Clone 4B5 Research Articles

Human Epidermal Growth Factor Receptor 2 Overexpression/Amplification in Primary Ovarian Endometrioid Carcinoma.

Human epidermal growth factor receptor 2 (HER2) expression has become increasingly helpful in predicting responses to anti-HER2 agents in gynecological cancers. This study retrospectively analyzed HER2 expression in 48 primary ovarian endometrioid carcinomas. HER2 immunohistochemistry was performed using the Ventana platform (Clone 4B5 monoclonal predilute) following the manufacturer's protocol. HER2 expression was equivocal (score 2+) by image analysis in 2 cases (4.17%) based on the breast cancer criteria. Fluorescence in situ hybridization was negative for HER2 amplification in one case (International Federation of Gynecology and Obstetrics, grade 1) and positive in the other (International Federation of Gynecology and Obstetrics, grade 3). Our findings contribute to the growing evidence that HER2 is overexpressed in a small proportion of ovarian endometrioid carcinoma, and thus may serve as a potential therapeutic target in selected cases.

HER2 persistence after treatment with T-DXd in breast and gastrointestinal cancers.

1052 Background: T-DXd is a HER2-targeted antibody drug conjugate approved for treatment of advanced HER2-positive breast and gastroesophageal cancers and HER2-low breast cancers. The prevalence of HER2 loss after exposure to T-DXd is unknown and has implications for treatment strategies in refractory patients. Methods: We investigated clinically reported HER2 immunohistochemistry (IHC) scoring on post-treatment tissue biopsies from patients who received at least 2 cycles of T-DXd as of 2/2023. IHC was performed using mAB clone 4B5 (Ventana) and scored by ASCO/CAP guidelines on a scale of 0 to 3+. MSK-IMPACT next generation sequencing (NGS) was performed on paired pre- and post-treatment samples when available. Statistics are descriptive. Results: A total of 62 patients with breast, gastroesophageal, or colon cancer had available post-treatment biopsies. The majority (n = 51) had breast cancer, including 32 with HER2-positive and 19 with HER2-low disease. Median time on therapy was 30 weeks in HER2-positive and 21 weeks in HER2-low breast cancer. All 32 patients with HER2-positive breast cancer had detectable HER2 expression by IHC on post-treatment biopsies (median IHC score 2+; range 1+ to 3+). Of those with HER2-low breast cancer, 12 (63.1%) patients had detectible IHC after treatment. Of the 7 with IHC scores of zero, 3 also had scores of zero on the most proximal pre-treatment biopsies. Seven patients with gastroesophageal and 3 with colorectal cancer were included, with a median time on therapy of 12 and 9 weeks respectively, of which 1 (9%) exhibited HER2-loss after treatment. Among all patients with HER2-positive cancers, the rate of complete HER2-loss by IHC after exposure to T-DXd was 2.3%. Thirty-two patients had paired genomic analysis, including 25 breast and 7 gastrointestinal cancers. No change was observed in the fraction of genome altered (p = 0.0736, q = .327) or tumor mutational burden (p = 0.139, q = .487) with T-DXd exposure. Changes in ERBB2 copy number did not show clear directionality, with 15 patients (46.7%) exhibiting ERBB2 amplification pre-treatment and 12 (37.5%) post-treatment, the sum of 3 temporal gains and 6 losses of ERBB2 amplification across the threshold of 1.8. Conclusions: HER2 remained detectable by IHC in the majority of patients treated with T-DXd in this cohort, especially those with HER2-positive cancers. These findings suggest that resistance to T-DXd may occur via target-independent factors, and that HER2 could still be exploited therapeutically in these populations. Further prospective studies using quantitative assays are needed to confirm these hypotheses. [Table: see text]

Abstract 5388: A unified computational pathology method to quantify HER2 expression from raw IHC and IF images in breast cancer

Abstract Background: The development of automated quantitative methods to measure biomarker expression such as HER2 aims at reducing the subjective variability of pathologist-performed biomarker assessment of immunohistochemically (IHC) stained tissue slides. As an example, the deep learning-based Quantitative Continuous Scoring (QCS) algorithm [1] enables the objective measurement of biomarker expression based on the detection of individual tumor cells in the tumor regions and - for each cell, on the instance segmentation of its nucleus, cytoplasm, and membrane compartments. Methods: In order to extend the QCS image analysis to similarly analyze tissue slides stained with immunofluorescence (IF), we re-trained the fully supervised deep-learning models, adjusted the normalization of images on tissue controls to account for variability between different staining batches, and finally used the normalized signal in IF instead of the Optical Density (OD) signal in IHC as a 8-bit grayscale image on which the biomarker expression is estimated. We performed QCS on HER2 IF images (HER2 clone 29D8 [CST], imaged with Vectra [Akoya] in parallel with HER2 IHC clone 4B5 [Roche Tissue Diagnostics]) performed on 26 primary and metastatic breast cancer samples representing the full range of HER2 expression, from null to highly overexpressed. Results: Our analysis demonstrated that the QCS-based scoring on IHC-HER2 images correlates with the QCS-based scoring on IF-HER2 images. We observed a Pearson correlation of R=0.92 between the median membrane OD in IHC and the median normalized membrane signal in IF. Defining a positive cell as having an estimated membrane expression higher than a given so-called positivity threshold, we found a median Pearson correlation of R=0.85 between the percentage of positive cells detected in IHC and the percentage of positive cells detected in IF for increasing values of positivity thresholds. Correlation of the QCS median normalized membrane signal in IF was R=0.91 with mRNA (ERBB2 transcript levels [Nano String]) and R=0.88 with IHC-based H-scores, against R=0.83 and R=0.87 respectively for the QCS median membrane OD signal in IHC. Conclusion: We describe the extension of a computational pathology-based approach for biomarker quantification in IHC to IF stained tissue slides. The consistency of the image analysis method translates into the consistency of the measurements in the two imaging methods. The use of IF could enable the improved quantification of expression, co-localization and spatial distribution of multiple proteins on the tissue sample.

