Central HER2 Research Articles

Abstract P2-03-03: A multicenter clinical study of Xpert® breast cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues

Abstract The Xpert® Breast Cancer STRAT4 (STRAT4) is a CE-IVD marked, semi-quantitative, cartridge-based RT-qPCR assay for the detection of ESR1, PGR, ERBB2 (HER2), and MKi67 mRNAs from formalin fixed, paraffin embedded (FFPE) breast tumors. The assay is fast (< 2 hrs), reproducible, robust, and easy to perform. The aim of this multicenter clinical study was to assess the performance characteristics of the STRAT4 assay relative to central lab immunohistochemistry (IHC) for ER, PgR, HER2, and Ki67 and to fluorescence in situ hybridization (FISH) for HER2 gene amplification. Methods: A total of 200 archived primary invasive breast cancer FFPE blocks were sourced from Indivumed for this study. From each block, twelve (12) adjacent tissue sections (4-µm thickness) on slides were prepared for pathological H&E confirmation to define tumor area, and for testing by STRAT4, IHC (ER, PgR, HER2,Ki67), and HER2 FISH. Standard STRAT4 lysate preparation using a single unstained slide per specimen and testing on N=84, N=68, and N=48 samples was performed at 3 independent sites, respectively (2 US and 1 EU). A single slide from each specimen was also processed using the recommended concentrated lysate procedure for STRAT4 testing at Cepheid. All IHC and FISH testing was performed by a central academic reference laboratory in the US. For a given sample, STRAT4 data generated using the standard lysate procedure was included for concordance analysis when all target gene test results were valid. In cases where the standard lysate preparation yielded indeterminate test results for any target, data from the concentrated lysate preparation was used for the data analysis. Receiver Operating Characteristic (ROC) analysis, overall percent agreement (OPA), positive percent agreement (PPA), and negative percent agreement (NPA) between STRAT4 and IHC (IHC/FISH for HER2) were determined for ESR1,PGR, ERBB2, and MKi67. Results: Of the 200 samples tested by STRAT4, all samples generated valid results for ESR1 and ERBB2, 199 of 200 samples were valid for PGR, and 198 of 200 samples were valid for MKi67 using the standard or concentrated lysate preparation protocol. One sample failed to generate results for both ER and PgR IHC. Twelve samples failed to yield HER2 FISH results. The STRAT4 success rate and results concordance with IHC were comparable across study sites. OPA between STRAT4 and IHC was 97% for ESR1, 88.9% for PGR, 93.3% for HER2 (92.4% for IHC and FISH), and 90.7% for MKi67 (excluding IHC 10-20% staining). Areas under the ROC curves were 0.9922 for ESR1, 0.9509 for PGR, 0.9958 for ERBB2, and 0.9395 for MKi67. Conclusion: STRAT4 measurements for ESR1, PGR, ERBB2 and MKi67 mRNA expression are robust and highly concordant with IHC (IHC/FISH for HER2). The technical portion of the assay is easily performed in < 2 hrs including hands-on time using standard FFPE tissue sections. Xpert STRAT4 offers local pathology labs an alternative to centralized, subjective IHC/FISH tests that require a higher level of expertise. Further investigations correlating STRAT4 markers directly with clinical outcomes in independent cohorts are in progress. Citation Format: Wu NC, Wong E, Acca B, Birkmeier J, Tran L, Zhao S, Wong W, Chu VC, Ho K, Malek M, Lu C, Ge G, David K, Quigley NB, Beqaj SS, Davenport S, Weidler J, Bates M, Press M. A multicenter clinical study of Xpert® breast cancer STRAT4 demonstrates high concordance with central lab ER, PgR, HER2, and Ki67 IHC and HER2 FISH tests in FFPE breast tumor tissues [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-03-03.

Clinical relevance and concordance of HER2 status in local and central testing-an analysis of 1581 HER2-positive breast carcinomas over 12 years.

Human epidermal growth factor receptor 2 (HER2) is a central predictive biomarker in breast cancer. Inaccurate HER2 results in different laboratories could be as high as 20%. However, this statement is based on data generated more than 13 years ago and may not reflect the standards of modern diagnostic pathology. We compared central and local HER2 testing in a total of 1581 HER2-positive tumors from five clinical trials. We evaluated the clinical relevance for pathological complete response (pCR) and disease-free survival in a subgroup of 677 tumors, which received an anti-HER2 therapy. Over the period of 12 years, the discordance rate for HER2 decreased from 52.4 (GeparTrio) to 8.4% (GeparSepto). Discordance rates were significantly higher in hormone receptor (HR)-positive tumors (26.6%), compared to HR-negative tumors (16.3%, P<0.0001), which could be explained by a different distribution of HER2 mRNA levels in HR-positive and HR-negative tumors. pCR rates were significantly lower in discordant tumors (13.7%) compared to concordant tumors (32.2%, GeparQuattro and GeparQuinto, P<0.001). In survival analysis, tumors with discordant HER2 testing had a reduced overall survival (OS) in the HR-negative group (P=0.019) and a trend for improved OS in the HR-positive group (P=0.125). The performance of local HER2 testing was considerably improved over time and has reached a 92% concordance, which shows that quality initiatives in diagnostic pathology are working. Tumors with discordant HER2 testing had a reduced therapy response and different survival rates.

Abstract P2-05-07: Comparison of the Xpert breast cancer stratifier mRNA assay with central ER, PR, HER2, and Ki67 immunohistochemistry (IHC) for rapid biomarker analysis in developing countries

