Review Article The assessment of HER2 status in breast cancer: the past, the present, and the future

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1 Pathology International 2016; 66: doi: /pin Review Article The assessment of HER2 status in breast cancer: the past, the present, and the future Hiroaki Nitta, 1 Brian D. Kelly, 2 Craig Allred, 1 Suzan Jewell, 1 Peter Banks, 1 Eslie Dennis 1 and Thomas M. Grogan 1 1 Medical & Scientific Affairs and 2 Technology & Applied Research, Ventana Medical Systems, Inc., Tucson, Arizona USA Humanized monoclonal anti-human growth factor receptor 2 (HER2) antibody trastuzumab was approved for HER2 positive breast cancer patient treatment 11 years after the demonstration of HER2 gene amplification associated with the HER2 protein overexpression in breast cancer in HER2 positive status of breast cancer patients is assessed by HER2 gene amplification with in situ hybridization (ISH) and/or HER2 protein overexpression with immunohistochemistry (IHC). Because the discordance between quantitative HER2 ISH and subjective, semiquantitative HER2 IHC assay results is a well-recognized issue of HER2 testing, we developed an assay combining HER2 ISH and HER2 IHC assays (HER2 gene-protein assay; HER2 GPA) as one test on the same tissue section. HER2 GPA allows pathologists to score the HER2 gene and HER2 protein status simultaneously at the individual cell level. The possibility that HER2 GPA may become the next generation of HER2 testing is discussed, particularly for cases in which it is difficult to assess the HER2 status of breast cancer patients due to the HER2 heterogeneity. Key words: breast, gene-protein assay, heterogeneity, human epidermal growth factor receptor 2 (HER2), immunohistochemistry (IHC), in situ hybridization (ISH) INTRODUCTION The proto-oncogene erb-b2 receptor tyrosine kinase 2 (ERBB2) or human epidermal growth factor receptor 2 (HER2) was discovered independently in the same time period by three USA research groups 1 3 and a Japanese research group. 4 Correspondence: Hiroaki Nitta, PhD, MBA, Medical Affairs, Medical & Scientific Affairs, Ventana Medical Systems, Inc., 1910 E Innovation Park Drive, Tucson, Arizona, 85755, USA. hiro.nitta@roche.com Received 23 November Accepted for publication 9 March Pathology International published by Japanese Society of Pathology and John Wiley & Sons Australia, Ltd. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. Amplification of the HER2 gene that is located in region q21 of chromosome 17 was originally observed in a mammary carcinoma, 2 a salivary gland adenocarcinoma, 4 and the MKN- 7 gastric cancer cell line 5 in early gene discovery studies. The important biological role of HER2 gene in cancer was hypothesized due to the correlation observed between overall survival and time to relapse of breast cancer patients with HER2 gene amplification. 6 Overexpression of HER2 protein was revealed in HER2 gene amplified breast cancer tissues by immunohistochemical (IHC) staining and Western blotting applications. 7 The first demonstration of the HER2 targeted therapy concept was reported with a xenograft tumor model in 1986 using a monoclonal anti-her2 antibody that successfully showed anti-tumor effects in vivo. 8 Trastuzumab (Herceptin, Genentech, San Francisco, CA, USA) is a recombinant humanized monoclonal immunoglobulin G1 kappa antibody against the extracellular domain of HER2 protein. 9 This anti- HER2 antibody was approved by the Food and Drug Administration (FDA) on September 25, 1998 as the first HER2-targeted therapy drug for metastatic breast cancer patients whose tumors overexpressed the HER2 protein. Then, trastuzumab with chemotherapy showed clinical benefit as the first-line treatment in HER2 positive metastatic breast cancer cases determined by the over-expression of HER2 protein. The results showed a longer progression-free survival time, a higher response rate, longer response duration, and improved overall survival. 10 On November 16, 2006, the FDA approval of trastuzumab was expanded to all HER2 positive breast cancer patients with node positive status as an adjuvant therapy. The results of two combined trastuzumab adjuvant therapy trials showed significantly improved clinical outcomes among the patients with surgically removed HER2 positive breast cancer. 11 However, it was reported that about 10 % of breast cancer patients without obvious amplified HER2 gene status also showed benefit from adjuvant trastuzumab therapy. 12 Finally in 2008, the use of trastuzumab was approved as a single agent for the treatment of HER2 positive breast cancer patients with