Abstract P2-09-03: Quantification of HER2 expression and spatial biology using computational pathology: A cross-assay validation study in breast cancer

Abstract Background Conventional pathologic scoring of HER2 by IHC is proven to distinguish potential responders to trastuzumab but has not been effective for next generation antibody drug conjugates (ADCs) such as trastuzumab deruxtecan (T-DXd), which is capable of bystander killing. Several alternative approaches have been deployed to measure HER2, including immunofluorescence and mRNA sequencing. We have developed a novel and fully automated computational pathology technique, Quantitative Continuous Scoring (QCS), to quantify the level and distribution of HER2 from digitized HER2 IHC slides in an objective, quantifiable, and reproducible manner on a per-cell basis [Gustavson et al., SABCS 2020]. To further validate this approach, we performed a systematic multi-omic comparison of QCS to orthogonal methods of HER2 quantitation on a cohort of primary and metastatic breast cancer cases (N=30). Methods HER2 was evaluated using three independent methods on serial tissue sections obtained from 30 archival FFPE breast cancer samples distributed over the full range of HER2 expression, from 0 to 3+. HER2-IHC staining (clone 4B5, Roche Tissue Diagnostics) was performed using standard methods and cases were scored by two pathologists using CAP/ASCO guidelines and H-scores were assigned. We performed FISH (HER2 IQFISH pharmDx [Dako]; PathVysion HER-2 DNA Probe Kit [Vysis]), mRNA quantification of ERBB2 transcript levels (Nano String), and immunofluorescence (IF; HER2 clone 29D8, CST). Imaged with Vectra (Akoya) and analyzed with Halo (Indica). QCS readouts were generated from the above-mentioned digital images of IHC slides by using a fully automated image analysis pipeline; readouts included per-cell staining intensity measurements of membranes and cytoplasmic sub-compartments in terms of optical density (OD) [Van der Laak, JQCS 2000], which were aggregated to a single slide-level score. Additionally, using the OD measurements and the cell locations, a Spatial Proximity Score (SPS) was computed, summing the percentage of cells with OD≥10 (corresponding to the limit of visual detection of IHC staining) as well as the percentage of cells with OD< 10 within a prespecified radius (25µm) of a neighboring cell with OD≥10. Results Our analysis demonstrated that QCS-based scoring correlates with orthogonal measurements used in this study. Comparing protein-based assays, the observed Pearson correlation was R=0.88 between QCS median membrane OD and IHC H-scores, R=0.86 with IF-based HER2 mean cell expression intensity, and R=0.85 with IF-based H-scores. Correlation with transcriptomic profiling was R=0.81 for OD vs. mRNA, however ERBB2 transcript levels did not distinguish between HER2 0, 1+, and 2+ FISH negative cases, while QCS was able to do so. Correlation between protein-based and nucleic-acid based assays were numerically worse, with R=0.64 for OD vs. FISH. All samples (including those with HER2 IHC scores of 0 and H-Scores < 10) had at least ~20% of cells with quantifiable HER2 expression by OD, the presence of which was confirmed using IF. For cases in the lowest quartile of HER2 expression by OD, SPS identified 20-50% additional HER2-null cells that were in close proximity to HER2-expressing cells that may be vulnerable to bystander killing. Conclusion QCS-based scoring is consistent with orthogonal protein-based measurements across the range of HER2 expression. Most importantly, QCS derived-spatial analysis features identify additional patients in the lower end of HER2 expression that might be highly relevant for ADC response prediction, particularly if a drug exerts bystander activity. Further clinical verification and validation on large cohorts is needed. Footnote: This study was approved by the IRB at MSKCC. Citation Format: Joshua Drago, Zonera Hassan, Jan Zaucha, Ansh Kapil, Fatemeh Derakhshan, Fresia Pareja, Shimulov Anatoliy, Fanni Ratzon, Travis J Hollman, Claire Myers, Jessica Chan, Andrea Spitzmuller, Mark Gustavson, Danielle Carroll, Dara Ross, Jorge Reis-Filho, Carl barrett, Sihem Khalifa, Schmidt Guenter, Hadassah Sade, Sarat Chandarlapaty. Quantification of HER2 expression and spatial biology using computational pathology: A cross-assay validation study in breast cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-09-03.

