CHO Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea
Yoon N, Do I-G, Cho EY. Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH. APMIS 2014; 122: 755–760. Easy and accurate HER2 testing is essential when considering the prognostic and predictive significance of HER2 in breast cancer. The use of a fully automated, quantitative FISH assay would be helpful to detect HER2 amplification in breast cancer tissue specimens with reduced inter-laboratory variability. We compared the concordance of HER2 status as assessed by an automated FISH staining system to manual FISH testing. Using 60 formalin-fixed paraffin-embedded breast carcinoma specimens, we assessed HER2 immunoexpression with two antibodies (DAKO HercepTest and CB11). In addition, HER2 status was evaluated with automated FISH using the Leica FISH System for BOND and a manual FISH using the Abbott PathVysion DNA Probe Kit. All but one specimen were successfully stained using both FISH methods. When the data were divided into two groups according to HER2/CEP17 ratio, positive and negative, the results from both the automated and manual FISH techniques were identical for all 59 evaluable specimens. The HER2 and CEP17 copy numbers and HER2/CEP17 ratio showed great agreement between both FISH methods. The automated FISH technique was interpretable with signal intensity similar to those of the manual FISH technique. In contrast with manual FISH, the automated FISH technique showed well-preserved architecture due to low membrane digestion. HER2 immunohistochemistry and FISH results showed substantial significant agreement (j = 1.0, p < 0.001). HER2 status can be reliably determined using a fully automated HER2 FISH system with high concordance to the well-established manual FISH method. Because of stable signal intensity and high staining quality, the automated FISH technique may be more appropriate than manual FISH for routine applications. Key words: Genes; erbB-2; in situ hybridization; fluorescence; carcinoma; ductal; breast; automation. Eun Yoon Cho, Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, #50 Ilwon-dong, Kangnam-gu, Seoul 135-710, South Korea. e-mail: [email protected]
N. Yoon and I.G. Do contributed equally to this work.
Human Epidermal Growth Factor Receptor 2 (HER2) is a proto-oncogene that encodes a transmembrane receptor tyrosine kinase of the epidermal growth factor receptor family. HER2 protein is overexpressed in about 10–25% of breast cancers (1, 2). Patients with HER2 amplification usually also overexpress HER2 protein and have a worse prognosis than patients with normal HER2 levels (3, 4). Furthermore, HER2-positive tumors are less sensitive to tamoxifen and non-anthracycline/ Received 13 March 2013. Accepted 21 October 2013
non-taxane-based chemotherapy, but are more sensitive to anthracyclines and paclitaxel than HER2negative tumors (1, 5, 6). In addition to its role as a prognostic and predictive marker, HER2 is an important therapeutic target for breast cancer treatment. Monoclonal humanized antibodies against HER2, such as trastuzumab (Herceptin; Genentech, South San Francisco, CA, USA) and lapatinib (GlaxoSmithKline, London, UK), improve the overall survival of HER2-positive patients when used alone or combined with chemotherapy (4, 7). HER2-positive patients who received neoadjuvant 755
YOON et al.
chemotherapy with trastuzumab also demonstrated high pathological complete responses in several studies (8–10). Considering its significance as a prognostic and predictive factor, the ability to accurately evaluate HER2 status is essential. Fluorescence in situ hybridization (FISH) is considered the gold standard for evaluating HER2 status, which has become essential in the era of personalized cancer therapy. Therefore, the need for an easy and accurate HER2 test and the demand for fully automated methods will increase in the future. Many laboratories are equipped for automated immunohistochemistry (IHC) and silver in situ hybridization (SISH). Fully automated IHC and SISH methods have been shown to save time and labor, reduce inter-observer and inter-laboratory variability, and improve reproducibility (1, 11, 12). In this study, we assessed the HER2 status of 60 formalin-fixed, paraffin-embedded breast cancer tissue specimens with a fully automated, quantitative FISH assay (Leica FISH System for BOND, Leica Microsystems, Newcastle upon Tyne, UK) and compared the results with a manual FISH assay (Abbott PathVysion HER-2 DNA Probe Kit, Abbott Laboratories, Des Plains, IL, USA). In addition, we performed an IHC study using the Leica Bond Oracle HER2 IHC System (clone CB11, Leica Microsystems, Newcastle upon Tyne, UK) and the DAKO HercepTest (DAKO, Glostrup, Denmark) to compare HER2 protein expression with different immunohistochemical antibodies to verify the correlation with FISH results. MATERIALS AND METHODS Patients and samples Formalin-fixed, paraffin-embedded blocks were retrieved from 60 patients who had invasive ductal carcinoma that was surgically resected between 2009 and 2011 at Samsung Medical Center. Hematoxylin and eosin (H & E)-stained sections and IHC slides for HER2, ER, and PR from all specimens were reviewed by light microscopy. Tumor type and histologic grade were also evaluated. HER2 expression status was as follows: 26 cases had a score of 0/1+, 8 had a score of 2+, and 26 had a score of 3+. The patient ages ranged from 38 to 67 years, with a mean age of 51.4 years. The Institutional Review Board at Samsung Medical Center approved this study. The clinicopathological findings according to HER2 IHC status are presented in Table 1.