2 314 H. Nitta et al. node negative status. After almost two decades of HER2- targeted therapy with trastuzumab, there is still room for improvement with appropriate patient selection for treatment and also further optimization for the best practice usage of trastuzumab, namely dosing, timing, and duration. Currently, in addition to trastuzumab, another anti-her2 antibody drug pertuzumab, small molecule intracellular tyrosine kinase inhibitors lapatinib and neratinib, and an antibody-drug conjugate ado-trastuzumab emtansine (T-DM1) are available for treating HER2 positive breast cancer patients. HER2 status assessment of breast cancer patients Over new invasive breast cancer cases are diagnosed among women in the US and about women die from breast cancer each year. 13 Approximately 15 % to 20 % of these patients have HER2 positive breast cancers and should be treated with a HER2-targeted therapy to improve their clinical outcome. The American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) released updated recommendations for HER2 testing in breast cancer in The recommendation states that all newly diagnosed early-stage or metastatic breast cancer patients should be tested for HER2 status. The breast cancers are classified as HER2 positive, HER2 equivocal, or HER2 negative by validated HER2 immunohistochemistry (IHC) and/or HER2 in situ hybridization (ISH) assays (Table 1). HER2 equivocal cases by either HER2 IHC or HER2 ISH assays must be retested with an alternative HER2 testing method or on different tissue blocks with the same testing method. However, both HER2 IHC and HER2 ISH assays have their own advantages and disadvantages in achieving optimum test results. Table 1 Summary of ASCO/CAP HER2 testing recommendations 2007 and 2013 HER2 status 2007 recommendations 2013 recommendations HER2 positive IHC 3+ >30 % of invasive tumor cells with uniform intense membrane staining or FISH Dual probes HER2/CEN17 of >2.2 or Single probe >6 HER2 gene copies HER2 equivocal IHC 2+ FISH Dual probes HER2/CEN17 of or Single probe Average HER2 gene copy number 4 6 per cell HER2 negative IHC 1+ <10 % of invasive tumor cells with weak complete membrane staining or Weak incomplete membrane staining in any portion of tumor cells IHC 0 No staining FISH Dual probes: HER2/CEN17 of <1.8 or Single probe: Average HER2 gene copy number < 4 per cell IHC 3+ >10 % of invasive tumor cells with uniform intense membrane staining or ISH Dual probes HER2/CEN17 of 2 with any copy numbers or HER2/CEN17 of <2.0withanaverage HER2 copy number 6.0 per cell Single probe Average HER2 copy number 6.0 per cell IHC 2+ >10 % of invasive tumor cells with incomplete or weak membrane staining or 10 % of invasive tumor cells with intense membrane staining ISH Dual probes HER2/CEN17 of <2.0withanaverage HER2 copy number 4.0 and <6.0/cell or Single probe Average copy number 4.0 and <6.0 per cell IHC 1+ >10 % of invasive tumor with faint incomplete membrane staining IHC 0 No staining or 10 % of invasive tumor cells with faint staining ISH Dual probes: HER2/CEN17 < 2.0 with average HER2 copy number < 4.0 per cell Single probe: Average HER2 copy number < 4.0 per cell ASCO/CAP, American Society of Clinical Oncology/College of American Pathologists; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; FISH, fluorescence in situ hybridization; CEN17, chromosome 17 centromere; ISH, in situ hybridization

3 HER2 status assessment of breast cancer 315 HER2 immunohistochemical analysis HER2 IHC assays are designed to analyze preserved HER2 protein with a bright field microscope on formalin-fixed, paraffin-embedded (FFPE) clinical tissue sections. HER2 IHC assays are technically easier to perform compared to HER2 ISH assays. However, HER2 IHC assays are subjective and semi-quantitative for determining the expression levels of HER2 protein in breast cancer. HER2 IHC equivocal cases must be reexamined with a different tissue block, if available, or by HER2 ISH assay. The specificity and sensitivity of HER2 IHC assays are influenced by selections of pretreatment conditions, antibody clones, 15,16 and signal detection systems. Furthermore, due to the lack of internal control within a tissue section, there is a reasonable concern with the reliability of HER2 IHC assays, especially when HER2 IHC results are negative. When the threshold for HER2 IHC 2+ cases was reduced from 30 % to 10 % of the tumor cells having incomplete membrane staining, HER2 ISH reflex test frequencies were increased by 59 % and the HER2 gene amplification detection of breast cancer cases was increased by 33 %. 