Discordance of HER2 expression in distant metastatic tumors (MTs) versus primary tumors (PTs) in urothelial carcinoma (UC).

550 Background: Available HER2 targeted agents have limited benefit for UC, but exceptional tumor responses are occasionally seen. HER2 expression has been tested as a biomarker of response to these drugs. Trial eligibility is often based on HER2 expression in PTs. Previous studies found moderate to high concordance of HER2 expression in PTs and synchronous regional nodes at cystectomy, but the concordance rate with distant MTs is poorly defined and further complicated by shifting definitions of HER2 positivity. Methods: Immunohistochemical (IHC) staining for HER2 (Ventana Pathway clone 4B5) was performed on PTs and matched MTs in 79 patients with UC. IHC staining was scored (0-3+) using intensity and % positive tumor cells using 2018 ASCO/CAP guidelines for breast cancer. Fisher’s Exact Test assessed association of PT and MT HER2 scores, and Cohen’s kappa statistic (κ) assessed agreement in HER2 3+ between groups. MTs were defined as synchronous if biopsied <60 days after the PT biopsy. Results: There were 67 metachronous and 12 synchronous paired biopsies; median time between metachronous biopsies was 418 days. MT biopsy sites included lymph node (30%), bone (22%), lung (18%), pelvic/peritoneal soft tissue (11%), liver (8%), brain (4%), other (6%). HER2 2+ and 3+ expression was seen in 30.4% and 12.7% of PTs and 12.7% and 7.6% of MTs, respectively. Of HER2 3+ PTs (n=10), HER2 expression in paired MTs was 0 (n=3), 1+ (n = 3), 2+ (n = 2), and 3+ (n = 2). Of HER2 3+ MTs (n=6), HER2 expression in paired PTs was 0 (n=1), 2+ (n=3), and 3+ (n=2). An association was observed between PT and MT HER2 scores (p=0.004), however there was low agreement of HER2 3+ rates between PT and MT lesions (κ = 0.17, p=0.113). Discordance was noted in both synchronous and metachronous biopsies. Conclusions: Based on the current HER2 3+ definition, HER2 overexpression is spatially and temporally discordant in UC between PTs and distant MTs. These findings suggest that both PTs and MTs should be tested for HER2 expression in future clinical trials assessing the efficacy of HER2 targeted agents in UC. The high discordance may explain previously negative therapeutic studies.

INTERRELATION OF THE EXPRESSION OF PROTEINS ALK, HER2 AND AMPLIFICATION OF THE GENE OF HER2 WITH THE PROLIFERATIVE ACTIVITY AND SURVIVAL IN PATIENTS WITH STAGE I-II LUNG ADENOCARCINOMA

To investigate protein content of ALK, Her2 and gene of Her2, CEP17 in interrelation with proliferative activity and survival in I-II stages lung adenocarcinoma cases. There were investigated 82 surgery samples taken from I-II stage lung adenocarcinoma patients. With the use of immunohistochemical method ALK protein (clone D5F3, Ventana), Her2 protein (clone 4B5, Ventana), topoizomerase IIα (clone JS5B4, Ventana), Ki-67 (clone MIB-1, Dako) were determined, Her2 gene was investigated with the use of the SISH method (Ventana) also. The positive expression of ALK is detected in 4(5%) cases, Her2 - in 16(20%) cases and amplification of Her2 gene - in 7(9%) cases. In lung adenocarcinoma with positive expression of ALK protein and amplification of Her2 gene low proliferative activity was noted (on labeling index Ki-67 and topoizomerase IIα). The survival of lung adenocarcinoma patients was less in cases of a positive expression of ALK protein in comparison with patients with the negative expression (difference is not reliable). Statistically significant low survival rates of patients is found in cases with amplification of Her2 gene. According to data mono- and multifactor regression analysis the amplification of Her2 gene has been interrelated with the poor forecast. Mutation of ALK gene and amplification of Her2 gene are interrelated with the proliferative activity and survival rate of lung adenocarcinoma patients.

Abstract 3921: A screen of breast and colon cancers with HER2 antibody clone UMAB36 does not exhibit the cross-reactivity of clone 4B5 with HER4 protein

