HER2 Gene Protein Assay Is Useful to Determine HER2 Status and Evaluate HER2 Heterogeneity in HER2 Equivocal Breast Cancer

HER2 Gene Protein Assay Is Useful to Determine HER2 Status and Evaluate HER2 Heterogeneity in HER2 Equivocal Breast Cancer Yanjun Hou, MD, PhD, 1 Hiroaki Nitta, PhD, 2 and Zaibo Li, MD, PhD 1 From the 1 Department of Pathology, Wexner Medical Center at The Ohio State University, Columbus; and 2 Ventana Medical Systems, Tucson, AZ. Key Words: HER2 gene protein assay; Breast carcinoma; Immunohistochemistry; Fluorescence in situ hybridization; HER2 heterogeneity Am J Clin Pathol January 2017;147:89-95 DOI: 10.1093/AJCP/AQW211 ABSTRACT Objectives: Approximately 15% of breast cancers show equivocal human epidermal growth factor receptor 2 (HER2) results on HER2 immunohistochemistry (IHC) and are reflexed for fluorescence in situ hybridization (FISH). However, some cases remain equivocal. In this study, we evaluated these double-equivocal cases by using a novel gene protein assay (GPA), which can simultaneously assess HER2 gene copy number and protein on a single slide using bright-field microscopy. Methods: GPA was performed on 42 HER2 IHC and FISH double-equivocal cases. Results: GPA was negative for amplification in 28 cases, equivocal in three cases, and positive in 11 cases. The GPA results showed excellent concordance with either repeat FISH using a chromosome 17 centromere probe or FISH using an alternative probe. Furthermore, HER2 heterogeneity was identified in three of 11 GPA-positive cases. Conclusions: HER2 GPA performs accurately and is very useful to determine HER2 status in HER2 IHC and FISH double-equivocal breast cancer cases and identify HER2 heterogeneity. Human epidermal growth factor receptor 2 (HER2; ERBB2) gene amplification and/or protein overexpression occur in up to 20% of breast cancers, and HER2 protein overexpression stimulates downstream signaling and then promotes cell proliferation and survival. 1-5 HER2 status is both a prognostic factor and a predictive factor for the effects of HER2-targeted therapies. The first humanized monoclonal antibody against HER2 protein, trastuzumab, has been demonstrated to be effective in HER2-positive primary and metastatic breast cancers. 6-13 Subsequently, other HER2 targeting agents such as pertuzumab (monoclonal antibody that prevents dimerization of HER2), adotrastuzumab emtansine (antibody drug conjugate), and lapatinib (small molecule inhibitor of epidermal growth factor receptor and HER2) have been developed and found to produce responses in patients with HER2-positive breast cancer. However, anti-her2 therapies are usually ineffective for HER2-negative breast cancers and can cause severe side effects to patients. Furthermore, anti-her2 therapies are costly. Therefore, it is necessary to accurately determine HER2 status in every single breast cancer case. HER2 status is usually assessed by immunohistochemistry (IHC) for HER2 protein overexpression or by fluorescence in situ hybridization (FISH) for HER2 gene amplification. Both methods were approved by the US Food and Drug Administration (FDA). IHC is used primarily and FISH is used as a reflex test on IHC 2þ cases by most laboratories in United States. FISH results have been accepted as a gold standard in the assessment of HER2 status. However, FISH uses dark-field fluorescence microscopy and lacks morphology details. Furthermore, the fluorescent American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com 89 Am J Clin Pathol 2017;147:89-95 89

Hou et al /HER2GENE PROTEIN ASSAY signals fade over time; therefore, the slides cannot be stored for a long time. The bright-field in situ hybridization (BISH) methods, including chromogenic in situ hybridization, silver in situ hybridization, and dual in situ hybridization, have been developed to overcome some of the limitations of FISH. 14-19 However, sometimes it is difficult to differentiate tumor cells (low-grade tumor cells) from surrounding stromal cells or lymphocytes and recognize cell boundaries due to weak counterstaining. HER2 IHC and in situ hybridization (ISH) assays are used for the same purpose to select patients who benefit from anti-her2 therapies, but they detect biologically different targets, HER2 protein and HER2 gene, respectively. Each assay has its own advantages and disadvantages. The gene protein assay (GPA) described in this study merges HER2 IHC and BISH to assess HER2 status accurately in tumor cells by correlating with tissue morphology. A few pilot studies comparing GPA with IHC alone and FISH alone have shown concordance exceeding 90% by using tissue microarray (TMA) slides on both breast cancer and gastric cancer cases. 20,21 Our recent study had applied this method on HER2 IHC 2þ breast tumor whole sections and showed excellent concordance between GPA and FISH results. 