Performance of HER2 DAKO HercepTest and Ventana 4B5 immunohistochemical assays on detecting HER2 gene-amplification in uterine serous carcinomas.

We compared the performance of two commonly-used HER2 immunohistochemistry (IHC) assays in uterine serous carcinomas (USC), correlating with HER2 gene amplification by fluorescence in-situ hybridization (FISH). Sixty-five USCs were stained by both HercepTest™ and PATHWAY 4B5 assays. FISH was performed by HER2 IQFISH pharmDx. Consensus HER2 IHC scoring was performed, and HER2 testing results were evaluated using USC-specific criteria. Complete concordance between HercepTest and 4B5 assays was achieved in 44/65 tumors (68%). The overall HER2 IHC/FISH concordance was 94% (45/48) by HercepTest and 91% (42/46) by 4B5. All HER2 IHC 3+ cases with HercepTest (n = 6) and 4B5 (n = 4) were gene-amplified, corresponding to specificities of 100%. For cases with IHC 2+, 41% (7/17) by HercepTest and 42% (8/19) by 4B5 had HER2 gene amplification. The sensitivity for HercepTest and 4B5 were 38% and 25%, respectively, at a cut-off of IHC 3+ (P = 0.50), and were 81% and 75%, respectively, at a cut-off of IHC 2+ (P > 0.99). Among HER2 IHC 0-1+ cases, 3/42 cases by HercepTest and 4/42 cases by 4B5 showed amplified FISH results, corresponding to overall false negative rates of 19% for HercepTest and 25% for 4B5. By using USC-specific IHC scoring criteria, both HercepTest and 4B5 assays showed high specificities (100%) for HER2 gene amplification in IHC 3+ cases, high IHC/FISH concordance, and comparable sensitivity for detecting HER2 gene amplification. The notable false negative rates using IHC 2+ as a cut-off for reflexing FISH analysis may warrant consideration for performing FISH in IHC 1+ cases until more data become available.

Interobserver and Interantibody Reproducibility of HER2 Immunohistochemical Scoring in an Enriched HER2-Low-Expressing Breast Cancer Cohort.

OBJECTIVES: We assessed the interobserver and interantibody reproducibility of HER2 immunohistochemical scoring in an enriched HER2-low-expressing breast cancer cohort. METHODS: A total of 114 breast cancer specimens were stained by HercepTest (Agilent Dako) and PATHWAY anti-HER2 (4B5) (Ventana) antibody assays and scored by 6 breast pathologists independently using current HER2 guidelines. Level of agreement was evaluated by Cohen κ analysis. RESULTS: Although the interobserver agreement rate for both antibodies achieved substantial agreement, the average rate of agreement for HercepTest was significantly higher than that for the 4B5 clone (74.3% vs 65.1%; P = .002). The overall interantibody agreement rate between the 2 antibodies was 57.8%. Complete interobserver concordance was achieved in 44.7% of cases by HercepTest and 45.6% of cases by 4B5. Absolute agreement rates increased from HER2 0-1+ cases (78.1% by HercepTest and 72.2% by 4B5; moderate agreement) to 2-3+ cases (91.9% by HercepTest and 86.3% by 4B5; almost perfect agreement). CONCLUSIONS: Our results demonstrated notable interobserver and interantibody variation on evaluating HER2 immunohistochemistry, especially in cases with scores of 0-1+, although the performance was much more improved among breast-specialized pathologists with the awareness of HER2-low concept. More accurate and reproducible methods are needed for selecting patients who may benefit from the newly approved HER2-targeting agent on HER2-low breast cancers.

Comparison of HercepTest™ mAb pharmDx (Dako Omnis, GE001) with Ventana PATHWAY anti-HER-2/neu (4B5) in breast cancer: correlation with HER2 amplification and HER2 low status.