Abstract Increased sensitivity and specificity of immunohistochemical (IHC) detection of HER2 expression is crucial as the role of Trastuzumab have expanded in the treatment of HER2 positive non-breast cancer patients such as gastric cancer patients. Non-specific nuclear and cytoplasmic staining of the HER2 antibody clone 4B5 has been reported by other labs. In this study, we evaluated the specificity of HER2 clone 4B5 and a new HER2 antibody clone UMAB36 using a suite of methods including protein lysate arrays, western blots, FISH and IHC correlation screens on 129 breast and 158 colon cancer cases for HER2 expression. The protein lysate array and western results revealed that clone 4B5 recognizes three proteins: HER2, HER4, and ZSCAN18, a nuclear transcription protein. In comparison, clone UMAB36 recognized only HER2 protein in the same protein lysate array and western screen as clone 4B5. False negative results, based on correlation of IHC with FISH HER2 positives, were generated using clone 4B5 in 1 breast and 5 gastric of the total 287 cancer cases screened. Comparatively, no false negative results were observed using clone UMAB36 in which there was a 100% correlation between IHC and FISH screen. In this study, areas of normal gastric tissue often stained positive by HER2 clone 4B5, so we performed further analysis of 470 normal gastric cases using Ventana's BenchMark instrument. The results show that 278 normal gastric cases had positive stain with clone 4B5 compared to 3 cases with clone UMAB36. The high background by clone 4B5 may be due HER4 being upregulated in adjacent normal gastric tissue. Our results indicate clone UMAB36 has higher specificity and sensitivity than clone 4B5 in screening gastric tumors. Citation Format: Lixin Zhou, Kehu Yuan, Fangfang Ren, Lili Ki, Min Zhou, Wei Fu, Xiaozheng Huang, Rachel M. Gonzalez, Youmin Shu, Yi Shen, Guangli Wang, Donghui Ma, Wei-Wu He, Jian Chen. A screen of breast and colon cancers with HER2 antibody clone UMAB36 does not exhibit the cross-reactivity of clone 4B5 with HER4 protein. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3921.

Abstract P5-07-07: Fluorescence quantitative image analysis of HER2 evaluation against current clinical HER2 assays in breast cancer testing

Abstract BACKGROUND: Presently, therapy for treatment of breast cancer is based on the evaluation of formalin fixed, paraffin embedded (FFPE) pathological specimens using a combination of immunohistochemistry (IHC) and gene copy assessment by in-situ hybridization (-ISH) techniques, following current testing guidelines from the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP). Patients with specimens found to have marked overexpression and amplification of the human epidermal receptor 2 (HER2) are approved for treatment with trastuzumab. While IHC and –ISH assays represent the current clinical standard, these assays are subject to pre-analytical variation, which could lead to false negative results. There are novel laboratory technologies which may improve the accuracy of HER2 measurements and lead to improved patient selection for therapy. AIM: In this comparative study, we assessed the utility of testing HER2 expression by the fluorescence IHC using AQUA® - a novel computer assisted platform to enable quantitative assessment of protein expression in FFPE tissue specimens – against current clinical assays (IHC and –ISH). METHODS: Local cases from 2008-2010 clinically evaluated for HER2 were identified for further pathological review. FFPE tissue specimens were retrieved from a total of 207 cases with sufficient tumor present, and placed into a tissue microarray (TMA). TMA sections underwent assessment for IHC HER2 (Clone 4B5, Ventana), HER2/Chromosome 17 gene copy number (Inform HER2 dual-ISH DNA Probe Cocktail Assay, Ventana), and fluorescence IHC (Clone SP3, Thermo Fisher). RESULTS: HER2 results were available for 142 patients for IHC, and 134 patients for dual–ISH and fluorescence IHC. A comparison of the current clinical methods revealed 11 discordant cases. The average median fluorescence IHC cytoplasmic HER2 expression (cAQUA) was found to be 225.65, (70.65-419.95). HER2 cAQUA was strongly correlated with dual-ISH, and cases with low level amplification had low cAQUA expression. There were cases having high cAQUA expression that did not show amplification by dual-ISH. Only a few amplfied cases demonstrated low cAQUA expression. Dichotomizing cAQUA at the 256 showed an improvement of the receiver operating characteristic compared to the clinical HER2 IHC assay (cAQUA=0.903, p<0.001; IHC=0.833, p=0.006). CONCLUSIONS: Measurement of HER2 expression using human interpretation can be imprecise as there is still some discordance between HER2 IHC and dual-ISH assays. Evaluation of HER2 protein expression using the novel AQUA assay showed correlation with IHC and dual-ISH, and AQUA may present a more precise way to quantify HER2 protein expression. HER2 cAQUA used a different antibody clone than the clinical IHC assay; however, previous studies have shown strong correlation between these two antibodies. The AQUA assay may identify a previously unrecognized group of breast cancers with elevated HER2 expression. The significance of this finding requires further investigation, particularly in regards with cAQUA HER2 serving as a marker for response to anti-HER2 therapy. Citation Format: Kornaga EN, Feng X, Klimowicz AC, Dean ML, Guggisberg N, Morris DG, Magliocco AM. Fluorescence quantitative image analysis of HER2 evaluation against current clinical HER2 assays in breast cancer testing. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-07-07.

HER2 gene and protein expression status of breast carcinoma can be reliably tested on a single slide.

