Comparative Analysis of Equivocal (2+) and Positive (3+) HER2 Immunohistochemistry (IHC) and Bright-Field Dual-Color In Situ Hybridization (DISH) in Primary Breast Cancer From 1,307 Node-Positive Patients

To compare dual-color in-situ hybridization (DISH) with fluorescence in situ hybridization(FISH) in evaluating the human HER2 gene status in invasive breast cancer. HER2 gene status in 110 cases of breast invasive ductal carcinomas with a 2+ score on immunohistochemistry (IHC) was investigated by FISH and DISH. The 2007 and 2013 ASCO (American Society of Clinical Oncology)/CAP (College of American Pathologists) HER2 guideline were used respectively to evaluate the agreement between these two techniques. (1) Using the 2007 ASCO/CAP guideline, the overall concordance between FISH and DISH was 97.3% (107/110). Fifty of 51 samples with amplification by FISH were also detected by DISH and the remaining one sample was equivocal.Eight of 10 equivocal samples by FISH were equivocal by DISH and the remaining two samples were negative. Forty-nine samples with no amplification by FISH were all negative by DISH. The concordance was 98.0%, 8/10 and 100.0% respectively for the FISH positive, equivocal and negative groups. (2) Using the 2013 ASCO/CAP guideline, the overall concordance between FISH and DISH was 90.0% (99/110). Fifty-three of 55 samples with amplification by FISH were also detected by DISH and the remaining two were equivocal and negative respectively. Two of 12 equivocal samples by FISH were equivocal by DISH and the remaining ten samples were negative in 7 cases and equivocal in 3 cases. Forty-three samples with no amplification by FISH were all negative by DISH. The concordance was 96.4%, 3/12 and 100.0% respectively for the FISH positive, equivocal and negative groups. Pearson correlation analysis indicated that the HER2 status detected by FISH and DISH were significantly correlated with each other (R=0.584, P<0.01). There is a high concordance between DISH and FISH for assessing the HER2 gene status in invasive breast cancer. DISH is a new option for assessing HER2 gene status of breast cancer in clinical practice. The clinical significance of the discordance between DISH and FISH in equivocal cases warrants further study.

Human epidermal growth factor receptor 2 (HER2; ERBB2 gene) is of prognostic and predictive significance in breast carcinoma. Both fluorescence in situ hybridization (FISH) and dual-color in situ hybridization (DISH) methods are available. DISH and FISH are highly concordant in validation studies, but differences may be more prevalent in the equivocal range. Our goal was to compare FISH and DISH on a cohort enriched for equivocal cases, with respect to HER2 determination. The cohort was enriched for equivocal (2+) cases. DISH and FISH were evaluated using standard protocols and the results compared with respect to HER2 status, HER2 copy number, and HER2/chromosome 17 (Chr17) ratio. In total, 109 cases were identified. The agreement rate of DISH with FISH was 74%. The mean ± SD HER2/Chr17 ratio by DISH was 1.63 ± 0.08 vs 1.59 ± 0.26 by FISH (P = .45). The mean ± SD HER2 copy number by DISH was 4.56 ± 0.45 vs 4.75 ± 1.08 by FISH (P = .004). Individual signals were more easily resolved using FISH in cases with higher copy numbers. In our cohort enriched for equivocal cases, the numerical values of HER2 copy number were significantly lower using DISH, resulting in discordances. Although DISH is a valid method, variations with FISH may be expected in high-equivocal cases and in quality assurance activities.