Performance of the new CE-IVD-marked HercepTest™ mAb pharmDx (Dako Omnis) assay (HercepTest (mAb)) was compared against the PATHWAY® anti-HER-2/neu (4B5) (PATHWAY 4B5) assay using 119 pre-selected breast cancer samples covering the entire range of HER2 immunohistochemistry (IHC) expression scores (0, 1 + , 2 + , 3 +). The sensitivity and specificity of both assays were assessed based on consensus IHC scores and amplification status, as determined by fluorescence in situ hybridization (FISH) according to 2018 ASCO/CAP testing guidelines. There was a high concordance between results from the HercepTest (mAb) and PATHWAY 4B5 assays for HER2-negative (IHC 0, 1 + , 2 + and FISH negative) and HER2-positive (IHC 3 + , 2 + and FISH positive) breast carcinomas (98.2%). Regarding individual IHC scores, complete agreement was achieved in 69.7% (83/119) of cases, and all but one of the discordant cases were due to higher HER2-status scoring using the HercepTest (mAb). Thus, more tumors were overscored as IHC 2 + by HercepTest (mAb) (27 versus 15) as evidenced by their lower FISH positivity rate (48.1% versus 80%). However, two amplified tumors identified as IHC 2 + by HercepTest (mAb) were missed by PATHWAY 4B5 (IHC 1 +). Four additional cases identified as IHC 2 + by HercepTest (mAb), with FISH ratio < 2 but elevated gene counts (≥ 4 to < 6), were recorded negative by PATHWAY 4B5. The HercepTest (mAb) detects HER2 expression with higher sensitivity in tumors with gene amplification (ISH group 1) and increased gene counts (ISH group 4) as well as in HER2-low tumors (HER2 IHC2 + /FISH negative or IHC 1 +). Future studies will demonstrate whether this translates into improved patient selection especially for new HER2-directed therapies.

A Companion Diagnostic With Significant Clinical Impact in Treatment of Breast and Gastric Cancer.

The development of trastuzumab (Herceptin®) was one of the most significant cancer drug development projects of the 20th century. Not only was it a scientific and medical achievement but it also paved the way for the drug-diagnostic codevelopment model, where a predictive biomarker assay is developed in parallel to the drug. One of the challenges in the development of trastuzumab was to select the right patient population likely to respond and here, it was critical to have access to an accurate, robust and reliable assay for detection of HER2 overexpression in tumors. In the clinical development of trastuzumab, a clinical trial assay (CTA), developed by Genentech, was used for selection of HER2 positive patients. However, during the phase III trial with trastuzumab, a new optimized IHC assay, HercepTest™ was designed and developed by Dako. In the final stage of its development, a comparative study with the CTA was conducted in order to show concordance between the two assays. In September 1998, the Food and Drug Administration (FDA) simultaneously granted approval to trastuzumab and HercepTest™. The assay has been used for patient selection in a number of significant breast cancer clinical trials such as the HERA, CLEOPATRA, EMILIA and more. In these trials, HercepTest™ demonstrated its clinical utility in the neoadjuvant, adjuvant, and metastatic setting as well as in relation to different types of HER2 targeted therapies. Likewise, the assay was used for selection of HER2 positive gastric cancer patients in the important ToGA trail. HercepTest™ was the first companion diagnostic ever approved by the FDA, and more than 20 years of use has documented its clinical impact.

The FDA-Approved Breast Cancer HER2 Evaluation Kit (HercepTest; Dako) May Miss Some HER2-Positive Breast Cancers.

OBJECTIVES: Accurate evaluation of human epidermal growth factor receptor 2 (HER2) in breast cancer is critical. METHODS: HER2 fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) tests were performed on 52 cases using a US Food and Drug Administration (FDA)-approved kit (HercepTest, FDA kit) and a laboratory-developed test (LDT) with the HercepTest antibody and a Leica Bond automated stainer. RESULTS: By FISH, 22 were HER2 positive, 29 were negative, and one was equivocal. Of the 22 HER2 FISH-positive cases, five were negative by the FDA kit and none by LDT. The five discrepant cases were retested using the same FDA kit in another Clinical Laboratory Improvement Amendments-certified laboratory, and all five cases were still negative. None of the 29 HER2 FISH-negative cases were positive by the FDA kit or LDT. The overall IHC-FISH concordance rate was 90.4% for the FDA kit and 100% for the LDT. CONCLUSIONS: The FDA kit may miss some HER2-positive cases. The LDT has a higher sensitivity and a higher concordance rate with FISH results.

Analysis of outpatient HER2 testing in New York state using the statewide planning and research cooperative system.