17 Because of the subjective nature of HER2 status assessment using HER2 IHC assays, it is possible that HER2 IHC equivocal cases might be determined to be HER2 negative and would not receive further HER2 status assessment. HER2 in situ hybridization analysis HER2 ISH assays are aimed to visualize the HER2 gene with or without a reference target chromosome 17 centromere (CEN17) in FFPE tissue sections of clinical samples. There are multiple signal detection systems approved by the FDA for HER2 ISH assays: (i) fluorescence in situ hybridization (FISH); (ii) chromogenic in situ hybridization (CISH); and (iii) dual in situ hybridization (DISH). HER2 FISH assays are performed using manual or semi-automated protocols and the subsequent analysis requires a specialized fluorescence microscope in a darkroom. HER2 CISH assays are also manual or semiautomated protocols and the HER2 CISH assay results are analyzed with a regular bright field microscope. HER2 DISH assays are performed only with automated protocols and analyzed with a bright field microscope. Signal preservation in HER2 CISH and DISH slides are superior to HER2 FISH slides and the assessment of the tissue morphology is also easier in chromogenic assays. A major advantage of HER2 ISH assays compared to HER2 IHC assays is that the HER2 status assessment is conducted by quantitative analyses and also includes internal control cells adjacent to tumor cells in the same tissue section. However, it has been reported that HER2 equivocal cases may result in different HER2 status assessment dependent on which ISH assays are used and the scoring criteria selected. 18 Thus, counting ISH signals in 40 cells instead of 20 cells per field of view was recommended to allow for a more accurate reflection of the HER2 gene status. 18 It should be noted that the level of HER2 gene amplification significantly affected pathological complete response to trastuzumab-based neoadjuvant therapy. 19 However, because an increase in HER2 gene copy numbers can be induced by chemotherapy, the assessment of HER2 status with HER2 ISH assays can be a challenging task for pathologists with those patients who have been treated with chemotherapy. 20 Cold ischemia time effects on HER2 testing The 2013 ASCO/CAP HER2 testing recommendations state that the time from excision to the initiation of tissue fixation (cold ischemic time) should be as short as possible and the duration of tissue fixation should be 6 72 hours. 14 The duration of formalin fixation appears to be less of an issue with HER2 testing results, as up to 96 hours of tissue fixation exposure (more than an over weekend length) did not significantly affect the HER2 testing scores. 21 However, the exact effects of cold ischemic time on HER2 IHC and HER2 ISH assays are still controversial. A study comparing the performance of HER2 IHC, HER2 FISH, and HER2 DISH with respect to cold ischemia time in breast cancer showed that there were no adverse effects on HER2 assessment in all HER2 testing methods with up to 3 hours of a delay to tissue fixation. 22 This was supported in data published by Neumeister et al 23 which also indicated that there was no evidence for the loss of HER2 protein antigenicity in a 4-hour cold ischemic time window. However, another study showed that decreased HER2 IHC staining was observed as early as within 1 hour delay of fixation 24 while HER2 FISH assay was less affected by the cold ischemic time Yet another study showed significant reduction of HER2 IHC staining after 4 hours with refrigerated tissue samples and 2 hours with non-refrigerated tissue samples of cold ischemic time. 21 Statistically significant differences with the quality of HER2 FISH and DISH assay results after one hour cold ischemic time point were also observed. 28,29 Furthermore, negative effects of cold ischemia time on HER2 ISH assay performances were more detrimental with HER2 FISH than HER2 DISH. 22 Thus, HER2 IHC assays should be performed at institutions that also perform HER2 ISH assay as the primary method of HER2 status assessment, particularly on HER2 ISH negative cases. It is clear that the cold ischemic time affects the quality of HER2 testing. However, we speculate that variation in published data was caused by, not only the cold ischemic time, but also the warm ischemic time and also the use of various HER2 testing methods. Because of the difficulties associated with pre-analytical process standardization, pathologists need to obtain both HER2 IHC and HER2 ISH assay results in breast cancer cases with

4 316 H. Nitta et al. indeterminate HER2 status assessments so that fewer patients will miss an opportunity to receive a HER2 targeted therapy. Thus, further improvements in HER2 testing approaches are required for the accurate diagnosis of breast cancer patients. Current HER2 testing approach Because previous references suggested that approximately 20 % of HER2 testing may not be accurate among breast cancer patients, the ASCO/CAP HER2 testing guideline was introduced in order to improve the quality of HER2 testing in and it was updated in Bright field HER2 ISH assays were included as a recommended HER2 gene amplification testing method in the latest guideline. However, there are still unresolved issues with HER2 testing in selecting breast cancer patients for HER2-targeted therapy. The ASCO/CAP HER2 testing guideline states cancer patients with