Background: HER2-targeted therapies such as trastuzumab neoadjuvant chemotherapy for patients with HER2-positive breast cancer can increase pathological complete response (pCR) better than non-HER2-targeted therapies. Previous studies have suggested that trastuzumab may convert HER2-positive (HER+) primary breast tumors to HER2 negative (HER−) after neoadjuvant chemotherapy. We compared the HER2 status in breast cancer patients before and after neoadjuvant treatment by using dual-color in situ hybridization (DISH). Methods: We retrospectively identified 46 patients from the Breast Cancer database at Tokai University Hospital, in whom HER2-positive primary breast cancer was diagnosed between 2000 and 2010, and who were treated with neoadjuvant chemotherapy or endocrine therapy with or without trastuzumab. Surgical specimens from patients achieving less than pCR were assessed to determine if there was enough residual tissue to evaluate the post-treatment HER2 status by DISH. HER2 status was defined as positive if DISH demonstrated a gene copy ratio of HER2:CEP17 &amp;gt;2.0. Paraffin tissue sections (4-μm thick) were mounted on glass slides (New Sliane III, Catalog No. 5126–25; MUTO PURE CHEMICALS, CO. LTD., Tokyo, Japan) and stained using the newly developed, fully automated HER2 Dual ISH assay on a BenchMark® XT slide stainer according to the recommended procedure (Ventana Medical Systems Inc., Tucson, AZ). Thereafter, the stained slides were rinsed with tap water containing neutral detergent and then rinsed again with distilled water. The slides were dried at room temperature for at least 60 min and cover-slipped with cover grass for SGC (MUTO PURE CHEMICALS). HER2 Dual ISH and H&amp;E images were obtained using an Olympus BX51 microscope. Results: A pCR was achieved in 9 of the 46 patients (19.6%). Specimens from core needle biopsy were not sufficient to assess the pretreatment HER2 status in 3 patients. In 9 patients, the post-treatment HER2 status could not be assessed because residual tumors were DCIS only in 4 patients and there were less than 20 invasive cells in 5 patients. Residual tumor was sufficient to assess the post-treatment HER2 status in 25 patients. In pretreatment specimens, of the 25 patients identified as HER2 + by immunohistochemical analysis, 3 patients were identified as HER2− by DISH. No post-treatment specimens were found to be HER2− by DISH among the tumors identified as HER2+ by DISH at pretreatment. Among the 3 pretreatment tumors identified as HER2− by DISH, 1 tumor was found to be HER2+ by DISH at post-treatment, and 2 showed a stable HER2 status. Conclusion: DISH revealed a stable HER2 status in pretreatment breast tumors and in residual tumors. However, we found a discrepancy in the HER2 status between the immunohistochemical analysis and DISH. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-08.

FISH in triple-negative breast cancer: a possible strategy for the future?

Although human epidermal growth factor receptor 2 (HER2) is a significant clinical biomarker for breast cancer, the HER2 testing involves a complicated evaluation process. We devised an easy method for HER2 gene testing and investigated its utility. HER2 testing was performed by immunohistochemistry (IHC) and dual-color in situ hybridization (DISH) on surgical specimens from 50 patients with invasive breast cancer. DISH was evaluated by two methods. One was the DISH count method in which the HER2 and CEP17 signals were counted and the HER2/CEP17 signal ratio was calculated. The other was the DISH-easy method in which pathologists only observed slices without counting the signals. The DISH-easy method was performed by two pathologists. We analyzed the correlations between the DISH-easy method and the DISH count method by each pathologist and calculated the inter-observer concordance rate of the DISH-easy method. The results from two pathologists using the DISH-easy method corresponded to the IHC results (P < 0.01) and the DISH count method results (P < 0.01). All cases determined to be negative using the DISH-easy method were also negative via the DISH count method with a corresponding rate of 100 % for both pathologist A (34/34) and B (32/32). Most of the results for the positive cases also corresponded; the correspondence rate of pathologist A was 100 % (8/8) and 89 % for B (8/9). The inter-observer concordance rate was 81 % (κ = 0.65). The DISH-easy method is simple and useful for HER2 testing regardless of proficiency of the evaluators.

A recent randomized controlled trial (Trastuzumab for Gastric Cancer [ToGA] study) established standard scoring criteria of human epidermal growth factor receptor 2 (HER2) for gastric cancer and demonstrated the efficacy of trastuzumab for treating metastatic gastric cancer. The aim of the present study was to evaluate the frequency of HER2-positive cases by application of the standard criteria in patients with resectable gastric cancer and to examine the relationships between HER2 expression and prognosis, mucin phenotype, p53 status, and clinicopathological features. A total of 213 patients were included in this retrospective study. All tumor samples were examined for HER2 expression by immunohistochemistry (IHC), HER2 amplification by in situ hybridization, and mucin and p53 expression by staining for CD10, MUC2, MUC5AC, MUC6, and p53. HER2-positive tumors were identified in 25 patients (11.7%). HER2-positive cases were more frequently found in men, older patients, and in the intestinal histological type (P=0.0048, 0.0309, and <0.0001, respectively). Although no association was found between HER2 overexpression and mucin phenotype, the expression of CD10 and p53 was significantly correlated with HER2 positivity (P=0.0079 and 0.013). The overall survival of HER2-negative and -positive patients was not significantly different. However, in patients with stage III/IV, overall survival was worse in HER2-positive patients (P=0.0149). In a comparison between dual-color in situ hybridization (DISH) and fluorescence in situ hybridization (FISH), four IHC2+/3+ cases that were DISH-positive were judged as negative by FISH. Our study indicated that HER2 expression was less frequent in resectable gastric cancer than in metastatic gastric cancer. The impact of HER2 expression on survival was limited. DISH was superior to FISH for evaluating cases with limited HER2 expression.