Abstract Breast cancer care in the developing world is limited by access to quality ER and HER2 IHC diagnostic assays needed to justify hormone and HER2 therapeutics. Shipping pathology specimens to a central testing site often out of country delays therapy and is costly. The Xpert Breast Cancer Stratifier assay makes quantitative measurements of ESR1, PGR, ERBB2, and MKi67 mRNAs from FFPE specimens in &amp;lt;2 hours on an easy-to-use automated diagnostic platform, the GeneXpert (GX). 10,000 GX machines are currently in use in 182 countries offering the possibility of a point-of-care solution. We compared concordance in tumor samples between IHC and mRNA intending to challenge the limits of the GX mRNA assay. 83 breast tumor samples were chosen including those with low cellularity, small volume disease, unusual subtypes, ER- tumors with surrounding benign epithelium, and low level HER2+ tumors. mRNA, IHC and FISH assays were performed. Slides were tested following macrodissection of invasive carcinoma and as non-macrodissected whole sections. GX measurements for Ki67 were compared with mitotic rate as an alternative to Ki67 IHC. Overall percent agreement following macrodissection was 95% for ER, 89% for HER2, 76% for PR, and 80% for Ki67 (&amp;gt;20% positive cut), and using whole section, 99% for ER, 80% for PR, 92% for HER2, and 73% for Ki67. Concordance was 92% for both macrodissection and whole section using mitotic rate to assess proliferation. Ignoring HER2 2+ calls which represented low level amplified tumors by FISH, the concordance rates were 95% for macrodissection and 99% for whole section. Discordance when testing long-term stored 4μm sections was resolved in a number of cases by using a fresh cut from the FFPE block. Half the ER discrepancies were in very small volume tumors ≤25mm2 and 75% were classified as ER-ve by IHC, and positive by Stratifier. 80% of ER IHC- cases were appropriately identified as ER- by the Stratifier in the presence of benign breast epithelium. HER2+ DCIS adjacent to HER2- invasive tumor resulted in a discrepant HER2 mRNA result even with macrodissection. No ER or HER2 discrepancies occurred in low cellularity tumors (≤30% cellularity) nor in lobular and mucinous subtypes. In a study intended to challenge an mRNA breast biomarker assay, concordance between mRNA results and IHC was high for ER and HER2, the two most important prognostic markers needed for therapeutic decision making. Use of whole sections rather than tumor macrodissection did not decrease concordance. Discrepant ER cases were more prevalent when analyzing low volumes of tumor and in this setting were seen in ER IHC- tumors surrounded by ER+ normal epithelium, or with weak IHC expression, highlighting predictable limitations of the assay. Concordance was better between Ki67 mRNA and mitotic rate than with IHC. Re-test data suggested that a fresh cut of the FFPE block yields the best results by GX, perhaps due to mRNA degradation in stored 4μm sections. The Xpert Breast Cancer Stratifier may provide a rapid, cost-effective solution to the problem of obtaining accurate diagnostic results at the point-of-care in low resource settings, and deserves further evaluation in developing countries. Citation Format: Brock JE, Milner DA, Ho K, Natalie W, Victor C, Annaliza R, Teresa B, Kathryn G-F, Edwin LW, Jodi W, Wendy W, Michael B. Comparison of the Xpert breast cancer stratifier mRNA assay with central ER, PR, HER2, and Ki67 immunohistochemistry (IHC) for rapid biomarker analysis in developing countries [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-05-07.

Abstract P6-07-06: Effect of serum biomarkers (activin A, CAIX, HER2, TIMP-1, and uPA) on outcome in HER2+ metastatic breast cancer patients treated in first line with lapatinib or trastuzumab combined with taxane: CCTG MA.31

Abstract Background: In MA.31, the lapatinib-taxane combination led to shorter PFS than trastuzumab-taxane in HER2+ metastatic breast cancer. We investigated the prognostic and predictive effects of pretreatment serum biomarkers. Methods: MA.31 accrued 652 patients; 537 (82%) were centrally-confirmed HER2+. Biomarkers were categorized for univariate and multivariate predictive investigations with a median cut-point, ULN cut-points (15 ng/ml- HER2; 506 pg/ml- CAIX; 454 pg/ml- TIMP-1; 1940 pg/ml– uPA; 600 pg/ml- activin A), and custom cut-points (30 and 100 ng/ml for HER2). Stratified step-wise forward Cox multivariate analysis used continuous and categorical biomarkers for PFS in the ITT and central HER2+ populations; central HER2+ biomarker results are shown. Results: Serum was banked for 472 (72%) of 652 patients. Higher serum activin A (&amp;gt;median; &amp;gt;ULN; p&amp;lt;0.0001); higher CAIX (&amp;gt;median; &amp;gt;ULN; p=0.02; p=0.001); higher HER2 (&amp;gt;median; &amp;gt;15; &amp;gt;30; or &amp;gt;100 ng/ml; p=0.05-0.002) and higher TIMP-1 (&amp;gt;median; &amp;gt;ULN; p=0.001; p=0.02) had shorter univariate PFS. In multivariate analysis for PFS: higher continuous activin A (HR=6.75 with Box-Cox transformation, P&amp;lt;0.0001) was associated with significantly shorter PFS, along with treatment arm, prior adjuvant anthracyclines, and higher central EGFR status. In multivariate analysis for OS: higher continuous activin A (HR=85.9, with Box-Cox transformation, P&amp;lt;0.0001) was associated with significantly shorter OS, along with treatment arm and higher central EGFR status. The interaction terms of serum biomarkers with treatment were not significant. Elevated serum activin A was also significant at the median cutpoint for PFS (HR 1.79, p=0.0002) and OS (HR 2.39, p=0.006) in multivariate analysis. Conclusions: Higher serum activin A was a significant independent prognostic biomarker of shorter progression-free and overall survival. No serum biomarker was predictive of differential response to lapatinib vs. trastuzumab. Evaluation of activin A and CAIX-targeted therapy in addition to HER2-targeted therapy may be warranted in patients with elevated serum levels of these biomarkers. *AK, MH, DH, &amp; JH contributed equally Grant: PA Breast Cancer Coalition. Citation Format: Kang A, Hupp M, Ho D, Huang J, Leitzel K, Ali S, Shepherd L, Parulekar WR, Ellis CE, Rocco CJ, Zhu L, Virk S, Nomikos D, Aparicio S, Gelmon KA, Truica C, Al-Marrawi Y, Rizvi S, Vasekar M, Nagabhairu V, Polimera H, Marks E, Richardson A, Ali AS, Krecko L, Carney WP, Downs S, Chen BE, Lipton A. Effect of serum biomarkers (activin A, CAIX, HER2, TIMP-1, and uPA) on outcome in HER2+ metastatic breast cancer patients treated in first line with lapatinib or trastuzumab combined with taxane: CCTG MA.31 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-06.