HER2 negative status determined by HER2 IHC or HER2 ISH testing are not subjected to confirmation of HER2 status by an alternate assay. Nevertheless, there are still various factors that may cause false negative results (cases that would be eligible for HER2-targeted therapy) as well as false positive results (unnecessary expensive therapy with possible side effects) in breast cancer. It was calculated that reflex testing for HER2 IHC negative (IHC 0 and 1+) or HER2 FISH negative early stage breast cancer patients is cost-effective at $ per quality-adjusted life years among approximately 4700 women in the US. 31 Because of inaccurate HER2 testing results, approximately early stage breast cancer patients are treated without optimal cancer therapies and the estimated annual total economic societal loss is about $1 billion. 32 Based on the results of retesting a clinical study cohort of 637 patients with HER2 status established by HER2 IHC and HER2 FISH assays, it was estimated that approximately 7330 women would receive false negative results in the US because of the current single HER2 testing approach and the tumor heterogeneity. 33 The currently conducted single HER2 test for determining the HER2 status of breast cancer patients is not advisable 33 and HER2 tumor heterogeneity is an unavoidable factor for accurate HER2 status assessment with the current HER2 testing approach. 34 Furthermore, some of HER2 negative patients may benefit from a HER2-targeted therapy 12,35 and it could be due to HER2 false negative results of those patients with the current HER2 testing approach. Thus, it is clear that we still need to improve the accuracy of HER2 testing in order to advance breast cancer patient care with optimal cancer therapies. HER2 tumor heterogeneity Breast cancer is a heterogeneous disease and the intratumoral heterogeneity poses an immense challenge to accurate breast cancer diagnostics. Along with the pre-analytic and assay variables discussed previously, HER2 tumor heterogeneity is an unavoidable factor for accurate HER2 status assessment. 34 HER2 tumor heterogeneity or HER2 genetic heterogeneity describes a cancer with different tumor cell populations (including obvious HER2 amplification status) in the same patient 36 and the heterogeneity creates difficulty in treating breast cancer patients with a single targeted therapy. 37 There are two main types of HER2 genetic heterogeneity: (i) clustered HER2 amplification showing a congregated HER2 amplified tumor cell population bordered by non-her2 amplified tumor cells; and (ii) mixed HER2 amplification showing dispersed HER2 amplified tumor cells among non-her2 amplified tumor cells (Fig. 1). 36 HER2 genetic heterogeneity is defined as more than 5% but less than 50% of infiltrating tumor cells with a HER2/CEN17 ratio higher than 2.2 based on HER2 ISH assay results with dual probes while >5% but <50% of infiltrating tumor cells with >6 HER2 signals per cell with an ISH assay without a control probe. 38 HER2 heterogeneity that affects accurate assessment of HER2 status was observed in 11 % 13 % of invasive breast cancer. 39,40 Another study showed that the genetic heterogeneity determined by HER2 FISH results was detected in one quarter of consecutive breast cancer cases: among these cases HER2 genetic heterogeneity was most often observed in HER2 negative cases, with the overall distribution being 4 % in HER2 gene amplified cases, 31.5 % in HER2 equivocal cases, and 64.5 % in HER2 negative cases. 41 Thus, it is apparent that HER2 tumor heterogeneity is a challenge for accurate HER2 status assessment of breast cancer as well as other cancers, particularly gastric cancer that is notorious for HER2 tumor heterogeneity. However, the HER2 heterogeneity guidelines are based on expert opinions, but not based on clinical outcome data. A few studies have reported that HER2 positive breast cancer patients with HER2 genetic heterogeneity had significantly shorter disease-free survival times compared to without heterogeneity 40 and HER2 heterogeneity was an independent predictor of breast cancer clinical outcome to trastuzumab therapy. 27 Future studies will define the significance of HER2 heterogeneity for treating breast cancer patients with HER2-targeted therapy. A welldescribed HER2 heterogeneity guideline is required for clinically relevant heterogeneity data collections to improve breast cancer patient care Even though 95 % concordance between non-equivocal HER2 FISH and HER2 IHC assay results in breast cancer is possible, 45 it is a well-known fact that the HER2 genetic heterogeneity is a factor in discrepant HER2 status assessments between HER2 IHC and HER2 ISH testing methods. 38 Significant HER2 genetic heterogeneity is detected among lowgrade HER2 amplification (2.2 < HER2/CEN17 ratio < 4.0) compared to high-grade (HER2/CEN17 ratio 4.0) HER2 IHC 3+ cases. 39 A high concordance in assessing HER2 status between core biopsies and surgical specimens was achieved unless breast cancer cases were HER2 equivocal or exhibited HER2 genetic heterogeneity. 46 One published case study