Background: Human epidermal growth factor receptor 2 (HER2) gene expression is an important predictive and prognostic biomarker in breast cancer (BC), which also guides treatment recommendations. The expression of HER2 is a continuum from null to positive and includes HER2-low and ultra-low as targets for anti-HER2 antibody–drug conjugates (ADC). However, HER2-low and ultra-low have not been studied in male BC. Here, we analyze whether there are any differences in molecular and immunological features between HER2-low, ultra-low and HER2-null/negative expression in males with BC. Methods: 199 male breast tumor samples were included in this study. HER2-null expression was defined as infiltrating cancer cells completely free of HER2 immunohistochemistry (IHC) staining. HER2 ultra-low expression was defined as ≤10% cancer cell showing incomplete and faint/weak membrane staining. HER2-low expression was defined as HER2 (IHC) 1+ or 2+ with negative chromogenic in situ hybridization (CISH) assay. HER2-positive expression was defined as HER2 IHC 3+ staining or 2+ with positive CISH assay. Mutations and gene expression were detected by next-generation sequencing (NextSeq; WES, NovaSeq) and Whole Transcriptome Sequencing (WTS; NovaSeq) (Caris Life Sciences, Phoenix, AZ), respectively; tumor mutational burden (TMB) totaled somatic mutations per tumor (high&amp;gt;10 mt/MB). Immune cell fractions were calculated by deconvolution of WTS:Quantiseq. Statistical significance was determined using chi-square and Mann-Whitney U test and p-value &amp;lt;0.05 was considered significant. Results: Of 199 samples, there were 70 (35.2%) HER2-null tumors, 49 (24.6%) HER2 ultra-low tumors, 64 (32.2%) HER2-low tumors, and 16 (8.0%) HER2-positive. The proportion of HR+ was 81.4% in HER2-null, 93.8% in HER2 ultra-low, 87.5% in HER2-low and 81.2% in HER2-positive tumors. HER2 ultra-low male BC had higher frequency of PIK3CA (44.19% vs 23.64%) compared to HER2-null and KMT2D (7.5% vs 0%) compared to HER2-low, all p&amp;lt;0.05. HER2-low male BC had numerically lower frequency of TP53 (8.7% vs 16.6%, p=0.2) and ESR1 (1.6% vs 5.4%, p=0.2) compared to HER2-null. No significant differences were noted in TMB-high frequency (7.1% vs 5.0% vs 6.9%) and PD-L1 positivity (22C3) (8.11% vs 9.0% vs 10.6%) between HER2 ultra-low, HER2-low and HER2-null (all p=1.0). Analysis of inferred immune cells revealed that HER2 ultra-low and HER2-low male BC had higher infiltration of B cells (6.6% vs 5.8% vs 4.9%) but lower infiltration of neutrophil (2.4% vs 2.0% vs 3.8%) (all p&amp;lt;0.05). HER2-low had lower expression of immune checkpoint gene LAG3 (fold change (FC): 1.6), stem cell-related genes (KLF4, POU5F1; FC: 1.3-1.7) and drug-efflux gene ABCB5 (FC: 2.2) compared to HER2-null tumors (all p&amp;lt;0.05). HER2 ultra-low had lower expression of drug-efflux gene ABCG2 (FC: 1.5) compared to HER2-null tumors (p&amp;lt;0.05). Data adjusted for HR-subtype will be presented at the meeting. Conclusions: Our findings add valuable information to the current understanding of the HER2 spectrum in the male breast cancer, including frequency distribution and molecular characterization. With some exceptions, HER2-low, ultra-low and null breast cancer in men shared genomic features, suggesting that the disease biology may not be different across the spectrum of what historically has been considered HER2-negative disease. Interestingly, HER2 ultra-low, HER2-low and HER2-null had differential tumor immune microenvironment that warrant further exploration. Citation Format: Dario Trapani, Sachin Kumar Deshmukh, Sharon Wu, Joanne Xiu, Priya Jayachandran, Nancy U. Lin, Giuseppe Curigliano, Milan Radovich, Maryam Lustberg, Stephanie L. Graff, George W. Sledge Jr, Sara M. Tolaney, Jose P. Leone. Molecular and immunological characterization of HER2-low, HER2 ultra-low, and HER2-null male breast cancer [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr P3-01-25.