Human epidermal growth factor receptor 2 (HER2) status in breast carcinomas serves as a predictor of benefit from anti-HER2 therapy. In providing clinicians with the information necessary to decide whether or not to treat with targeted therapy, it might be necessary to choose between methods assessing HER2 protein overexpression or gene amplification. A new diagnostic approach could be a combination of both tests on the same slide. If accurate and reproducible, this approach might optimize patient stratification for therapy. In this study, formalin-fixed paraffin-embedded tumor samples from 77 breast cancer patients were examined for HER2 by immunohistochemistry (IHC) and silver in situ hybridization (SISH) using HER2 IHC (clone 4B5), HER2/CEN17SISH, and combined IHC and SISH assay, called gene protein (GP). Cases were selected to ensure a sufficient number of borderline cases on the basis of IHC scores (0, 1+, 2+, 3+), obtained during diagnostic histopathological workup. The concordance between the HER2 IHC score obtained during diagnostic histopathological workup and GP was 93 %. Discordances had no influence on therapy decisions. The concordance between ISH results using dual ISH (DISH) and GP was 96 %. Of the 77 cases studied by GP, three cases with a ratio close to 2 would have been called amplified by DISH. The use of GP reduced the time for slide reading for a trained pathologist by up to 25 %, relative to sequential reading of IHC followed by SISH. For cases with an IHC score of 2+, the final result was obtained in 1 day, while the sequential technique would have required retesting by ISH on a second day. In conclusion, assessment of HER2 status by GP is an improvement for pathologists and facilitates clinical decision-making for breast cancer management.

Non-small cell lung cancer: HER2 oncogene status

to study HER2 protein and HER2 gene, their heterogeneity in non-small cell lung cancer. 218 intraoperative non-small cell lung samples were examined using tissue matrix methods. HER2 protein was determined by immunohistochemistry (clone 4B5, <<Ventana>> and HER2 gene and CEP1 7 were evaluated by in situ hybridization (SISH, <<Ventana>>). Positive and indefinite statuses were found in 59 (27%) and 47 (22%) cases, respectively; intratumor heterogeneity was detected in 32 (30%) cases. Amplification of the HER-2 gene was found in 12 (6%) cases; that of the HER2 gene along with an increase in CEPI 7 was observed in 7 (3%) cases; elevated CEP1 7 levels were seen in 19 (9%) cases. Intratumor heterogeneity of HER2 gene amplification was not found; however, one case of adenocarcinoma showed high-level HER2 gene amplification in the gland-like areas and low-level HER2 gene amplification in the solid areas. HER2-positive status and amplification were more common in adenocarcinoma than in squamous cell carcinoma (p<0.001). There was a moderate correlation between HER2 immunohistochemical status and amplification (r=0.38; p<0.001). Thus, in non-small cell lung cancer, there is an elevated HER2 protein level and, well less frequently, altered activity in the HER2 gene (amplification) as a cause of enhanced protein synthesis.

Abstract P1-02-03: Clinical utility of combined brightfield HER2 gene-protein assay in IHC equivocal cases

Abstract The combined gene protein assay (GPA) for HER2 (Ventana Medical Systems Inc, Tucson AZ) is a newly developed technique where both HER2 gene and protein can be simultaneously assessed on a single slide using routine bright field microscopy. This is made possible by combining HER2 immunohistochemistry (using clone 4B5 from Ventana) with the HER2 Dual in-situ hybridization (DISH) DNA probe cocktail. The main goal of the study was to compare this new GPA methodology with standard fluorescence in situ hybridization (FISH) on the most challenging group of breast cancers, i.e. HER2 immunohistochemical (IHC) 2+ cases. This validation study included whole tissue sections of 36 invasive breast cancer cases with equivocal HER2 IHC results (IHC score 2+) on clinical testing. FISH and GPA were performed on the same tumor blocks. The GPA assay staining results were enumerated by counting at least 20 nuclei by a pathologist blinded to FISH results. Similar to FISH, areas with stronger IHC staining were counted in heterogeneous cases. For both FISH and GPA, HER2 amplification was defined as HER2/CEP17 ratio ≥ 2.0. Cases with HER2/CEP17 ratio &amp;lt;2.0 were considered as non-amplified. In addition, 25 known HER2 IHC 3+ invasive carcinoma cases (represented on a tissue microarray) and 22 known IHC negative (scores 0 and 1+ also represented on tissue microarray) were also evaluated by GPA. GPA was successful in all cases in the first attempt. All previously tested IHC 2+ cases were again scored as 2+ on GPA. The agreement for IHC 2+ cases between FISH and GPA with respect to HER2 gene status was 94.4% (see table). The 2 discordant cases were amplified by GPA and not amplified by FISH. The HER2 to CEP17 ratio on these 2 cases by GPA was 2.0 and 2.34 with average HER2 copies/cell of 5.7 and 5.5 respectively. For the 36 IHC 2+ cases, the mean HER2 copies per cell for FISH were 3.09 (std dev: 1.8; range: 1.9 to 11.07) versus 3.52 (std dev: 1.8; range: 1.8 to 9.95) for GPA. The mean CEP17 signals per cell were 2.24 (std dev: 0.6; range: 1.65 to 4.2) for FISH versus 2.3 (std dev: 0.6; range: 1.6 to 3.5) for GPA. The mean for HER2:CEP17 ratio was 1.34 (std dev: 0.6; range: 1.03 to 4.32) by FISH and 1.50 (std dev: 0.7; range: 1.02 to 4.72) by GPA. GPA versus FISH for IHC 2+ cases GPA amplifiedGPA not amplifiedTotalFISH amplified202FISH not amplified23234Total43236FISH: Fluorescence in-situ hybridization; GPA: Gene protien assay All 25 (100%) of IHC 3+ cases showed clusters of HER2 gene consistent with HER2 amplification by GPA. These 25 cases also showed strong membranous (“chicken wire”) reactivity consistent with 3+ IHC score. Similarly, the 22 known IHC negative cases on the TMA, were again IHC negative, and lacked HER2 gene amplification by GPA. The HER2 GPA shows excellent concordance with both HER2 IHC assay and FISH assay. It performs accurately not only for unequivocally positive and negative cases but also on the most challenging (IHC 2+) breast cancer cases. This new assay combines the advantages of both IHC and FISH, i.e. accuracy similar to FISH for IHC equivocal cases, faster turn around time (less than 24 hours) like IHC and evaluation under bright-field microscope where the tissue morphology is well preserved and heterogeneity is better appreciated. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P1-02-03.