5 HER2 status assessment of breast cancer 317 Figure 1 Human epidermal growth factor receptor 2 (HER2) heterogeneity types in breast cancer. Homogenous breast cancer cell populations are HER2 (a) negative or (b) positive. Heterogeneity of HER2 positive breast cancer cells are clustered (c) or mixed (d) populations with HER2 negative tumor cells. (Modified from Hanna et al, 2014). reported that a HER2 positive breast cancer patient, determined by a core biopsy sample, who received an adjuvant trastuzumab therapy after a mastectomy, relapsed without HER2 amplified tumor cells. 47 It was revealed that the tumor had a focal HER2 amplification in the patient s mastectomy sample. Thus, the detection of HER2 heterogeneity in breast cancer may be important in providing appropriate therapies to breast cancer patients. HER2 genetic heterogeneity is frequently detected among HER2 equivocal (HER2 IHC 2+) cases. 43,48,49 Individual breast tumor cells with HER2 gene amplification are also often found in HER2 negative breast cancer cases classified by the current HER2 testing guidelines. 48 HER2 FISH negative cases with HER2 gene amplified tumor cells exhibited an aggressive characteristic of breast cancer. 49 HER2 negative cases containing obvious HER2 gene amplified tumor cells are not eligible for HER2-targeted therapy by the current criteria regardless of HER2 positive tumor cell populations. A model study for intratumoral heterogeneity in HER2 positive xenograft tumors demonstrated the correlation between the efficacy of anti-her2 antibody therapy and the degree of HER2 tumor heterogeneity. 50 If these findings can be applied to human breast cancer treatments, then HER2 heterogeneity status will be a major decision making factor in selecting an appropriate breast cancer therapy. Impact of updated 2013 ASCO/CAP HER2 testing guideline The 2013 ASCO/CAP HER2 testing guidelines altered the threshold for both HER2 IHC and HER2 FISH assays and the window for HER2 equivocal cases. 14 Substantial numbers of breast cancer patients were scored differently for HER2 status assessment (7.7 % of a cohort) by the updated HER2 testing guideline. 51 After implementing the new HER2 ISH scoring criteria, the number of HER2 FISH equivocal and positive 51 cases increased significantly because HER2 gene copy numbers became an important decision making factor regardless of HER2/CEN17 ratio of breast cancer patients. HER2 IHC equivocal case numbers also increased with the updated HER2 testing guidelines and reflex HER2 ISH testing identified more HER2 positive patients among the equivocal cases. 54,55 Thus, there are more HER2 equivocal cases diagnosed with both HER2 IHC and HER2 ISH assays with the updated ASCO/CAP HER2 testing guideline and pathologists are facing significant increase in reflex HER2 testing. Development of HER2 gene-protein assay HER2 IHC and HER2 ISH assays are used for the same purpose: selecting breast cancer patients for HER2-targeted therapy. However, HER2 IHC and HER2 ISH assays detect biologically different targets, namely HER2 protein overexpression for the target of HER2 therapy and HER2 gene amplification (with or without a CEN17 control probe) correlated to HER2 protein overexpression, respectively. Each assay has advantages and disadvantages as stated earlier in this publication. The major discordant results of HER2 protein and HER2 gene statues are caused by tumor heterogeneity, mainly detected in HER2 equivocal cases. Furthermore, the updated ASCO/CAP HER2 testing guidelines have increased the

6 318 H. Nitta et al. number of HER2 equivocal cases. In general, HER2 status analyses of equivocal cases require both HER2 IHC and HER2 ISH assays unless different tissue blocks of breast cancer patients are available for retesting with the same IHC or ISH test. Therefore, our concept was to combine HER2 IHC and HER2 ISH assays as one assay so that HER2 protein and HER2 gene status can be determined more accurately at the individual cell level in heterogeneous breast cancer. Previously, in order to assess the HER2 heterogeneity, the entire ISH slide needed to be scanned before analyzing the HER2 gene status while comparing it to the HER2 IHC slide. 