Abstract P1-03-09: Highly reproducible decentralized gene expression analysis of ESR1, PGR, ERBB2 and MKi67 on an automated, standardized molecular diagnostics platform, GeneXpert

Abstract Background: Accurate assessment of ER, PgR, HER2, and Ki67 status using FFPE samples from patients with breast cancer is critical for appropriate patient management, yet immunohistochemistry (IHC), the most common method of assessing these markers, suffers from inherent variability due to pre-analytical/analytical factors and subjective interpretation by pathologists. Here we describe the GeneXpert (GX) Breast Cancer Stratifier RUO Assay (BC Strat), a real time quantitative PCR assay (RT-qPCR) kit which exhibits robust, highly reproducible mRNA measures of ESR1, PGR, ERBB2 (HER2) and MKi67. The aims of this study were: 1) To assess the impact of variability contributed by pathologist-to-pathologist differences in the selection of the tumor area to be assayed, inter-laboratory assay performance, and macrodissection (MAC) vs. no macrodissection (nonMAC) on decentralized BC Strat results; and 2) to assess preliminary concordance of BC Strat with central IHC and FISH results. Materials &amp; Methods: The GX BC Strat is a cartridge-based RT-qPCR assay performed on the GeneXpert® Instrument (Cepheid) that automates RNA purification, RT-qPCR amplification and detection of mRNA of target genes (ESR1, PGR, ERBB2, and MKi67) and a control gene (CYFIP1) after sample preparation. Results are reported as delta cycle threshold (dCt) measurements (CYFIP1 Ct - target gene Ct) in less than 2 hrs. Thirty-two invasive ductal carcinoma FFPE blocks were sourced based on varying levels of ER, PgR, HER2, and Ki67 expression and % tumor cell content/tumor area. Adjacent sections from each block were prepared as slides and sent to 3 external GX testing sites and a reference lab. Each site used its own pathologists/technicians to determine the % tumor cell content/tumor area, perform MAC or nonMAC, prepare lysates, and perform GX testing. Reference IHC/FISH was performed by Geneuity/MPLN (Maryville, TN, USA). Site-to-site concordance in GX results for MAC or nonMAC samples using pre-defined assay cutoffs per marker were analyzed, as were % tumor cell content/tumor area assessments between pathologists. Results: BC Strat testing of 32 FFPE breast cancer samples with MAC demonstrated excellent GX site-to-site concordance in positive/negative status calls for ESR1 (100%), PGR (100%), ERBB2 (97%), and MKi67 (97%). In most cases, MAC vs. nonMAC had minimal impact on final positive/negative calls for GX, resulting in high overall concordance for MAC vs. nonMAC for ESR1 (91%), PGR (99%), ERBB2 (99%), and MKi67 (95%). The assay also demonstrated a strong overall concordance with IHC for ESR1 (97%), PGR (81%), ERBB2 (98%, IHC/FISH), and MKi67 (89%). Conclusion: Decentralized performance of the GX BC Strat Assay is feasible and minimally affected by differences in tumor area selection and MAC techniques across tumors with a range of sizes, invasive tumor cell contents, and expression levels of ER, PgR, HER2, and Ki67. GX BC Strat dCt results across sites are highly reproducible and show good concordance of results with central lab IHC and HER2 FISH results. These results suggest standardized, decentralized testing of ESR1, PGR, ERBB2 and MKi67 by the GX BC Strat in local pathology labs is feasible. Citation Format: Wong W, Ho KE, Wu N, Chu VC, Lalli P, Longshore JW, Klein J, Stonecypher M, Lykke C, Sherwood T, Davenport S, Weidler J, Bates M, Press MF. Highly reproducible decentralized gene expression analysis of ESR1, PGR, ERBB2 and MKi67 on an automated, standardized molecular diagnostics platform, GeneXpert [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-03-09.

Central validation of HER2 status to determine heterogeneity of marker expression in HER2-positive gastric cancer (GC).

12 Background: 10-20% of GC overexpress HER2, a membrane-bound receptor tyrosine kinase (RTK) which belongs to the epidermal growth factor receptor (EGFR) family. Drugs directed against HER2 have shown mixed success in the treatment of advanced GC. While trastuzumab, a monoclonal antibody addressing HER2 has been approved for 1st-line treatment of stage IV HER2+ GC, trastuzumab-emtansine failed to improve outcomes in 2nd-line and lapatinib, a small molecular RTK inhibitor of HER2 and EGFR was not effective in 1st- and 2nd-line. Until now, primary and secondary resistance against HER2-directed treatment of GC is not well understood. The VARIANZ study aims to assess mechanisms influencing efficacy of trastuzumab in HER2+ GC. Methods: In this multicenter study, patients who receive medical treatment for advanced GC are recruited in 31 sites. The HER2 status is verified centrally by two dedicated GI pathologists using immunohistochemistry (IHC, DCS, HI608C0I) and chromogenic-in-situ hybridization (CISH, Zytomed Systems, C-3022-40). Results: From May 2014 to August 2016, we have enrolled 316 patients in this ongoing project (72% male, median age 64 years). At present, 281 samples were fully characterized for the HER2 status. According to criteria from the Trastuzumab for Gastric Cancer (ToGA) study, 53 of 281 samples were characterized HER2+ by central testing. In 38 samples that were diagnosed as HER2+ by local pathologists the HER2 status could not be verified centrally. 7 HER2- probes in local testing were characterized as HER2+ by central testing. The overall deviation rate between local and central testing is 27%. HER2 gene amplification in HER2+ tumors with deviating local report (mean HER2/CEP17: 2.8 ± 0.9, range between 1.9 and 4.5) is lower compared to HER2+ tumors and confirmed local report (mean HER2/CEP17: 5.5 ± 2.6; range between 2.2 and 11.0; p = 0.014). Conclusions: HER2-expression in GC is heterogeneous and still not easy to assess. Variability between local and central HER2 assessment is significant. Robust biomarkers predicting response or resistance to HER2 and other target therapies are needed. Clinical trial information: NCT02305043.

How to fill in and interpret an observation chart.