22 The major discordant results of HER2 protein and HER2 gene statuses are caused by tumor heterogeneity, mainly detected in HER2-equivocal cases. Furthermore, the updated American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) HER2 testing guidelines have increased the number of HER2 double-equivocal cases (HER2 IHC 2þ and HER2 FISH equivocal). 23-25 Therefore, our study aimed to apply this novel GPA method on the most challenging group of breast cancers (HER2 double-equivocal cases) to assess its clinical utility in this specific group of breast cancers. Materials and Methods Case Selection This study included whole tissue sections of 42 consecutive invasive breast cancer cases (32 core needle biopsy specimens, eight lumpectomy specimens, and two mastectomy specimens) with both equivocal HER2 IHC (IHC score 2þ) and initial HER2 FISH results on clinical testing at The Ohio State University Wexner Medical Center. In addition, five HER2 IHC 3þ/HER2 FISH-amplified and five HER2 IHC-negative/HER2 FISH nonamplified cases were also included in the study. The use of human materials was approved by the institutional review board at The Ohio State University. GPA The same tissue blocks with HER2 IHC and HER2 FISH performed were selected for GPA. GPA was performed on the BenchMark XT platform (Ventana Medical Systems, Tucson, AZ) according to the manufacturer s recommendation. The gene and protein detection combines PATHWAY anti-her2/neu (4B5) rabbit monoclonal primary antibody (Ventana Medical Systems) for IHC and the INFORM HER2 Dual ISH DNA probe cocktail (Ventana Medical Systems) for ISH on a single slide. As recommended, HER2 IHC was performed first using the iview DAB IHC Detection Kit (Ventana Medical Systems). Thereafter, hybridization was performed using a cocktail of the 2,4 dinitrophenyl (DNP) labeled HER2 probe and digoxigenin (DIG) labeled chromosome 17 centromere (CEN17) probe. The HER2 gene and CEN17 signals were detected using the ultraview Silver ISH DNP Detection Kit and ultraview Red ISH DIG Detection Kit (Ventana Medical Systems), respectively. The HER2 gene signals were detected prior to CEN17 detection. The slides were counterstained with Hematoxylin II (Ventana Medical Systems). Signal visualization was performed via light microscopy in which the HER2 gene appeared as discrete black signals and CEN17 as red signals in the nuclei, and the HER2 protein showed brown staining in the cell membranes. Representative GPA images are illustrated Image 1. FISH The FISH analysis with the CEN17 probe was performed at our institution using the dual-color Vysis FDAapproved PathVysion HER2 DNA Probe Kit (Abbott Molecular, Des Plaines, IL). The signals for the HER2 gene and CEN17 were visualized under a fluorescence microscope using appropriate filters. The average numbers of HER2 and CEN17 signals per cell were recorded for at least 50 cells, and the HER2/CEN17 ratio was calculated for each case. The results were interpreted by specialized molecular pathologists using the 2013 HER2 ASCO/CAP updated guidelines 26 and signed out by case pathologists specialized in breast pathology. The FISH with alternative probe (D17S122) was performed at The Mayo Clinic as a send-out test. The FISH results were reported back to our institution and entered into the record as an addendum. Interpretation GPA slides were interpreted for both HER2 IHC and HER2 ISH according to the 2013 HER2 ASCO/CAP guidelines. 26 For the ISH portion of the GPA, the staining results were enumerated by counting at least 50 nuclei by two 90 Am J Clin Pathol 2017;147:89-95 American Society for Clinical Pathology 90

A B C Image 1 Representative gene protein analysis (GPA) images. A, One case with GPA immunohistochemistry (IHC) negative and GPA in situ hybridization (ISH) negative results. B, One case with GPA-IHC positive and GPA-ISH amplified results. C, One case with GPA-IHC equivocal and GPA-ISH negative results. D, One case with GPA-IHC equivocal and GPA-ISH amplified results. pathologists (Y.H. and Z.L.). In cases with HER2 intratumoral heterogeneity, the areas with stronger IHC staining were selected for counting. Results GPA Results in Five HER2-Negative and HER21 Cases All five previously tested HER2-negative cases were again scored as 0 or 1þ on GPA-IHC and did not show increased HER2 signals per cell on GPA-ISH. Similarly, all five previously tested HER2-positive cases showed intense D complete membranous staining (3þ) on GPA-IHC and showed increased HER2 signals per cell on GPA-ISH. GPA Results in 42 HER2 Double-Equivocal Cases In 42 HER2 IHC and FISH double-equivocal cases, GPA-IHC was equivocal (2þ) for each case, but GPA-ISH was negative for amplification in 28 cases, equivocal in three cases, and positive in 11 cases. For 28 GPA-ISH negative cases, 12 had repeat FISH (CEN17) with 10 as negative and two as equivocal. Both cases with equivocal repeat FISH (CEN17) and negative GPA-ISH results were negative by FISH with alternative probes Figure 1. American Society for Clinical Pathology Am J Clin Pathol 2017;147:89-95 91 91