BACKGROUNDThe needs for human epidermal growth factor receptor 2 (HER-2) and/or programmed death-ligand 1 (PD-L1) evaluations in gastric cancer are dramatically increasing. Although the importance of standardization of sample fixation has been widely recognized, most of the evidence regarding the fixation duration or type of fixing solution are based on breast cancer.AIMTo investigate the real effects of fixation conditions on HER-2 testing or PD-L1 testing for gastric cancer using gastrectomy specimens.METHODSThirty-two patients who underwent gastrectomy for gastric cancer were enrolled. Their resected specimens were each divided into four pieces and fixed in four strictly controlled different durations (6 h, 24 h, and 48 h, and 1 wk) by 10% formalin (n = 22) or 10% neutral buffered formalin (NBF) (n = 10). Immunohistochemistry (IHC) of HER-2 and PD-1 was performed, and a pathology examination was conducted. In the HER-2-immunoreactive cases, all four specimens were subjected to dual-color in situ hybridization (DISH). Five cases were assessed as HER-2-positive by IHC and DISH. We used the cut-off values of 1%, 10%, and 50% to assess the IHC findings of PD-L1.RESULTSNo significant difference was observed in comparisons between the shorter fixation period groups (6 h, 24 h, and 48 h) and the prolonged fixation period (1 wk) group in the HER-2 and PD-L1 analyses. Although no significant difference was observed between 10% formalin and 10% NBF within 1 wk of fixation, the superiority of 10% NBF was confirmed in a long-term (> 3 mo) fixation in both the HER-2 and PD-L1 analyses.CONCLUSIONIn this small-numbered pilot study, prolonged fixation within 1 wk showed no inferiority in HER-2 or PD-L1 testing. However, a large-numbered prospective study is needed to obtain conclusive results.

Tumor human epidermal growth factor receptor 2 (HER2) status is defined by either protein expression using immunohistochemistry (IHC) or gene amplification using fluorescence in situ hybridization (FISH). Approximately 20% of HER2-positive breast cancer is HER2 IHC 2+/FISH-positive. Unlike trastuzumab, it has not been studied whether the response to trastuzumab emtansine (T-DM1) differs according to HER2-positive status. We retrospectively identified and reviewed medical records of all patients with HER2-positive advanced breast cancer (ABC) who received T-DM1 in our hospital from October 2013 to December 2016. We compared the objective response rate (ORR) and progression-free survival (PFS) between patients in the HER2 IHC 3+ group and those in the HER2 IHC 2+/FISH-positive group. A total of 39 patients (IHC 3+: n = 32; IHC 2+/FISH-positive: n = 7) were analyzed. Nineteen (48.7%), 13 (33.3%), and 29 (74.4%) patients had received at least one prior chemotherapy, more than three lines of chemotherapy, and prior pertuzumab for ABC, respectively. ORR was significantly higher in the IHC 3+ group than in the IHC 2+/FISH-positive group (53.3% vs. 0%, P = 0.024). Median PFS was 7.9 months in the IHC 3+ group versus 3.9 months in the IHC 2+/FISH-positive group (hazard ratio 0.68; 95% confidence interval 0.28-1.69, P = 0.408). Among the HER2-positive ABC patients treated with T-DM1, ORR was significantly worse in HER2 IHC 2+/FISH-positive than in HER2 IHC 3+ patients. Median PFS tended to be shorter in patients with HER2 IHC 2+/FISH-positive.