Is HER2 amplification predictable by digital immunohistochemistry?

HER2 positive breast cancers can benefit from trastuzumab therapy based on a validated immunohistocemical reports. HER2-negative and strong positive cases are easy to interpret, but equivocal cases should be analyzed with FISH-technique to reveal HER2 amplification. Image analysis methods have been recently developed such as MembraneQuant by 3DHISTECH to support this process. We validated MembraneQuant software on HER2-immunostained (clone 4B5) tissue microarrays of 100 breast cancers covering all positivity groups and tested if semi-automated software analysis of HER2 immunostaining can discriminate between FISH-positive and negative equivocal cases. The renowned 4-tiered evaluation guidelines were used. The HER2 gene status of the 15 equivocalcases was also assessed with FISH. Detailed MembraneQuant analysis in the 9 FISH- and 6 FISH+ cases was used to predict HER2 amplification status.

Immunohistochemical analysis of the monoclonal antibody 4B5 in breast tissue expressing human epidermal growth factor receptor 4 (HER4)

A recent study examining the specificity of human epidermal growth factor receptor (HER) 2 pharmacodiagnostic antibodies demonstrated that CB11 and 4B5 stain both HER2-transfected and HER4-transfected cell lines. However, there has been no evidence showing that 4B5 has affinity for HER4 in clinically obtained tissues, and, if so, whether this has any impact on the assessment of HER2. We therefore sought to determine the expression of membrane-bound HER4 in clinical breast carcinomas, and evaluate its impact on the clinical utility of 4B5 in determining HER2 status. Breast carcinomas were assessed by immunohistochemistry (IHC) for membrane-bound HER4 using anti-HER4 clone E200. HER2 expression in these cases was then assessed using anti-HER2 clone 4B5, and a reference clone, SP3. In all 117 membrane HER4-positive cases (out of 241), 4B5 scored equal to or less than the reference anti-HER2 clone SP3. Eighteen cases were positive for membrane-bound HER4 by E200 and negative by 4B5, including a membrane HER4 level 3+ case. No cross-reactivity of 4B5 with membrane-bound HER4 was identified in the clinical IHC analysis of formalin-fixed paraffin-embedded breast carcinoma cases as evidenced by the HER4 antibody clone E200.

A gene-protein assay for human epidermal growth factor receptor 2 (HER2): brightfield tricolor visualization of HER2 protein, the HER2 gene, and chromosome 17 centromere (CEN17) in formalin-fixed, paraffin-embedded breast cancer tissue sections

BackgroundThe eligibility of breast cancer patients for human epidermal growth factor receptor 2 (HER2)-directed therapies is determined by the HER2 gene amplification and/or HER2 protein overexpression status of the breast tumor as determined by in situ hybridization (ISH) or immunohistochemistry (IHC), respectively. Our objective was to combine the US Food and Drug Administration (FDA)-approved HER2 & chromosome 17 centromere (CEN17) brightfield ISH (BISH) and HER2 IHC assays into a single automated HER2 gene-protein assay allowing simultaneous detection of all three targets in a single tissue section.MethodsThe HER2 gene-protein assay was optimized using formalin-fixed, paraffin-embedded (FFPE) samples of the xenograft tumors MCF7 [HER2 negative (non-amplified gene, protein negative)] and Calu-3 [HER2 positive (amplified gene, protein positive)]. HER2 IHC was performed using a rabbit monoclonal anti-HER2 antibody (clone 4B5) and a conventional 3,3'-diaminobenzidine IHC detection. The HER2 & CEN17 BISH signals were visualized using horseradish peroxidase-based silver and alkaline phosphatase-based red detection systems, respectively with a cocktail of 2,4-dinitrophenyl-labeled HER2 and digoxigenin-labeled CEN17 probes. The performance of the gene-protein assay on tissue microarray slides containing 189 randomly selected FFPE clinical breast cancer tissue cores was compared to that of the separate HER2 IHC and HER2 & CEN17 BISH assays.ResultsHER2 protein detection was optimal when the HER2 IHC protocol was used before (rather than after) the BISH protocol. The sequential use of HER2 IHC and HER2 & CEN17 BISH detection steps on FFPE xenograft tumor sections appropriately co-localized the HER2 protein, HER2 gene, and CEN17 signals after mitigating the silver background staining by using a naphthol phosphate-containing hybridization buffer for the hybridization step. The HER2 protein and HER2 gene status obtained using the multiplex HER2 gene-protein assay demonstrated high concordance with those obtained using the separate HER2 IHC and HER2 & CEN17 BISH assays, respectively.ConclusionsWe have developed a protocol that allows simultaneous visualization of the HER2 IHC and HER2 & CEN17 BISH targets. This automated protocol facilitated the determination of HER2 protein and HER2 gene status in randomly selected breast cancer samples, particularly in cases that were equivocal or exhibited tumor heterogeneity. The HER2 gene-protein assay produced results virtually equivalent to those of the single FDA-approved HER2 IHC and HER2 & CEN17 BISH assays.Virtual slidesThe virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2041964038705297