Background: The expression of ER, PR and HER2 in breast cancer is determined by routine pathology assessment to indicate systemic treatment. Here, we investigated the ability of a standardized mRNA-based assay to predict ER, PR and HER2 status and treatment response. Methods: ESR1, PGR and ERBB2 expression was analyzed from the standardized nCounter-based PAM50 assay in 1,544 FFPE breast tumors obtained from 13 independent studies (NeoEribulin, GEICAM 2012-09, TBCRC023/006, LPT109096, EGF117165, PAMELA, PerELISA, GEICAM 2003-11, VENTANA, IBIMA and HCB). All immunohistochemical and in situ hybridization analyses followed ASCO/CAP criteria and were performed in central labs except for Neoeribulin and VENTANA studies. To explore the best cutoff for each gene, we used Monte Carlo cross validation (repeated 1000 times, 2/3 training, 1/3 testing) to achieve the highest kappa values when gene expression was compared to pathology assessment. Receiver operating characteristic analysis and area under the ROC curve (AUC) was used to evaluate the performance of each gene to predict ER, PR and HER2 status. Finally, the association of each gene (using the pre-established cutoffs) with pathological complete response (pCR) was evaluated using univariate logistic regression analyses in 2 neoadjuvant cohorts: 1) A combined HER2+ cohort using 191 tumor samples from PAMELA/PerELISA phase II trials, where patients received neoadjuvant dual HER2 blockade without chemotherapy for 15-18 weeks and 2) a combined cohort of hormone receptor-positive/HER2-negative using 205 tumor samples from IBIMA/HCB consecutive series, where patients received anthracycline/taxane-based chemotherapy. Results: Concordance between ESR1 and ER was 95.3% (95% confidence interval [95CI] 94.0-96.4%; AUC=0.98). ER+ and ER- cases were classified as ESR1- and ESR1+ in 5.5% and 4.8% of the cases, respectively. Concordance between PR and PGR was 85.2% (95CI 83.1-87.1%; AUC=0.95), and between ERBB2 and HER2 was 92.8% (95CI 91.4-94.1%; AUC=0.95). HER2+ and HER2- cases were classified ERBB2- and ERBB2+ in 16.7% and 2.9% of the cases, respectively. In the neoadjuvant HER2+ cohort, the pCR rates were 44.6% in ESR1- and 18.8% in ESR1+ (odds ratio [OR] 3.9; 95CI 1.9-8.4; p Conclusion: ESR1, PGR and ERBB2 standardized mRNA levels show high concordance with ER, PR and HER2, and might provide a more objective and quantitative prediction of response to chemotherapy and anti-HER2 therapy. The level of concordance between GE and pathology-based assessments is similar to 2 FDA 510(k) cleared pathology-based ER assays (e.g. SP1 versus 6F11 antibody clones), local versus central HER2 testing, and ER or PR status when determined by the ligand-binding assay versus immunohistochemistry. Citation Format: Pascual T, Tsai YS, Martin M, Lluch A, Cortes J, Llombart A, Conte P, Guarneri V, Rimawi MF, Alba E, Ruiz-Borrego M, Rojo F, de la Haba J, Schiff R, Adamo B, Vidal M, Pare L, Chic N, Munoz M, Galvan P, Gonzalez-Farre B, Brauer HA, Sullivan A, Nuciforo P, Parker JS, Prat A. Standardized nCounter-based determination of estrogen receptor (ER), progesterone receptor (PR) and HER2 receptor status in breast cancer [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 P2-08-06.

The evolution of high complexity companion testing for targeted and immuno-oncology.

The evolution of high complexity companion testing for targeted and immuno-oncology.

Abstract S5-02: Neratinib after trastuzumab-based adjuvant therapy in early-stage HER2+ breast cancer: 3-year analysis from a phase 3 randomized, placebo-controlled, double-blind trial (ExteNET)

Abstract Background: Neratinib (Puma Biotechnology Inc), an irreversible pan-HER tyrosine kinase inhibitor, was evaluated in a phase 3 randomized, placebo-controlled, double-blind trial (ExteNET) to assess for improved invasive DFS (iDFS) in women with early-stage HER2+ breast cancer (BC) after trastuzumab-based therapy (clinicaltrials.gov NCT00878709). Primary efficacy analysis at 2-yrs demonstrated iDFS of 93.9% and 91.6% neratinib and placebo, respectively (HR 0.67, 95% CI 0.50–0.91; P=0.009). Greater benefit was observed in hormone-receptor (HR)-positive and centrally- confirmed HER2 subsets – HR 0.51, p=0.001 and HR 0.51 p=0.002, respectively [Chan et al. ASCO 2015]. We report an exploratory analysis of efficacy after 3-yrs of follow-up. Methods: Women with HER2+ BC were randomly assigned to oral neratinib 240 mg/day or matching placebo for 1 year, stratified by nodal status, HR-status and prior trastuzumab regimen. The study was initiated in April 2009 with recruitment ceased in October 2011, limiting follow-up to 2 years. In January 2014, follow-up was restored to 5 years. During year 1, physical examinations were performed at 3-monthly intervals, at 4-monthly intervals in year 2 and then 6-monthly to 5-years; with mammography performed annually. Patients were asked to re-consent following ethics approval of the January 2014 protocol amendment. During years 3 to 5, disease-free survival (DFS) and survival events were identified from medical records. A descriptive analysis of iDFS, to investigate the durability of neratinib effect was performed after 3-yrs of follow-up. Data for overall survival remains blinded until 248 events have occurred. Hazard ratios (HR) with 95% confidence intervals (CI) were estimated using stratified Cox proportional-hazards models. Results: The intention-to-treat population included 2840 patients (neratinib, N 1420; placebo, P=1420). Central HER2 testing has now been performed in 2041 pts (72%) - HER2-positive 1709 (84%) and HER2-negative 332 (16%). Pts were randomized within 12-months of completion of prior adjuvant trastuzumab in 2297 (81%) pts. Three-yr iDFS for ITT population demonstrated HR 0.74, [95% CI 0.56-0.96]. In the ITT pts who were randomized within 1-yr of trastuzumab completion, iDFS HR 0.72 [0.54-0.95]. For pts who had centrally-confirmed (cc) HER2-positive disease , iDFS HR 0.70 [0.50-0.98]. Pts with HR-positive disease had 3-yr iDFS HR 0.57 [0.39-0.82] and HR-negative pts HR 0.96 [0.67-1.45]. Overall survival data are not yet mature. Conclusions: Exploratory analysis at 3-years supports the significant benefit seen in the primary analysis of 12-months of neratinib following adjuvant trastuzumab-based therapy. Patients who consistently derived the greatest benefit were 1) patients who received neratinib within 12 months of completing adjuvant trastuzumab; 2) centrally confirmed HER2 positive patients and 3) HR positive patients. Citation Format: Chan A, Delaloge S, Holmes FA, Moy B, Iwata H, Harker G, Masuda N, Neskovic Konstantinovic ZB, Petrakova K, Guerrero Zotano AL, Iannotti N, Rodriguez GI, Tassone P, von Minckwitz G, Ejlertsen B, Chia SKL, Mansi J, Barrios CH, Buyse M, Wong A, Bryce R, Ye Y, Martin M. Neratinib after trastuzumab-based adjuvant therapy in early-stage HER2+ breast cancer: 3-year analysis from a phase 3 randomized, placebo-controlled, double-blind trial (ExteNET). [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 S5-02.