Background. The ASCO-CAP guidelines for HER2 testing by fluorescence in situ hybridization (FISH) have a category, referred to as “equivocal” (average HER2 copies per tumor cell &amp;gt;4-6 with HER2/CEP17 ratio &amp;lt;2·0), which is neither “HER2-positive” nor “HER2-negative”. Approximately 4% - 12% of invasive breast cancers are “HER2-equivocal” based on FISH. Cancers in this category may be resolved as “negative” or “positive” by FISH alternative control probes (2013/2014 guidelines) or HER2 immunohistochemistry (IHC) (2018 update). Our objectives were to evaluate the following hypotheses: 1.) Genetic loci used as alternative controls show heterozygous deletion in a substantial proportion of breast cancers; 2.) Use of these loci for assessment of HER2 by FISH leads to false-positives; 3.) HER2 FISH false-positive breast cancer patients have outcomes that do not differ from clinical outcomes for HER2-negative breast cancer patients; and 4.) HER2-equivocal breast cancers seldom show HER2 protein overexpression (IHC 3+). Methods. We retrospectively assessed the use of chromosome 17 p-arm and q-arm alternative control genomic sites (TP53, D17S122, SMS, RARA, TOP2A), as recommended by the 2013/2014 ASCO-CAP guidelines, in patients whose data were available through the Molecular Taxonomy of Breast Cancer International Consortium (METABRIC)(N=1980) or whose tissues were available from the BCIRG-005 clinical trial (N=3298). We used either FDA-approved HER2 IHC (HercepTest) or laboratory-developed HER2 (10H8) IHC assays to assess HER2 protein expression. Results. Using METABRIC we found heterozygous deletions, particularly in specific p-arm sites, were common in both HER2-amplified and HER2-not-amplified breast cancers. Use of alternative control probes from these regions to assess HER2 by FISH in “HER2 equivocal” as well as HER2-not-amplified breast cancers resulted in high rates of false-positive ratios (HER2-to-alternative control ratio &amp;gt;2·0) due to heterozygous deletions of control p-arm genomic sites used as ratio denominators. Misclassifications of HER2 status was observed not only in breast cancers with ASCO-CAP “equivocal” status but also in breast cancers with an average of &amp;lt;4·0 HER2 copies per tumor cell. These deletions were also identified by FISH. IHC demonstrated &amp;lt;1% of FISH “HER2-equivocal” breast cancers in BCIRG-005 had IHC3+ immunostaining, consistent with HER2-not-amplified status. Clinical outcomes of “HER2-equivocal” breast cancer patients with HER2-to-alternative control ratio &amp;gt;2·0 did not differ significantly from clinical outcomes of those with HER2-to-alternative control ratio&amp;lt;2·0. Conclusion. Using chromosome 17 p-arm alternative controls, as recommended by 2013/2014 ASCO-CAP guidelines, instead of CEP17 for resolution of “HER2 equivocal” cases, is problematic due to frequent heterozygous deletions of these loci in breast cancers. The indiscriminate use of alternative control probes to calculate a HER2 FISH ratio in “HER2-equivocal” breast cancers leads to false-positive interpretations of HER2 status resulting from unrecognized heterozygous deletions in one or more of these alternative control genomic sites and incorrect HER2 ratio determinations. Citation Format: Press MF, Seoane JA, Curtis C, Quinaux E, Guzman R, Sauter G, Eiermann W, Mackey JR, Robert N, Pienkowski T, Crown J, Martin M, Valero V, Bee V, Ma Y, Villalobos I, Slamon DJ. HER2/ERBB2 status in “HER2 equivocal” breast cancers by FISH and ASCO-CAP guidelines: False-positives due to heterozygous deletions of alternative control loci [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD3-11.

BackgroundDespite great progress in surgery and other treatments, the prognosis of patients with esophageal squamous cell carcinoma (ESCC) is still very poor. HER2 has strong therapeutic implications in certain cancers, such as breast cancer and gastric cancer. However, literature on the frequency of HER2 expression in ESCC is scarce. In the present study, HER2 protein expression, HER2 gene amplification and the relationship between HER2 status and clinicopathological characteristics were evaluated in a large cohort of Chinese ESCC patients.MethodsA total of 857 consecutive ESCC patients who received radical esophagectomy without neoadjuvant therapy between January 2014 and October 2015 were included in this retrospective study. HER2 protein expression was analyzed by immunohistochemistry (IHC), and its correlation with clinicopathological parameters was assessed. In addition, 65 cases, including 13 HER2 overexpression (3+) cases and 52 HER2 equivocal (2+) cases from the 857-case cohort, and another 104 ESCC cases, including 1 HER2 overexpression (3+) case, 3 HER2 equivocal (2+) cases and 100 HER2 negative (1+/0) cases, were selected to construct tissue microarrays (TMAs). Dual-color in situ hybridization (DISH) was performed on the TMAs to assess HER2 gene amplification and the relationship with clinicopathological parameters.ResultsWe found HER2 overexpression (3+) status in 1.5% (13/857) of cases and HER2 equivocal (2+) status in 6.1% (52/857) of cases. HER2 IHC expression was significantly associated with gender (P = 0.028). However, there were no significant correlations between HER2 IHC expression and age, tumor differentiation, pT stage, pN stage, pM stage and pTNM stage (P > 0.05). Regarding the 169 cases analyzed by DISH, 14 (of 14, 100%) HER2 overexpression (3+) cases, 10 (of 55, 18.2%) HER2 equivocal (2+) cases, and 0 (of 100, 0%) HER2 negative (1+/0) cases showed HER2 gene amplification. HER2 gene amplification was not significantly associated with clinicopathological characteristics such as age, gender, tumor differentiation, pT stage, pN stage, pM stage and pTNM stage (P > 0.05).ConclusionsApproximately 1.5% of the Chinese ESCC patients had HER2 overexpression based on IHC. IHC and DISH had a high concordance rate. These results provide valuable insight for the future treatment of ESCC.