Abstract 1258: High-density protein lysate arrays: an essential tool in assessing antibody specificity for clinical applications

Abstract Antibodies for clinical applications require rigorous scrutiny qualifying them for diagnostics or therapeutic use. However, this is a lengthily and complicated process that provides only limited conformation. Protein microarrays are new tools that can determine the specificity and reactivity of Abs and help identifying their target in less than a day. We created a high-density protein array comprised from more than 10,000 over-expressed protein lysates made in human HEK293T cells. We tested more than 15 available antibodies to two cancer targets; the excision repair cross-complementation group 1 (ERCC1) and the receptor tyrosine kinase ERBB2 (Her2/neo). Our results demonstrated the ability of the lysate array in determining the specificity of antibodies with a single fast and easy assay. ERCC1 is a predictor of resistance to platinum based drugs and for poor prognosis in cancer. The commonly used anti ERCC1 antibody, mAb 8F1, is known to give non-specific cytoplasmic staining and to cross-react with an unknown protein of similar size. We tested 10 commercially available Abs to ERCC1 and identified four mono-specific ones (TrueMAb OriGene). Most of these specific Abs gave higher reactivity than clone 8F1. With this test we also identified a cytoplasmic protein of appropriate molecular weight that is cross-reacting with mAb 8F1. The above results were corroborated by WB analysis and IHC assays supporting the validity of this type of assays. We extended our tests to include anti ERBB2 Abs as this important biomarker is indicative for the use and response to the drug Herceptin. Our tests included mono and poly clonal antibodies and clone 4B5, a rabbit mAb currently used for IHC diagnostic. We found clone 4B5 to be reactive with two additional proteins besides ERBB2, one of them is ERBB4. We also tested a rabbit polyclonal anti ERBB2 serum and found it to be reactive with additional proteins unrelated to those recognized by mAb 4B5. Three out of the four mouse mAbs tested (TrueMAb OriGene) were determined to be specific. We submit that the over-expression protein lysate arrays can establish the reactivity and specificity of Abs and identify unknown targets. The speed and convenience of these assays make them an important tool in developing Abs for clinical use. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1258. doi:1538-7445.AM2012-1258

Human epidermal growth factor receptor 2 (HER2) immunoreactivity: specificity of three pharmacodiagnostic antibodies

AimsThe availability of specific antibody-based test systems is essential to testing of HER2 protein expression. Here, we mapped epitopes recognized by three pharmacodiagnostic HER2 antibodies and investigated their specificity towards peptides and fusion proteins homologous to the intracellular domains of HER1, HER2, HER3 and HER4. The investigated antibodies were PATHWAY® HER2 (clone 4B5; Ventana Medical Systems Inc., Tucson, AZ, USA), HercepTest™ (Dako Denmark A/S, Glostrup, Denmark), and Oracle® HER2 (clone CB11; Leica Microsystems GmbH, Wetzlar, Germany).Methods and resultsEpitopes were mapped using the alanine scanning method. Specificity was investigated in immunohistochemical stainings, competitive enzyme-linked immunosorbent assay (ELISA) and immunoblotting. All three antibodies reacted with HER2 proteins and peptides in immunohistochemical stainings, ELISA and immunoblotting. PATHWAY® HER2 also stained HER4-expressing cells, reacted with HER4 peptide in ELISA and detected HER4 fusion protein in an immunoblot. Oracle® HER2 weakly detected HER4 in immunohistochemical stainings, whereas the HercepTest™ antibody showed no cross-reactivity with other HER proteins.ConclusionOur study shows that the PATHWAY® HER2 antibody can bind HER4 peptides and fusion proteins in three different experimental settings. This should be investigated further to determine whether binding of HER4 also occurs in tissue samples and if such binding would have implications for therapy decisions for breast cancer patients.