FISH analysis Both FISH staining methods were applied to all 60 specimens. Manual FISH analysis was performed using the FDA-approved Abbott Molecular PathVysion HER-2 DNA Probe Kit (Abbott Laboratories, Des Plains, IL,
756
Table 1. Clinicopathological findings according to HER2 IHC status IHC 0/1+(26) 2+(8) 3+(26) Age (
APMIS 122: 755–760
Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH NARA YOON, IN-GU DO and EUN YOON CHO Department of Pathology, Samsung Medical Center, Sungkyunkwan University College of Medicine, Seoul, Korea
Yoon N, Do I-G, Cho EY. Analysis of HER2 status in breast carcinoma by fully automated HER2 fluorescence in situ hybridization (FISH): comparison of two immunohistochemical tests and manual FISH. APMIS 2014; 122: 755–760. Easy and accurate HER2 testing is essential when considering the prognostic and predictive significance of HER2 in breast cancer. The use of a fully automated, quantitative FISH assay would be helpful to detect HER2 amplification in breast cancer tissue specimens with reduced inter-laboratory variability. We compared the concordance of HER2 status as assessed by an automated FISH staining system to manual FISH testing. Using 60 formalin-fixed paraffin-embedded breast carcinoma specimens, we assessed HER2 immunoexpression with two antibodies (DAKO HercepTest and CB11). In addition, HER2 status was evaluated with automated FISH using the Leica FISH System for BOND and a manual FISH using the Abbott PathVysion DNA Probe Kit. All but one specimen were successfully stained using both FISH methods. When the data were divided into two groups according to HER2/CEP17 ratio, positive and negative, the results from both the automated and manual FISH techniques were identical for all 59 evaluable specimens. The HER2 and CEP17 copy numbers and HER2/CEP17 ratio showed great agreement between both FISH methods. The automated FISH technique was interpretable with signal intensity similar to those of the manual FISH technique. In contrast with manual FISH, the automated FISH technique showed well-preserved architecture due to low membrane digestion. HER2 immunohistochemistry and FISH results showed substantial significant agreement (j = 1.0, p < 0.001). HER2 status can be reliably determined using a fully automated HER2 FISH system with high concordance to the well-established manual FISH method. Because of stable signal intensity and high staining quality, the automated FISH technique may be more appropriate than manual FISH for routine applications. Key words: Genes; erbB-2; in situ hybridization; fluorescence; carcinoma; ductal; breast; automation. Eun Yoon Cho, Department of Pathology, Samsung Medical Center, Sungkyunkwan University School of Medicine, #50 Ilwon-dong, Kangnam-gu, Seoul 135-710, South Korea. e-mail: [email protected]
N. Yoon and I.G. Do contributed equally to this work.
Human Epidermal Growth Factor Receptor 2 (HER2) is a proto-oncogene that encodes a transmembrane receptor tyrosine kinase of the epidermal growth factor receptor family. HER2 protein is overexpressed in about 10–25% of breast cancers (1, 2). Patients with HER2 amplification usually also overexpress HER2 protein and have a worse prognosis than patients with normal HER2 levels (3, 4). Furthermore, HER2-positive tumors are less sensitive to tamoxifen and non-anthracycline/ Received 13 March 2013. Accepted 21 October 2013
non-taxane-based chemotherapy, but are more sensitive to anthracyclines and paclitaxel than HER2negative tumors (1, 5, 6). In addition to its role as a prognostic and predictive marker, HER2 is an important therapeutic target for breast cancer treatment. Monoclonal humanized antibodies against HER2, such as trastuzumab (Herceptin; Genentech, South San Francisco, CA, USA) and lapatinib (GlaxoSmithKline, London, UK), improve the overall survival of HER2-positive patients when used alone or combined with chemotherapy (4, 7). HER2-positive patients who received neoadjuvant 755
YOON et al.
chemotherapy with trastuzumab also demonstrated high pathological complete responses in several studies (8–10). Considering its significance as a prognostic and predictive factor, the ability to accurately evaluate HER2 status is essential. Fluorescence in situ hybridization (FISH) is considered the gold standard for evaluating HER2 status, which has become essential in the era of personalized cancer therapy. Therefore, the need for an easy and accurate HER2 test and the demand for fully automated methods will increase in the future. Many laboratories are equipped for automated immunohistochemistry (IHC) and silver in situ hybridization (SISH). Fully automated IHC and SISH methods have been shown to save time and labor, reduce inter-observer and inter-laboratory variability, and improve reproducibility (1, 11, 12). In this study, we assessed the HER2 status of 60 formalin-fixed, paraffin-embedded breast cancer tissue specimens with a fully automated, quantitative FISH assay (Leica FISH System for BOND, Leica Microsystems, Newcastle upon Tyne, UK) and compared the results with a manual FISH assay (Abbott PathVysion HER-2 DNA Probe Kit, Abbott Laboratories, Des Plains, IL, USA). In addition, we performed an IHC study using the Leica Bond Oracle HER2 IHC System (clone CB11, Leica Microsystems, Newcastle upon Tyne, UK) and the DAKO HercepTest (DAKO, Glostrup, Denmark) to compare HER2 protein expression with different immunohistochemical antibodies to verify the correlation with FISH results. MATERIALS AND METHODS Patients and samples Formalin-fixed, paraffin-embedded blocks were retrieved from 60 patients who had invasive ductal carcinoma that was surgically resected between 2009 and 2011 at Samsung Medical Center. Hematoxylin and eosin (H & E)-stained sections and IHC slides for HER2, ER, and PR from all specimens were reviewed by light microscopy. Tumor type and histologic grade were also evaluated. HER2 expression status was as follows: 26 cases had a score of 0/1+, 8 had a score of 2+, and 26 had a score of 3+. The patient ages ranged from 38 to 67 years, with a mean age of 51.4 years. The Institutional Review Board at Samsung Medical Center approved this study. The clinicopathological findings according to HER2 IHC status are presented in Table 1.
FISH analysis Both FISH staining methods were applied to all 60 specimens. Manual FISH analysis was performed using the FDA-approved Abbott Molecular PathVysion HER-2 DNA Probe Kit (Abbott Laboratories, Des Plains, IL,
756
Table 1. Clinicopathological findings according to HER2 IHC status IHC 0/1+(26) 2+(8) 3+(26) Age (