Human epidermal growth factor receptor 2 (HER2) protein overexpression and gene amplification are important biomarkers for trastuzumab treatment in breast and gastric cancer patients. Gastric cancer presents high rates of tumor heterogeneity, which may influence the results of HER2 testing. A novel gene-protein assay (GPA) can allow the simultaneous analysis of HER2 protein and gene status on a single slide. Using the tissue microarray technique, the HER2 status of each of 875 gastric cancer cases was evaluated by immunohistochemistry (IHC), brightfield dual-color in situ hybridization (DISH), and GPA. Intratumoral phenotypic and genotypic heterogeneity were evaluated by comparing the HER2 statuses of two tissue cores from each case. There was excellent concordance between GPA and IHC (99.2%), as well as between GPA and DISH results (99.3%). HER2 positivity obtained by GPA was almost identical (99.8%) to the results obtained by IHC and DISH assays. Intratumoral phenotypic heterogeneity was more frequently observed in IHC 2+ cases (63.5%) compared with IHC 3+ cases (28.3%). Phenotypic heterogeneity (48.8%) was more frequently observed than genotypic heterogeneity (26.8%). Tumor heterogeneity was consistently observed from early to advanced stages. HER2-positive gastric cancers presented different levels of HER2 protein expression and gene amplification statuses within the same lesion in almost half the cases examined. Evaluating both phenotypic and genotypic heterogeneity may contribute to a deeper understanding and improved prediction of clinical outcome in gastric cancer patients treated with trastuzumab. This newly established GPA technology may also be useful for developing biomarkers for other molecularly targeted therapies.

The assessment of human epidermal growth factor receptor 2 (HER2) status is crucial for selecting patients with gastric cancer who may benefit from HER2‐targeted therapy. Accurate assessment using biopsy specimens is important for patients with advanced‐stage cancer. Intratumoral heterogeneity of HER2, however, is a major challenge in HER2 testing. Here, we aimed to examine whether assessment of HER2 status could be accurately carried out with currently used methods, namely, immunohistochemistry (IHC), FISH, and dual‐color in situ hybridization (DISH). Human epidermal growth factor receptor 2 status was evaluated in 108 biopsy tissues from patients with gastric adenocarcinoma and 70 matched surgical specimens by IHC, FISH, and DISH; HER2 amplification was detected in 11 (10.2%) out of 108 biopsy specimens. The IHC and FISH results were well correlated, and FISH and DISH results were consistent for all cases. The overall concordance rate of HER2 status between biopsy tissues and surgical specimens was 91.4%. All six discordant cases were false negative on biopsy; of these cases, five showed HER2 heterogeneity on surgical resection. Assessment of the HER2 status of biopsy tissues could predict the status of the whole tumor; however, a proportion of these cases may be discordant because of intratumoral heterogeneity.