58P - Concordance of local and central HER2 status in 1597 patients participating in German neoadjuvant breast cancer studies

58P - Concordance of local and central HER2 status in 1597 patients participating in German neoadjuvant breast cancer studies

Abstract PD5-7: PTEN and PIK3CA but not p4EBP1 are associated with low rates of pathological complete response (pCR) to trastuzumab based chemotherapy in primary HER2-overexpressing breast cancer

Abstract Background: Phosphatidylinositol 3-kinase (PI3K)/AKT pathway and phosphatase and tensin homolog deleted on chromosome 10 (PTEN) aberrations are common in breast cancer (BC). PIK3CA mutation is associated with lower pCR rates especially in patients treated with double anti-HER2 therapy (Loibl et al. JCO accepted). Another mechanism of resistance for trastuzumab-based treatment could be loss of PTEN, resulting also in downstream activation of the pathway. We investigated the correlation between PIK3CA, PTEN, p4EBP1 (phosphorylated E4 binding protein 1) and pCR in pts receiving neoadjuvant therapy. Methods: In addition to PIK3CA we retrospectively evaluated PTEN and p4EBP1 by immunohistochemistry in HER2+ patients who received EC followed by docetaxel within the G4 study (Untch et al. 2011). The G4 study demonstrated a higher pCR rate by adding trastuzumab to chemotherapy. HER2 and hormone receptors (HR) were centrally assessed. PTEN was assessed using the automated quantitative immunofluorescence analysis (Aqua) using an antibody (Cell Signalling Technology). p4EBP1 assessed by immunohistochemistry with an immunreactive score ranging from 0-12. PTEN was categorized by an optimized cut-off determined by the cut-off finder software (http://molpath.charite.de/cutoff/) and p4EBP1 was measured as a continuous variable and correlated with PIK3CA genotype and pCR (ypT0, ypN0). Central HER2+ve cases with a tumor cell content of ≥20% were selected (n=181). Results: Median age was 48years (22-77); HR+ve 51%; Grade 3, 47.4%; pCR rate 32%. p4EBP1 analysis was available from 137 and PTEN from 108 patients. PIK3CA genotype was available in 83 of these patients. 58 pts had PIK3CA and PTEN assessable, 14/58 had a PIK3CA mutation (mut) (25%). Overall, pCR rate in PTEN low tumors was 27.6% vs 57.1% in PTEN high (p=0.010). Within the PIK3CA mut cohort 13/14 (92.9%) tumors were PTEN low. Within the PIK3CA wild-type (wt) cohort 30/44 (68.2%) were PTEN low (p=0.066). The tumours were grouped into 4 subsets using PIK3CA (mut vs wt) and PTEN (low vs. high). pCR rate was 57.1% (8/14) in PTEN high/PIK3CA wt cohort and decreased to 15.4% in the PTEN low/PIK3CA mut cohort. The group with either PTEN low and PIK3CA wt or PTEN high and PIK3CA mut had a pCR rate of 32.2% (p=0.015). In multivariable analysis after adjustment for baseline parameters PTEN was an independent predictor for pCR in the complete cohort (OR 12.3 [95% 1.82-82.9] p=0.010) and in PIK3CA wt cohort (OR 10.3 [95% CI 1.31-81.62] p=0.027). Within the HR+ve group PTEN low tumors had a pCR rate of 22.2% vs 61.5% in the PTEN high group (p=0.007). In multivariable analysis PTEN was independently predictive for pCR in the HR+ve group (OR 49.5 [95% 3.4-707] p=0.004). p4EBP1 correlated weakly with PIK3CA (p=0.049) but not with PTEN. There was no association of p4EBP1 with pCR. Conclusion: Low PTEN was significantly associated with lower pCR and added independent predictive information not only in the overall HER2+ve cohort but also in the PIK3CA wt and HR+ve cohort. It will be confirmed in a larger sample size if PTEN assessment adds information to PIK3CA genotype to select patients with low pCR rates after trastuzumab therapy. Citation Format: Sibylle Loibl, Silvia Darb-Esfahani, Alexander Klimowicz, Gunter von Minckwitz, Bianca Lederer, Jens Huober, Arndt Hartmann, Holger Eidtmann, Berit Maria Pfitzner, Peter A Fasching, Katharina Tiemann, Christian Jackisch, Keyur Mehta, Michael Untch, Carsten Denkert. PTEN and PIK3CA but not p4EBP1 are associated with low rates of pathological complete response (pCR) to trastuzumab based chemotherapy in primary HER2-overexpressing breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr PD5-7.

Abstract P4-11-04: Intrinsic subtypes and BCL2 as predictive and prognostic biomarkers in the TACT2 trial (CRUK/05/019)

Abstract Introduction: TACT2, a multicentre randomized phase III trial in patients with node +ve or high risk node-ve invasive EBC with E-CMF as control tested two hypotheses in a 2x2 factorial design, presented results showing: i) no evidence of benefit from accelerated 2-weekly epirubicin (aE) compared to standard 3-weekly epirubicin (E) (Cameron 2012); and ii) capecitabine (X) gives equivalent efficacy but preferential side-effect profile to CMF (Canney 2014). Here we present prognostic and potential predictive value of translational biomarkers. We address two main hypotheses: i) aE is less effective in patients with luminal A than patients with other subtypes (Coates 2012) and ii) BCL2 is an independent prognosis marker (Callagy 2006). We also explore the relationship between CK5/6, EGFR and BCL2 and residual risk following chemotherapy. Methods: Tumour samples were collected prospectively from 3803 patients (86.6% of the 4391 TACT2 patients and 94.5% of those consenting). Tissue microarrays were constructed as per published guidelines and central ER, PgR, HER2, Ki67, CK5/6, EGFR and BCL2 staining performed and quantified by imaged analysis for ER, PgR, HER2, Ki67 and BCL2. EGFR and CK5/6 were dichotomised by light microscopy evaluation of cores. 94.5-97.6% of cases were stained and successfully analysed for individual biomarkers by IHC. Patients were categorised into 4 BC subtypes by central ER/PgR/HER2/Ki67 (Luminal A, B, HER2+ve, Triple-ve (TN)) with TN further divided into basal-like (CK5/6 or EGFR +ve) and 5-marker-ve (both CK5/6 and EGFR -ve). Log-rank tests assessed prognostic effect of each marker individually and cancer subtypes on time to tumour recurrence (TTR). Cox-regression models tested independent prognostic value of BCL2 in the presence of tumour size, grade, nodal status, and biological subtype. Results: No evidence of a difference in the efficacy of aE compared with E between the 4 patient subtypes was observed (Luminal A: n=608, HR (for aE compared with E) 0.80 (95% CI 0.47-1.38); Luminal B: n=1804, HR=0.97 (95% CI 0.76-1.23); HER2+ve: n=219, HR=1.03 (95% CI 0.53-1.99); TN: n=638, HR=1.00 (95% CI 0.72-1.39); Test for heterogeneity p=0.84). When subdividing the TN group into basal-like and 5-marker-ve, HRs were 0.66 (95% CI 0.45-0.98) and HR 1.56 (95% CI 0.82-2.96) respectively, post-hoc analysis for heterogeneity between these 2 subtypes p=0.025. No differential effect between subtypes was observed for the comparison of CMF and X. 82/434 (18.9%) patients with low BCL2 expression (≤10%) had a TTR event compared with 444/3158 (14.1%) with high BCL2 (&amp;gt;10%), univariate HR 1.39, 95% CI 1.10-1.76, p=0.006. However this difference was no longer seen after adjustment for clinical factors and biological subtype (HR 1.17 95% CI 0.90-1.52, p=0.25). Conclusion: We found no statistical evidence that luminal A cancers are associated with reduced benefit from aE vs E. A hypothesis generating observation that benefit from aE vs E might be different between basal-like and 5-marker-ve cancers should be interpreted with caution due to the small numbers of cases and the retrospective nature of the analysis. In this study BCL2 did not provide independent prognostic information when corrected for conventional histopathological features. Citation Format: James Morden, Judith Bliss, Jane Bayani, Robert Laing, Rajiv Agrawal, Jeremy Thomas, Andrew Goodman, Vivienne Loo, Peter Clark, Peter Canney, Peter Barrett-Lee, John Bartlett, David Cameron. Intrinsic subtypes and BCL2 as predictive and prognostic biomarkers in the TACT2 trial (CRUK/05/019) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-04.