56 Thus, it also makes sense to merge HER2 IHC and bright field HER2 ISH assay, not dark field HER2 ISH assay, so that the HER2 status assessment can be achieved accurately with tumor cells by correlating with tissue morphology. There are several technical publications for bright field HER2 GPA applications To our knowledge the first publication for a combination of HER2 IHC and HER2 ISH assays for breast cancer was by Downs-Kelly et al. in The exact protocol was not described in the publication, but HER2 gene and HER2 protein were detected with silver detection (black dots) and fast red detection (red color), respectively without a CEN17 control probe using a semi-automated protocol. The first HER2 GPA performance was compared to dual color HER2 FISH and HER2 IHC assays. The weaknesses of this HER2 GPA approach were: (i) HER2 IHC detection was not the standard 3,3 -diaminobenzidine (DAB) signal visualization for HER2 protein; (ii) HER2 ISH assay did not include the control probe that is used in the most common HER2 FISH assay; and (iii) the protocol was complicated and not totally automated. Pathologists are accustomed to scoring DAB stained HER2 protein and the use of an alternate IHC detection system creates a different dynamic range of IHC staining. Thus, accurate HER2 protein evaluation of HER2 IHC 2+ and 1+ cases might be challenging. CEN17 ISH evaluation, if it is required, must be conducted on a different tissue section than the dual color HER2 GPA detection for HER2 protein and HER2 gene targets. It is impossible to score the HER2 gene and CEN17 ISH signals in the same cell when two separate tissue sections are used, and it becomes significantly more difficult with HER2 tumor heterogeneity cases. In general, multiplex assays are rather complex and an automated protocol is recommended to generate reproducible assay results. We presented feasibility study data of automated tricolor HER2 GPA application at the joint meeting of the Japan Society of Histochemistry and Cytochemistry and the Histochemistry Society in This version of HER2 GPA application was for HER2 IHC with NBT/BCIP detection (purple), HER2 gene ISH with DAB detection (brown dots), and CEN17 with fast red (red dots). This was the first attempt to perform an automated tricolor HER2 GPA with breast cancer cases. The next full publication of a dual HER2 GPA application was by Ni et al in This HER2 GPA protocol was partially automated for HER2 IHC with a manual HER2 ISH assay for HER2 gene targets. HER2 protein was detected with fast red (red color), not DAB, and HER2 gene signal was visualized with DAB (brown dots) detection. The assay performance of this HER2 GPA was compared to the dual HER2 FISH assay. Their HER2 GPA algorithm used in this study was rather cumbersome as wet HER2 IHC assay slides were scored first followed by a manual HER2 CISH staining for quantitative analyses of HER2 gene copy numbers on HER2 IHC 2+ cases. The third publication of dual HER2 GPA with DAB detection (brown color) for both HER2 gene and HER2 protein was published by Reisenbichler et al in These investigators compared two versions of their HER2 GPA protocols: (i) HER2 IHC followed by HER2 CISH; and (ii) HER2 CISH followed by HER2 IHC. They reported that HER2 CISH signal was not obtainable when the CISH was performed after HER2 IHC. Thus the authors focused on optimizing GPA in which HER2 CISH assay was conducted before HER2 IHC assay. However, the HER2 IHC sensitivity of HER2 GPA was lower than the sensitivity of HER2 IHC only. In order to compensate for the decreased HER2 IHC sensitivity, the length of the HER2 antibody incubation period was extended from 30 minutes to 45 minutes. We published the first bright field tricolor HER2 GPA assay development and performance analyses with xenograft tumors and breast cancer tissue microarrays in This version of HER2 GPA allows the visualization of HER2 protein with DAB detection (brown), HER2 gene with silver detection (black dots), and CEN17 with fast red detection (red dots) (Fig. 2). This HER2 GPA is a fully automated protocol that uses two FDAapproved HER2 IHC and HER2 DISH assays. In order to develop the bright field tricolor HER2 GPA protocol, we first established a dual color ISH assay for HER2 gene and CEN17 targets 62 and this dual color ISH became the HER2 DISH assay. Like Reisenbichler et al. reported, 59 we encountered decreased sensitivity of HER2 IHC when the HER2 IHC assay was performed after the HER2 DISH assay. Decrease in HER2 IHC sensitivity is caused by an ISH protocol, particularly due to the denaturing agent formamide in hybridization buffer and the protease treatment required for DNA target retrieval. Our goal in developing HER2 GPA was solely for more accurate diagnosis of breast cancer patients for HER2-targeted therapy. Also, because the HER2 IHC assay was already approved by FDA, we decided not to alter the protocol for HER2 protein detection. Thus, our only choice was to perform HER2 IHC using DAB detection prior to HER2 DISH in order to maintain the sensitivity, dynamic range, and familiar brown staining of HER2 IHC. However, we encountered unacceptable silver background staining when HER2 DISH was simply conducted after DAB IHC staining (Fig. 3a, inserted photograph). We identified a chemical naphthol phosphate as a blocker in hybridization buffer to eliminate the silver background staining (Fig. 3b, inserted photograph). We hypothesize that