Hou et al /HER2GENE PROTEIN ASSAY GPA Repeat FISH with CEN17 FISH with alternative probes +/ + 28 3 11 NA +/ + NA +/ + NA +/ + 16 10 2 0 1 2 0 0 3 0 5 3 NA +/ + NA +/ + 0 2 0 0 2 0 0 3 cells, all with more than 10% of tumor cells with HER2 signal >6/cell) Image 2B. Intracellular HER2 heterogeneity (defined by discordant HER2 gene amplification and protein expression in same cells) was also identified in those two cases with genetic heterogeneity Image 2C. In all three cases with heterogeneity, HER2 signals from GPA-ISH were greater than 6, while in the other eight GPA-positive cases, HER2 signals from GPA-ISH were less than 6 but with a ratio more than 2. Figure 1 Gene protein analysis (GPA) results and repeat fluorescence in situ hybridization (FISH) results with either CEN17 or alternative probe in 42 human epidermal growth factor receptor 2 (HER2) double-equivocal cases. NA, not applicable. For 11 GPA-ISH positive cases, eight cases had repeat FISH (CEN17) with three as positive and five as equivocal. FISH with alternative probes was performed in three cases with equivocal repeat FISH (CEN17) and positive GPA results and was positive in all three cases (Figure 1). For three GPA-equivocal cases, two cases had repeat FISH (CEN17), and both cases were negative (Figure 1). In the group of 28 GPA-negative cases, the HER2 signals from original FISH were greater than 4 per cell but less than 5, with a mean of 4.44. All HER2 signals from GPA-ISH were less than 4 per cell. The mean HER2 signal from GPA-ISH was significantly less than the HER2 signal from original FISH (2.94 vs 4.44, P <.05), while the HER2/CEN17 ratio showed no difference between these two methods Table 1. In the group of 11 GPA-positive cases, the HER2 signals from original FISH were greater than 4 per cell but less than 6, with a mean of 4.79. The HER2 signals from GPA- ISH ranged from 3.84 per cell to 12.56, including three cases with HER2 signals greater than 6 per cell and eight cases with HER2 signals between 4 and 6 but HER2/CEN17 ratios greater than 2. The mean HER2 signal from GPA-ISH was significantly greater than that from original FISH (5.77 vs 4.79, P <.05), while there was no significant difference in the HER2/CEN17 ratio between these two methods (Table 1). HER2 Heterogeneity Identified by GPA Furthermore, HER2 heterogeneity were identified in three of 11 GPA-positive cases, including one with regional heterogeneity (defined as the existence of amplificationnegative or amplification-equivocal patterns in different areas of the tumor) Image 2A and two with genetic heterogeneity (defined as the presence of scattered/small clustered tumor cells with HER2 amplification in 5%-50% of tumor Discussion To our knowledge, this is the first study to examine GPA s performance in breast carcinoma specimens with HER2 double-equivocal results (IHC 2þ and FISH equivocal) using the 2013 ASCO/CAP HER2 testing update guidelines. The GPA in the current study combines the anti- HER2 antibody for IHC and the HER2 Dual ISH DNA probes for ISH on a single slide. Limited studies had evaluated GPA in breast and gastric carcinomas. Nitta et al 20 and Hirschmann et al 21 performed GPA on TMA slides, and both studies found an excellent agreement between FISH and GPA-ISH (up to 95%). However, most of the cohort cases in both studies were either clearly negative or unequivocally positive cases. Our recent study examined GPA s performance on HER2 IHC 2þ cases and found an agreement of 82% with FISH results. 22 The 2013 updated ASCO/CAP HER2 testing guidelines have increased the number of HER2 double-equivocal cases (HER2 IHC 2þ and HER2 FISH equivocal). 26 In our current study, we performed GPA on the most challenging group of breast cancers (HER2 double-equivocal cases) using whole slides and found that GPA was able to recategorize 93% (39/42) of HER2 double-equivocal cases into an unequivocally negative or positive category with excellent concordance with either repeated FISH results using the CEN17 probe or an alternative probe. In the group of 28 GPA-negative cases, no intratumoral heterogeneity was identified, and all cases were reclassified as HER2 negative possibly because of more accurate counting by improved visualization. In the group of 11 GPA-positive cases, three cases were reclassified as HER2 positive because of intratumoral heterogeneity, and the other eight cases were reclassified as HER2 positive possibly because of more accurate counting by improved visualization. HER2 intratumoral heterogeneity has been reported in up to 40% of breast carcinomas and has gained more attention because of its impact on treatment decisions and effect. 27-32 The 2013 ASCO/CAP HER2 testing Update Committee recently recommended reporting any discrete population of amplified cells more than 10% of the total 92 Am J Clin Pathol 2017;147:89-95 American Society for Clinical Pathology 92