Abstract P4-11-03: Androgen receptor expression is an independent marker of lower residual risk in the TACT2 trial (CRUK/05/019)

Abstract Introduction: TACT2 (CRUK/05/019), a multicentre randomized phase III trial in patients with node +ve or high risk node -ve invasive EBC with E-CMF as control tested two hypotheses in a 2x2 factorial design, with previously presented results showing: i) no evidence of a benefit from accelerated 2-weekly epirubicin (aE) compared to standard 3-weekly epirubicin (E) (Cameron 2012); and ii) capecitabine (X) gives equivalent efficacy but preferential side-effect profile to CMF (Canney 2014). Studies suggest that Androgen receptor (AR) expression may be associated with improved outcome in early breast cancer. We performed an analysis of AR expression in TACT2 patients to test the hypothesis that AR would represent an independent predictor of residual risk following adjuvant therapy. Methods: Tumour samples were collected prospectively from 3803/4391 TACT2 patients, Tissue micro-arrays were constructed as per published guidelines and central AR, ER, PgR, HER2, Ki67, CK5/6, EGFr and BCL2 staining performed and quantified by imaged analysis for ER, PgR, HER2, Ki67 and BCL2. EGFr and CK5/6 were dichotomised by light microscopy evaluation of cores. After planned biomarker analyses had been performed, a subset of TACT2 cases for whom remaining tissue was available were analysed for AR expression. Data for 1878 cases was available for this analysis (49% of those consenting to tissue collection). AR expression was dichotomised as AR-ve (&amp;lt;10%) vs AR+ve (&amp;gt;10%). Log-rank tests were used to explore the prognostic value of AR on time to recurrence (TTR). Cox-regression models were used to test the independent prognostic value of AR in the presence of tumour size, grade, nodal status, and biological subtypes. Marker by treatment interaction terms were included in models to test the predictive value of AR for both randomised treatment comparisons. Results: 1398/1878 (74%) patients were classed as AR+ve and 480/1878 (26%) as AR-ve. The proportion of AR+ve patients differed significantly between biological subtypes (Luminal A 269/293 (92%); Luminal B 784/928 (84%); HER+ve 86/111 (77%); Basal-like 37/216 (17%); 5-marker-ve 37/93 (40%); χ2 p&amp;lt;0.001) 117/480 (24%) AR-ve patients had a TTR event compared with 183/1398 (13%) AR+ve patients (HR for AR-ve compared to AR+ve =2.05 95%CI 1.63-2.59; p&amp;lt;0.001). AR expression remained independently prognostic following adjustment for nodal status, grade, tumour size and biological subtype (HR1.68, 95%CI 1.26-2.25; p&amp;lt;0.001). Exploratory analysis suggested the prognostic impact of AR might be predominantly in luminal B and 5-marker-ve breast cancers; however the interaction between subtype and AR was not statistically significant. No differential treatment effect between AR subtypes was observed for either randomised treatment comparison (E/aE or CMF/X). Conclusion: AR is an independent prognostic marker for residual risk following chemotherapy in this large study which included patients with both luminal and non-luminal cancers. AR expression patterns differ between molecular subtypes of early breast cancer, with evidence suggesting the impact of AR on residual risk may also differ between subtypes. AR is currently being explored as another potential target for therapy so these data could have future clinical relevance. Citation Format: Jane Bayani, James Morden, Sunil Skaria, Peter Bliss, Robert Grieve, Adrian Harnett, Chris Bradley, Diana Ritchie, Peter Barrett-Lee, Peter Canney, David Cameron, Judith Bliss, John Bartlett. Androgen receptor expression is an independent marker of lower residual risk in the TACT2 trial (CRUK/05/019) [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-11-03.

Quantitative measurement of HER2 expression in breast cancers: comparison with 'real-world' routine HER2 testing in a multicenter Collaborative Biomarker Study and correlation with overall survival.