7 HER2 status assessment of breast cancer 319 Figure 2 Tricolor human epidermal growth factor receptor 2 (HER2) gene-protein assay (GPA) signal detection scheme for HER2 protein, HER2 gene, and chromosome 17 centromere (CEN17). HER2 protein (brown color) is visualized with HER2 immunohistochemistry followed by the detection for HER2 gene (black dots) and CEN17 (red dots) signal with dual color HER2 in situ hybridization. (From Nitta et al., 2012). naphthol phosphate blocks the nonspecific binding of the DNP hapten labels on the HER2 probe to the deposited DAB in tissue sections (Fig. 3). We, like Reisenbichler et al, 59 also encountered difficulty of getting ISH signal, particularly with CEN17 signal during our assay development, after HER2 IHC assay. However, we could overcome the technical issue by increasing protease digestion time without obvious damage to tissue preservation. Thus, we successfully combined HER2 IHC and HER2 DISH as one assay on the same tissue section (Fig. 4). A similar tricolor HER2 GPA protocol and its performance were published by Varga et al in The performance of our tricolor HER2 GPA assay has been evaluated further on breast cancer, gastric cancer, 64,68,69 and lung cancer. 70 A publication by Li et al, 65 focused on HER2 equivocal cases, reported that HER2 GPA divided breast cancer cases accurately into HER2 positive, equivocal, and negative statuses and it aided in identification of HER2 heterogeneity well. Analyses by Chenard et al showed a 25 % time saving in scoring a breast cancer case by a single HER2 GPA slide scoring protocol compared to a typical IHC followed by HER2 DISH testing protocol. 66 Thus, the use of HER2 GPA approach can optimize laboratory workflow and deliver faster, more accurate HER2 status assessment for patient management. Stålhammar et al. identified another advantage with a single slide HER2 GPA approach as an IHC-guided ISH assay for selecting appropriate areas. 67 Apathologistcan determine an area for HER2 DISH scoring based on HER2 IHC staining at a lower magnification. All publications concluded that HER2 GPA is an ideal test for HER2 equivocal cases that require additional HER2 testing. Furthermore, we found that the HER2 GPA method allows us to find breast as well as gastric tumor cell populations that present amplified HER2 gene without overexpression of HER2 protein (Fig. 5) in HER2 positive, equivocal, and negative clinical cases. We propose to term this new type of HER2 gene and HER2 protein discordant phenomenon at individual cell level HER2 micro-heterogeneity and to include the HER2 micro-heterogeneity as a new category of HER2 heterogeneity. We have also confirmed that the HER2 micro-heterogeneity exists in xenograft tumors of a HER2 positive cell line (Fig. 6).

8 320 H. Nitta et al. Figure 3 Blocker mechanism hypothesis of naphthol phosphate for human epidermal growth factor receptor 2 (HER2) gene-protein assay (GPA). Unacceptable levels of silver background staining (black staining in the insert photograph, a) is caused by two steps: (i) DNP hapten on HER2 probe binds to DAB bound to genomic DNA and (ii) anti-dnp antibody of silver in situ hybridization (ISH) signal detection system recognizes DNP hapten on the probe (a). Naphthol phosphate blocks the binding of DNP hapten on the HER2 probe to DAB on genomic DNA and silver ISH detection only presents HER2 gene signal as shown in the insert photograph (b). Figure 4 Human epidermal growth factor receptor 2 (HER2) gene-protein assay (GPA) combining HER2 immunohistochemistry (IHC) and HER2 dual in situ hybridization (DISH). HER2 IHC staining shows complete membrane staining (brown color) of HER2 protein onaher2ihc3+case(a). HER2 DISH assay demonstrates amplified HER2 gene (black dots) and normal chromosome 17 centromere (CEN17, red dots) copy numbers (b). HER2 GPA that is a combination of HER2 IHC and HER2 DISH shows all HER2 protein, HER2 gene, and CEN17 with wellpreserved tissue section (c). Osamura et al suggested that the HER2 micro-heterogeneity might be a biological mechanism of HER2-targeted therapy resistance as the target of trastuzumab is HER2 protein, not the gene. 71 Thus, HER2 gene amplified tumor cells without HER2 protein overexpression might escape from HER2 targeted therapy. A HER2 micro-heterogeneity xenograft model study showed that the efficacy of an anti-her2 antibody therapy was dependent on the ratio of amplified HER2 gene tumor cells with overexpressed HER2 protein to amplified HER2 gene tumor cells without HER2 protein. 50 Even though future studies are required to examine the HER2 microheterogeneity hypothesis, the heterogeneity status may play a significant role in determining appropriate treatment choices for HER2 positive breast cancer patients.