AIM: HER2 testing is necessary in the context of therapy with trastuzumab, pertuzumab, lapatinib and neratinib. There is a paucity of reports describing the utilization rates of HER2 testing in large outpatient populations. METHODS: The Statewide Planning and Research Cooperative System (SPARCS) was used to examine HER2 testing across the state of New York (USA) during the 2012-2016 period. RESULTS: There was a linear increase in HER2 testing (r = 0.91, p = 0.030). There were increases in HER2 testing observed among minorities, including 0.5-fold and 3.5-fold increases in individuals identified as black and Asian, respectively. Major state population centers showed the highest HER2 testing. CONCLUSION: This study establishes a platform to further evaluate clinical utility, outcomes and equity of access for 'precision oncology' testing.

Clinicopathologic and molecular markers in cervical carcinoma: a prospective cohort study.

BACKGROUND: Cervical cancer is a major health problem worldwide. Identification of effective clinicopathologic and molecular markers is vital to improve treatment stratification. OBJECTIVES: The purpose of this study was to validate a set of well-defined clinicopathologic features in a large population-based, prospectively collected cervical cancer cohort to support their use in the clinic. Further, we explored p53 and human epidermal growth factor receptor 2 as potential prognostic markers in cervical cancer. STUDY DESIGN: Tissue was collected from 401 patients with cervical cancer. Clinical data that included follow-up evaluations were collected from patient journals. Histopathologic data were evaluated and revised by an expert pathologist. The prognostic impact of selected clinicopathologic variables was analyzed in the whole cohort. Tissue microarrays were prepared from 292 carcinomas, and p53 and human epidermal growth factor receptor 2 protein levels were evaluated by immunohistochemistry. Fresh frozen samples from overlapping cervical carcinomas previously were subjected to human papilloma virus typing (n=94), whole exome (n=100) and RNA (n=79) sequencing; the results were available for our analyses. RESULTS: Among the clinicopathologic variables, vascular space invasion, histologic type, and tumor size were verified as strong independent prognostic markers. High p53 protein levels were associated significantly with markers for aggressive phenotype and survival, also in multivariate survival analysis, but did not reflect TP53 mutational status. High human epidermal growth factor receptor 2 protein levels were identified in 21% of all tumors. ERBB2 amplification was associated with poor outcome (P=.003); human epidermal growth factor receptor 2 protein level was not. CONCLUSIONS: Our findings support that the Féderation Internationale de Gynécologie et d'Obstétrique s guidelines should include vascular space invasion and tumor size 2-4 cm and that careful selection of histologic type is essential for stratification of patient risk groups. High p53 levels independently predict poor survival yet do not reflect mutational status in cervical cancer. Amplified ERBB2 significantly links to poor survival, while HercepTest does not. With optimal stratification, human epidermal growth factor receptor 2-based therapy may improve cervical cancer treatment.

Interobserver reproducibility for HER2/neu immunohistochemistry: A comparison of reproducibility for the HercepTest™ and the 4B5 antibody clone.

BACKGROUND: IHC results for HER2/neu vary with replicate testing using the same antibody clone and when alternate clones are utilized. A number of factors appear to be responsible for this variability, including fixation times, equipment utilized and training and experience of staff. A number of studies have documented interobserver variability for a single antibody clone but few have evaluated reproducibility between antibody clones and which clones demonstrate the highest degree of interobserver reproducibility. DESIGN: We studied a series of 93 cases stained by both the HercepTest™ and the 4B5 clone for interobserver reproducibility. Formalin-fixed, paraffin-embedded sections were stained by the immunohistochemical technique using the manufactures directions for both the HercepTest™ and the 4B5 clone. FISH testing was performed on formalin-fixed paraffin embedded sections according to the PathVysion HER-2 DNA probe kit instructions. RESULTS: Absolute agreement rate for Hercep was 85%. Absolute agreement for 4B5 was 69%. This difference was statistically significant (p<0.0001). The chance-corrected agreement (weighted kappa) for the HercepTest™ was 79% and 71% for 4B5 (p<0.0001). Absolute agreement between antibody clones was 58% with the chance corrected agreement being 51%. Absolute agreement of 4B5 with FISH was significantly greater than that of the HercepTest™ (54% vs 35%). CONCLUSION: Agreement between evaluators was greater with the HercepTest. However, agreement with FISH results was superior for the 4B5 clone. Interobserver agreement was less than the 95% agreement threshold recommended by the ASCO/CAP guidelines for development of a new testing method for HER2 evaluation.