IntroductionAccurate assessment of HER2 status is critical in determining appropriate therapy for breast cancer patients but the best HER2 testing methodology has yet to be defined. In this study, we compared quantitative HER2 expression by the HERmark™ Breast Cancer Assay (HERmark) with routine HER2 testing by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and correlated HER2 results with overall survival (OS) of breast cancer patients in a multicenter Collaborative Biomarker Study (CBS).MethodsTwo hundred and thirty-two formalin-fixed, paraffin-embedded breast cancer tissues and local laboratory HER2 testing results were provided by 11 CBS sites. HERmark assay and central laboratory HER2 IHC retesting were retrospectively performed in a blinded fashion. HER2 results by all testing methods were obtained in 192 cases.ResultsHERmark yielded a continuum of total HER2 expression (H2T) ranging from 0.3 to 403 RF/mm2 (approximately 3 logs). The distribution of H2T levels correlated significantly (P <0.0001) with all routine HER2 testing results. The concordance of positive and negative values (equivocal cases excluded) between HERmark and routine HER2 testing was 84% for local IHC, 96% for central IHC, 85% for local FISH, and 84% for local HER2 status. OS analysis revealed a significant correlation of shorter OS with HER2 positivity by local IHC (HR = 2.6, P = 0.016), central IHC (HR = 3.2, P = 0.015), and HERmark (HR = 5.1, P <0.0001) in this cohort of patients most of whom received no HER2-targeted therapy. The OS curve of discordant low (HER2 positive but H2T low, 10% of all cases) was aligned with concordant negative (HER2 negative and H2T low, HR = 1.9, P = 0.444), but showed a significantly longer OS than concordant positive (HER2 positive and H2T high, HR = 0.31, P = 0.024). Conversely, the OS curve of discordant high (HER2 negative but H2T high, 9% of all cases) was aligned with concordant positive (HR = 0.41, P = 0.105), but showed a significantly shorter OS than concordant negative (HR = 41, P <0.0001).ConclusionsQuantitative HER2 measurement by HERmark is highly sensitive, accurately quantifies HER2 protein expression and correlates well with routine HER2 testing. When HERmark and local HER2 results were discordant, HERmark more accurately predicted overall survival.

P032 Central lab HER2 testing by RT-PCR, IHC and FISH in locally HER2-Neg, ER+ IBC with in situ carcinoma

P032 Central lab HER2 testing by RT-PCR, IHC and FISH in locally HER2-Neg, ER+ IBC with in situ carcinoma

HER2 expression status in diverse cancers: review of results from 37,992 patients.

Human epidermal growth factor receptor 2 (HER2) amplification/overexpression is an effective therapeutic target in breast and gastric cancer. Although HER2 positivity has been reported in other malignancies, previous studies generally focused on one cancer type, making it challenging to compare HER2 positivity across studies/malignancies. Herein, we examined 37,992 patient samples for HER2 expression (+/− amplification) in a single laboratory. All 37,992 patients were tested by immunohistochemistry (IHC); 21,642 of them were also examined for HER2 amplification with either fluorescent in situ hybridization (FISH) (11,670 patients) or chromogenic in situ hybridization (CISH) (9,972 patients); 18,262 patients had tumors other than breast or gastric cancer. All tissues were analyzed in a Clinical Laboratory Improvement Amendments (CLIA) laboratory (Caris Life Sciences) at the request of referring physicians. HER2 protein overexpression was found in 2.7 % of samples. Over-expressed HER2 was detected predominantly in malignancies of epithelial origin; for cancers derived from mesenchyme, neuroendocrine tissue, central nervous system, and kidney, HER2 expression and amplification were remarkably rare or non-existent. Bladder carcinomas, gallbladder, extrahepatic cholangiocarcinomas, cervical, uterine, and testicular cancers showed HER2 positivity rates of 12.4, 9.8, 6.3, 3.9, 3.0, and 2.4 %, respectively. HER2 overexpression and/or amplification is frequently found across tumor types. These observations may have significant therapeutic implications in cancers not traditionally thought to benefit from anti-HER2 therapies.Electronic supplementary materialThe online version of this article (doi:10.1007/s10555-015-9552-6) contains supplementary material, which is available to authorized users.

Abstract 2840: Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology

Abstract With the advent of multiple HER-2 targeting agents, the need for a diagnostic test that accurately predicts the levels of HER2 in newly diagnosed breast cancer patients has never been greater. The goal of this study was to develop an immunofluorescence based assay to objectively and reproducibly quantify HER-2 protein level in Formalin-Fixed Paraffin-Embedded (FFPE) specimens using AQUA Technology and to provide a binary readout, thus eliminating reflex testing and enabling quick treatment decisions. Methods: Three well known HER-2 antibody clones, A0485, CB11, and SP3 were evaluated across a three-log dilution series under four different antigen retrieval conditions on a tissue microarray (TMA) containing 80 breast cancer cases with known central HER2 immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) amplification status. The top six immunofluorescence assay combinations out of 108 conditions were quantified by AQUA scoring based on the their ability to accurately separate negative and positive cases using receiver operating characteristic (ROC) analysis. These top six assay conditions were then assessed on an independent TMA containing 94 breast cancer cases and their AQUA scores were compared to HER2 protein levels determined by reverse phase protein microarray (RPMA®), HER2 mRNA levels assessed by RNAscope® as well as to the current “gold standard” IHC and FISH amplification. Results: Top three conditions clearly segregating HER2 positive and HER2 negative breast cancer cases were selected and analyzed on an independent TMA. Conditions associated with both SP3 and A0485 antibodies demonstrated high correlations with both HER2 protein levels determined by RPMA analysis and HER2 mRNA levels assessed by RNAscope (spearman's rho &amp;gt;0.85). Conclusion: Assessment of HER2 status using AQUA Technology was confirmed using two expression platforms, including both protein (RPMA) and RNA (RNAscope) supporting the utilization of the AQUA methodology for diagnostic test development. Citation Format: Jennifer Bordeaux, Krupa Chandrasekaran, Sue Beruti, Mike Nerenberg, Corinne Ramos, David Rimm, Jelveh Lameh, Naveen Dakappagari. Evaluation of HER-2 RNA and protein levels in a large cohort of breast cancer specimens to support development of a diagnostic immunofluorescence assay quantified by AQUA® Technology. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2840. doi:10.1158/1538-7445.AM2014-2840

Preliminary HER2 expression data from NeuACT, the phase II randomized, open-label trial of DN24-02 in patients (pts) with surgically resected HER2+ urothelial cancer (UC) at high risk for recurrence.