Table 1 HER2 Signal, CEN17 Signal, and Ratio in 28 GPA-Negative Cases and 11 GPA-Positive Cases GPA-Negative Cases (n ¼ 28), Mean (Range) GPA-Positive Cases (n ¼ 11), Mean (Range) Characteristic Original FISH GPA-ISH Original FISH GPA-ISH HER2 4.44 (4.02-4.81) 2.94 (2.06-3.89) 4.79 (4.04-5.8) 5.77 (3.84-12.56) CEN17 3.3 (2.63-4.10) 2.08 (1.89-2.86) 3.24 (2.45-4.26) 2.12 (1.32-4.0) Ratio 1.35 (1.15-1.75) 1.43 (1.12-1.78) 1.51 (1.3-1.8) 2.79 (1.67-4.9) CEN17, chromosome 17 centromere; FISH, fluorescence in situ hybridization; GPA, gene protein analysis; HER2, human epidermal growth factor receptor 2; ISH, in situ hybridization. A B C Image 2 Representative gene protein analysis images of cases with human epidermal growth factor receptor 2 heterogeneity. A, One case with regional heterogeneity. B, One case with genetic heterogeneity. The outlined areas show tumor cells with HER2 gene amplification and protein overexpression. C, One case with intracellular heterogeneity. The outlined areas show tumor cells with HER2 gene amplification but no protein overexpression. tumor cell population. 26 The 2013 Update Committee also suggested scanning the entire slide prior to counting and/or using an IHC HER2 test to define areas of potential amplification. 26 One of the main advantages of BISH assays, like IHC, is the ease of assessment of HER2 intratumoral heterogeneity. Therefore, a simultaneous assessment American Society for Clinical Pathology Am J Clin Pathol 2017;147:89-95 93 93

Hou et al /HER2GENE PROTEIN ASSAY combining IHC and ISH on one slide would facilitate evaluation and enhance test efficiency, especially for cases with HER2 heterogeneity. Indeed, previous studies and our current study have found that HER2 intratumoral heterogeneity was appreciated much easier on GPA slides. In the current study, we identified that three of 11 HER2-postive cases on GPA had HER2 intratumoral heterogeneity, including one with regional heterogeneity and two with genetic and intracellular heterogeneity. Usually, regional heterogeneity can be highlighted by HER2 IHC, and then the potentially amplified areas can be circled for FISH. However, genetic heterogeneity is difficult to be assessed by FISH because HER2-amplified cells are intermixed with non HER2-amplified cells. However, GPA can overcome this difficulty because HER2 membranous staining is able to highlight the targeted cells for ISH assessment. In our institution, we perform both HER2 IHC and FISH at the initial biomarker evaluation. Occasionally, we have encountered breast carcinoma cases with negative HER2 IHC but positive HER2 FISH. This discordance between HER2 IHC and FISH may be explained by HER2 intracellular heterogeneity (HER2 gene amplification but no protein overexpression). As shown in one representative image (Image 2C), this intracellular heterogeneity is easily appreciated on the GPA slide. However, the intracellular heterogeneity will not be assessed by FISH. The GPA shows similar accuracy to the FISH method but a faster turnaround time (less than 24 hours) than FISH. Furthermore, GPA does not need a fluorescence microscope and does not require special training for interpretation. It is much easier to count HER2 signals and to differentiate tumor cells from surrounding stromal cells and lymphocytes on GPA slides because tumor cell boundaries are clearly noted by the IHC staining. Moreover, the IHC staining on GPA slides can be used to choose the interested area for counting. It is not uncommon that both HER2 IHC and FISH are performed simultaneously at initial breast biomarker assessment, like in our institution. GPA would be a perfect alternative in this setting because of its cost saving and faster turnaround time. Although many laboratories use HER2 IHC as a screening assay and reflex HER2 FISH for HER2 IHC 2þ cases, some outsource FISH to reference laboratories. GPA would be beneficial in such a setting where the IHC staining on GPA slides can be used to choose the interested area for counting. In summary, GPA performed accurately on the most challenging HER2 double-equivocal breast carcinoma cases and was able to recategorize up to 93% of HER2 doubleequivocal cases into an unequivocally negative or positive category with excellent concordance with repeated FISH results. Furthermore, HER2 intratumoral heterogeneity was easily assessed on GPA slides. 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