9 HER2 status assessment of breast cancer 321 Figure 5 Human epidermal growth factor receptor 2 (HER2) micro-heterogeneity demonstration by HER2 gene-protein assay (GPA). Homogeneous HER2 positive breast cancer population shows strong HER2 protein membrane staining while some tumor cells lack of amplified HER2 gene due to truncated artifact (a). HER2 GPA reveals the HER2 microheterogeneity that presents amplified HER2 gene breast tumor cells without overexpression of HER2 protein among amplified HER2 gene tumor cells with HER2 protein overexpression (b). Figure 6 Human epidermal growth factor receptor 2 (HER2) micro-heterogeneity demonstration in HER2 positive xenograft tumor by gene-protein assay (GPA). HER2 IHC 3+ staining is clearly presented on HER2 positive Calu-3 xenograft tumor at a low magnification (4, a). HER2 micro-heterogeneity that is seen with amplified HER2 gene tumor cells without HER2 protein overexpression (dotted red circles) at a higher magnification (20, b). Marcel Proust the French novelist quoted the real act of discovery consists not in finding new lands, but in seeing with new eyes 72 and HER2 GPA approach might be a new eye to see HER2 tumor heterogeneity in breast cancer. Because HER2 IHC and HER2 ISH test results are the decision making factors in selecting breast cancer patients for a targeted therapy, the simultaneous analyses of HER2 gene and HER2 protein status by HER2 GPA provides pathologists better eyes to assess the HER2 status at individual cell level in difficult cases. CONCLUSIONS Although HER2 IHC and HER2 ISH assays are used for the determination of patient selections for HER2 therapy, there is no real gold standard for HER2 status assessments in breast cancer today. Discordance of HER2 IHC and HER2 ISH results is a well-recognized issue with HER2 testing in breast cancer, predominantly due to the tumor heterogeneity among HER2 equivocal cases. Accurate HER2 heterogeneity identification is an important factor in deciding on an appropriate therapy approach for breast cancer patients. Despite the fact that the HER2 GPA will not solve the issue of non-standardized pre-analytical issues that can affect HER2 test results, the GPA method can allow a pathologist to examine the status of HER2 protein and HER2 gene at individual cell level while correlating it to the tissue morphology, even among equivocal cases. Because HER2 protein and HER2 gene status can be evaluated simultaneously, the HER2 intratumoral micro-heterogeneity can be easily evaluated. HER2 GPA might become the ultimate HER2 status assessment test not only for breast cancer, but also for gastric cancer that is known for HER2 tumor heterogeneity. ACKNOWLEDGMENTS The primary author Hiroaki Nitta, Ph.D., M.B.A., was invited to the 103rd meeting of The Japanese Society of Pathology in Hiroshima, Japan in 2014 and this manuscript is based upon the presentation given at the meeting. The authors would like to thank Ruediger Ridder, Ph.D. (Medical & Scientific Affairs, Ventana Medical Systems, Inc.) and Bryan Jones, J.D. (Legal Affairs, Ventana Medical Systems, Inc.) for their critical reviews of the manuscript. DISCLOSURE All authors are employees of Ventana Medical Systems, Inc., (Ventana) a member of the Roche Group, in Tucson, AZ, USA. Ventana is the tissue diagnostic division of Roche Diagnostics and is a manufacture for automated immunohistochemical and in situ hybridization assays. Roche manufactures HER2-targeted drugs Herceptin (trastuzumab), Perjeta (pertuzumab), and Kadcyla (ado-trastuzumab emtansine).

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