4527^ Background: HER2 overexpression may be a prognostic factor for poor outcomes in pts with high-risk UC. Publications report a wide variability of HER2 expression in UC, with ≥2+ HER2 expression by immunohistochemistry (IHC) reported in &lt;10% to &gt;50% of cases. DN24-02 is an investigational autologous immunotherapy targeting HER2, based on the same manufacturing platform used for sipuleucel-T. NeuACT (N10-1; NCT01353222) is designed to evaluate whether DN24-02 can prolong survival when given as adjuvant therapy following surgical resection in pts with high risk HER2-expressing UC (Bajorin, et al. ASCO 2012). Here we report preliminary data for HER2 expression on primary tumor and positive lymph node samples, and covariate analyses. Methods: Trial eligibility criteria include surgical resection of a primary UC, with either ≥pT2 or pN+ staging, and HER2 expression ≥1+ IHC. Surgical specimens are screened for HER2 expression by central pathology laboratory review and HER2 positivity is scored using the Dako HercepTest system. Results: As of December 2012, tumor specimens from 114 pts have been screened. Of these pts, 84 (74%; 95% CI: 65–82%) had a HER2 expression score ≥1+ in the primary tumor, with 36 (32%) having a 2+ score and 5 (4%) having a 3+ score. Thirty-eight pts also had HER2 expression levels evaluated in lymph node samples. Of these 38 pts, 35 (92%; 95% CI: 79–98%) had a HER2 expression score ≥1+ in the lymph nodes, with 17 (45%) having a 2+ score and 4 (11%) having a 3+ score. Gender, primary tumor site, nodal stage and prior neoadjuvant chemotherapy were not significantly correlated with HER2 expression (p≥0.10). Conclusions: To date, high frequencies (≥74%) of HER2 expression ≥1+ in primary tumor and lymph node samples of UC pts have been observed. No baseline variables, including prior neoadjuvant chemotherapy, appear to affect the rate of HER2 expression. Although preliminary, these data are consistent with prior studies that have noted a higher incidence of HER2 expression in UC lymph node tumor vs. primary tumor and suggest that HER2 protein expression is common in high-risk UC. Clinical trial information: NCT01353222.

HER2 expression in patients (pts) with surgically resected urothelial cancer at high risk of recurrence screened for the phase II randomized, open-label trial of DN24-02, an autologous cellular immunotherapy targeting HER2.

292 Background: HER2 overexpression has been suggested as a poor prognostic factor in pts with high-risk urothelial carcinoma (UC). Published data note a wide range of HER2 overexpression in UC, with some reports showing &lt;10% of cases and others exhibiting &gt;50% of cases with ≥2+ HER2 expression by immunohistochemistry (IHC). NeuACT (N10-1; NCT01353222) is a phase 2 trial to determine whether DN24-02, an autologous cellular immunotherapy targeting HER2 based on the same platform used for sipuleucel-T, given as adjuvant therapy following surgical resection can prolong survival (Bajorin, et al. ASCO 2012). Here we report preliminary data for HER2 expression on primary tumor and positive lymph node samples from this study. Methods: Trial eligibility criteria include surgical resection of a primary UC, with either ≥pT2 or pN+ staging, and HER2 expression ≥1+ IHC. Surgical specimens are screened for HER2 expression by central pathology laboratory review and HER2 positivity is scored using the Dako HercepTest scoring system. Results: As of August 2012, tumor specimens from 61 pts have been screened for HER2 expression. Of these pts, 49 (80%; 95% CI: 68–89%) had a HER2 expression score of ≥1+ in the primary tumor, with 27 (44%) having ≥2+ score and 3 (5%) having a 3+ score. Twenty-three pts (38%) also had HER2 expression levels evaluated in tumor from involved lymph nodes. A total of 20 (87%; 95% CI: 66–97%) of these pts had a HER2 expression score of ≥1+ in the lymph nodes, with 12 (52%) having a ≥2+ score and 3 (15%) having a 3+ score. Two pts had primary tumor samples that expressed HER2 but were negative for HER2 expression in the lymph nodes submitted for analysis. Conclusions: Although preliminary, a high frequency (≥80%) of HER2 expression score of ≥1+ in primary tumor and lymph node samples was observed. These data highlight the prevalence of HER2 protein expression in high-risk UC. Furthermore, the HER2 expression data are consistent with previously published data in pts with invasive UC. Clinical trial information: NCT01353222.

Abstract P2-05-06: Quantitative measurement of HER2 expression in breast cancers: comparison with “real world” HER2 testing in a multi-center Collaborative Biomarker Study (CBS) and correlation with clinicopathological features

Abstract Background: Accurate determination of HER2 status is critical in determining appropriate therapy for breast cancer patients. The HERmark® assay is a novel method to quantitatively measure HER2 total protein expression (H2T) in breast cancer. In this study, we compared HERmark H2T with central laboratory HER2 retesting and local (site reported) HER2 testing of formalin-fixed, paraffin-embedded (FFPE) breast cancer tissues. The quantitative total HER2 measurements (H2T) by HERmark and results of local HER2 tests were correlated with tumor pathohistological characteristics and overall survival of breast cancer patients. Methods: 232 FFPE breast cancer tissues were provided by 11 CBS study sites for HER2 testing by the HERmark assay and central laboratory IHC re-testing performed in blinded fashion. Local HER2 immunohistochemistry and/or fluorescence in situ hybridization (FISH) results and valid HERmark H2T and central HER2 IHC results were obtained in 192 cases for analysis. Results: H2T showed a significant correlation with central HER2 IHC staining intensity (P &amp;lt; 0.0001). The concordance rates of positive and negative HERmark status (excluding equivocal) with those of local HER2 status determined by the CBS sites, and with those of central HER2 IHC status were 84% (Kappa = 0.68) and 96% (Kappa = 0.91), respectively. Higher H2T levels significantly correlated with higher tumor grade (p = 0.007) and negative ER/PR status (p = 0.002). Twenty-six (14%) cases showed discordant (conversion of negative and positive) results between local HER2 status and HERmark status. Of the discordant cases, HERmark significantly agreed with H-score of central HER2 IHC retesting (p = 0.014), as compared with local HER2 status. The concordant negative group (local HER2 negative/H2T low) demonstrated better overall survival (OS) (HR = 0.198, p = 0.0001), compared to that of concordant positive group (local HER2 positive/H2T high). The concordant negative group also showed better OS than that of discordant local HER2 negative/H2T high group (HR = 0.065, p = 0.0003), but showed no significant difference in OS as compared to that of discordant local HER2 positive/H2T low group (HR = 1.774, p = 0.499).). In 24 cases (13%) considered to be “triple negative” by local HER2, ER and PR testing, HERmark re-classified 4 cases (17%) as HER2 positive. Conclusions: H2T by HERmark yields a continuum of quantitative HER2 protein measurements that shows an excellent correlation with central HER2 IHC retesting and confirms the known correlations between HER2 expression with tumor grade and ER/PR status. OS results of concordant HER2 positive or negative groups (between local HER2 testing and HERmark H2T) confirmed that HER2 positive patients (excluding adjuvant trastuzumab therapy) have worse OS than patients with HER2 negative disease. However, in the HERmark and local HER2 discordant groups, OS appeared to track better with H2T by HERmark and not with the local HER2 status. Novel quantitative HER2 measurements may identify patients with false (+) and (−) HER2 status by local HER2 testing and may provide added clinical value to routine “real world” HER2 testing. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P2-05-06.