Fixation Time Does Not Affect Expression of HER2/neu

It is said that HER2/neu expression by immunohistochemical analysis varies with the time of fixation. The purpose of this pilot study was to determine the impact of the length of fixation in 10% buffered formalin on the expression of HER2/neu by immunohistochemical analysis. We studied tissue samples from 10 invasive breast cancer cases after fixation for 3, 48, 72, 96, and 120 hours. The tissue was processed immediately after fixation, resembling routine practice. The 50 resulting blocks were then batch stained with PATHWAY HER2/neu clone 4B5 rabbit monoclonal antibody using the Ventana Ultraview DAB detection kit in a Ventana BenchMark XT processor (Ventana, Tucson, AZ). The stained slides were reviewed and scored. We found no significant difference in the intensity of the stain or the percentage of cells stained regardless of the time in fixation. Fixation times between 3 and 120 hours in 10% buffered formalin do not appear to have an impact on the expression of HER2/neu by immunohistochemical analysis.

Designed ankyrin repeat proteins: a novel tool for testing epidermal growth factor receptor 2 expression in breast cancer

Designed ankyrin repeat proteins are a novel class of specific binding molecules, which display increased thermodynamic stability, smaller size and at least equal target affinity compared to immunoglobulins, making them potentially powerful tools in diagnostic pathology and therapeutic oncology. Here, we investigated whether designed ankyrin repeat proteins can reliably identify the amplification status of the epidermal growth factor receptor 2 in breast cancer. Designed ankyrin repeat proteins specific for epidermal growth factor receptor 2 were tested in paraffin-embedded tissue sections. Detection using enzymatic biotinylation proved to be most specific and sensitive. The affinity of the designed ankyrin repeat proteins was found crucial, but for a picomolar binder no further gain was found by making it multivalent. The best designed ankyrin repeat protein, G3 (KD 90 pM) was compared on breast cancer tissue microarrays (n=792) to an FDA-approved rabbit monoclonal antibody against epidermal growth factor receptor 2 (clone 4B5; Ventana Medical Systems) and correlated with corresponding epidermal growth factor receptor 2 amplification status measured by fluorescent in situ hybridization. Amplification status and epidermal growth factor receptor 2 expression measured by designed ankyrin repeat protein and antibody correlated strongly with each other (P<0.0001 each), the correlation between designed ankyrin repeat protein and amplification status being the strongest (0.87 compared to 0.77 for the antibody, Kendall's tau-beta). Using a modified scoring system for the designed ankyrin repeat protein, we show that the designed ankyrin repeat protein detects a positive epidermal growth factor receptor 2 amplification status with similar sensitivity and significantly higher specificity than the antibody (P=0.0005). This study suggests that designed ankyrin repeat proteins provide a valuable alternative to antibodies for the detection of epidermal growth factor receptor 2 expression in breast cancer and adds further compelling evidence for the use of designed ankyrin repeat proteins in diagnostic pathology and therapeutic oncology.

HER-2/neu expression in glioblastoma multiforme (GBM)

e13035 Background: Glioblastoma multiforme (GBM) is a disease in which very few cytotoxic chemotherapy agents have been shown to have activity. This is partly due to their inability to cross the blood brain barrier. Trials with bevacizumab, a VEGF inhibitor that disrupts tumor angiogenesis, have demonstrated activity against this otherwise chemotherapy resistant disease. This has led to interest in other biologic agents that target angiogenic pathways for the treatment of GBM. Over-expression of HER-2/neu by human tumor cells is closely associated with increased angiogenesis and expression of VEGF. Lapatinib is a recently available low molecular weight immunotherapeutic agent that targets HER-2/neu proteins. In a recent study, breast cancer patients treated with lapatinib and capcitabine had decreased brain metastases indicating that lapatinib may cross the blood brain barrier and thus have potential in the treatment of malignant gliomas. Limited studies have evaluated HER-2/neu gene expression in GBM and the results are inconsistent. We evaluated the expression of Her-2/neu protein in 41 consecutive GBM cases to explore the potential utility of targeting this pathway. Methods: Archival histopathologic sections from 41 patients (age 26–89 years) with a diagnosis of GBM from 2004–2008 were reviewed. The diagnosis was confirmed and optimal sections were selected. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded tissue sections using the primary antibody against HER-2/neu (clone 4B5, Ventana). The results were evaluated by three independent investigators. Interpretation was performed using the semi-quantitative criteria (Score 0 to 3+) currently used for primary breast carcinomas. Results: 38 out of 41 cases showed no immunohistochemical staining with HER-2/neu antibody (score 0). Three cases demonstrated weak, incomplete membrane staining of rare tumor cells (score 1+) and were interpreted as negative. The positive and negative controls worked properly. Conclusions: Our study indicates that there is no significant immunohistochemical over-expression of HER-2/neu protein in GBM. This suggests that HER-2/neu over-expression is not a significant oncogenic pathway in GBM, and therefore may not be a potential therapeutic target in this disease. No significant financial relationships to disclose.