Introduction: Trastuzumab therapy has been given to the HER2 positive breast cancer (BC) patients (IHC 3+ and/or amplified HER2 gene). Heterogeneity of HER2 expression has been a problem when the recurrence is considered. Recently, the combined HER2 IHC and dual color ISH (DISH) method has been introduced as a gene-protein assay (GPA) (Roche Diagnostics Inc.). This method enables us to analyze heterogeneous expression of HER2 protein and HER2 gene at the individual cell level on the same tissue sections. This study is aimed to elucidate the relevance of GPA in studying the gene-protein(GP) heterogeneity of HER2 status at the individual cells of BCs and further to pursue the cellular GP heterogeneity as a possible cause of stage IV post-therapeutic recurrence. Materials and Methods: Formalin-fixed, paraffin-embedded sections from 17 HER2 IHC 3+ cases (5 immediate post-operative cases and 12 recurrence cases) and 11 HER2 IHC 2+ cases (9 immediate post-operative cases and 2 recurrence cases) were analyzed with HER2 GPA using BenchMark ULTRA (Roche Diagnostics K.K.). All slides were scored for HER2 protein expression (0 to 3+) and the ratio of HER2/CEN17 of 100 cells from the following areas: one hot spot and two non-hot spots. HER2 protein and gene status of individual cells were grouped from A to F according to the following definition: Group A: HER2 IHC positive (3+), DISH positive (HER2/CEN17 ratio ≥2.0) Group B: HER2 IHC positive (3+), DISH negative (&amp;lt;2.0) Group C: HER2 IHC equivocal (2+), DISH positive (≥2.0) Group D: HER2 IHC equivocal (2+), DISH negative (&amp;lt;2.0) Group E: HER2 IHC negative (0 or 1+), DISH positive (≥2.0) Group F: HER2 IHC negative (0 or 1+), DISH negative (&amp;lt;2.0) Results: HER2 GPA technology clearly demonstrated the HER2 heterogeneity at the individual cell level of invasive BCs. The GP heterogeneity was apparent in the HER2 3+ and 2+ immediate post-operative cases, particularly more obvious with HER2 IHC 2+ cases. For IHC 3+ cases (table 1), groups A and C are prominent with the HER2 GP heterogeneity compared with the other groups. For IHC 2+ cases (table 2), the HER2 GP heterogeneity was more obvious with prominent groups D and F than others. Table 1: Distribution of groups of HER2 status by GPA Cases with HER2 IHC 3+CasesABCDEFPost-operative79.22.88.407.02.6Recurrent50.83.017.06.17.68.9Post-operative and recurrent cases showing hetelogeneity as individual cell level For the recurrent cases, the GP heterogeneity was similar but more pronounced comparing to immediate post-operative tumors in IHC 3+ cases, and GP heterogeneity in recurrent cases was more pronounced in IHC 2+ cases. Conclusions: HER2 GPA system is an appropriate and essential technology to study the GP heterogeneity at the individual tumor cell level of BCs. We hypothesize that the GPA system may possibly predict the recurrence of post-therapeutic stage IV tumors by the presence of more pronounced HER2 GP heterogeneity. Table 2: Distribution of groups of HER2 status by GPA Cases with HER2 IHC 2+CasesABCDEFPost-operative01.714.944.78.630.2Recurrent007.00.548.044.5 Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-06-12.

Human epidermal growth factor receptor 2 (HER2)-positive breast cancers are known to have significant clinical and pathological response to neoadjuvant systemic therapy (NST). The aim of this study was to identify factors associated with pathological complete response (pCR), defined as no residual invasive carcinoma in the breast and axillary lymph nodes (ypT0/is ypN0), among patients with HER2-positive breast cancer and to compare pCR rates between breast cancers with HER2 protein overexpression by immunohistochemistry (IHC) versus HER2 gene amplification by fluorescence in situ hybridization (FISH) in the absence of protein overexpression by IHC. We conducted a retrospective review of HER2-positive breast cancer patients treated with NST and surgery at Memorial Sloan Kettering Cancer Center between January 2013 and May 2018. Estrogen receptor (ER), progesterone receptor (PR), and HER2 status were assessed according to the 2018 ASCO/CAP guidelines. During the study period, 560 patients were identified. Of 531 patients with IHC results available, 455 patients had HER2 IHC 3+, and 76 had IHC < 3+ but HER2 amplification detected by FISH. The overall pCR rate was 59% (330/560). The pCR rate among patients with HER2 protein overexpression (IHC 3+) was 67%, compared to 17% among patients with HER2 amplification by FISH (IHC < 3+). On univariate and multivariate analyses, HER2 protein overexpression by IHC (IHC 3+) was a significant predictor of pCR, along with grade 3 histology, PR-negative status, and dual anti-HER2 therapy. Although both HER2 IHC and FISH are standard HER2 testing methods in breast cancer, achievement of pCR is associated with HER2 IHC expression level, among other factors.