HER2 mRNA Levels Research Articles

A Comparison Between Immunohistochemistry and mRNA Expression to Identify Human Epidermal Growth Factor Receptor 2–Low Breast Cancer

Context.— Breast cancers (BCs) with low levels of human epidermal growth factor receptor 2 (HER2) expression (HER2-low) have become a targetable subset because of novel antibody-drug conjugates. HER2 immunohistochemistry (IHC) is the recommended assay for HER2 classification but is associated with limited interobserver agreement concerning HER2-low identification. Objective.— To investigate whether mRNA expression quantified via quantitative reverse transcription–polymerase chain reaction (RT-qPCR) could serve as a valuable complementary and more objective method to identify HER2-low BCs. Design.— We selected all cases from a previously published interobserver study, which included 105 needle biopsies from HER2 nonamplified BC cases. HER2 IHC was evaluated by 16 pathologists. For the current study, mRNA was extracted from microdissected invasive tumor cells. RT-qPCR was performed for quantitative evaluation of HER2, using the cutoff values of the MammaTyper assay. We compared the mRNA expression levels with the IHC scores of the majority agreement (IHC 0, IHC >0, <1+ [ultralow], 1+, 2+) and the following HER2 subcategories: HER2 0/ultralow and HER2-low (IHC 1+ and 2+/fluorescence in situ hybridization negative). Results.— In total, 88 nonamplified HER2 cases could be analyzed. Based on IHC, 17 cases were HER2 0/ultralow and 71 were HER2-low. The mean rank HER2 mRNA level was significantly higher in HER2-low cases than in the HER2 0/ultralow group (P < .001). However, 10 of 17 HER2 0/ultralow cases by IHC (58.8%) were classified as HER2-low by MammaTyper, 2 of 71 cases (2.8%) were HER2-low by IHC and HER2 0/ultralow by MammaTyper, and 2 (2.8%) were HER2-low by IHC and HER2-positive by RP-qPCR. Conclusions.— Our findings indicate a strong agreement between mRNA expression quantified by RT-qPCR and HER2 IHC scores, although there was a substantial proportion of discordant HER2 results between both methods owing to overestimation of HER2 expression by MammaTyper compared to IHC. Future large-scale trials should determine which technique is best associated with clinical outcome.

Quantitative HER2 mRNA assay in breast cancer with HER2 immunohistochemistry 0

Objective: To investigate HER2 mRNA expression in breast cancer with HER2 immunohistochemistry (IHC) 0 and to analyze the feasibility of distinguishing between the tumor with HER2 μltra-low expression and the one without expression of HER2 (no staining by IHC) by HER2 mRNA level preliminarily. Methods: HER2 mRNA was analyzed by reverse transcription digital PCR in 41 cases of formalin-fixed paraffin-embedded surgical tissue samples of invasive breast cancer obtained between January 2020 and March 2023 at Peking Union Medical College Hospital. The cohort included 21 HER2 IHC 1+ and 20 IHC 0 (12 ultra-low and 8 non-expression of HER2). HER2 mRNA expression level was quantitatively evaluated by the FAM (HER2)/VIC (reference gene) ratio. Results: The expression of HER2 mRNA for the cases with 1+, ultra-low, and non-expression of HER2 by IHC was 0.30 to 1.78 (average 0.90, median 0.82), 0.55 to 1.51 (average 0.93, median 0.90) and 0.22 to 0.78 (average 0.41, median 0.36), respectively. For the mean and median HER2 mRNA levels, there was no significant difference between HER2 IHC 1+ and HER2 ultra-low expression diseases (P=0.757). A remarkable difference in HER2 gene expression was found between the tumors with 1+ and non-expression of HER2 by IHC (P=0.002). And, HER2 ultra-low cases contained statistically higher levels of HER2 mRNA compared with non-expression of HER2 subgroup by IHC (P=0.001). Conclusions: Based on HER2 mRNA, HER2 non-expression and HER2 weak expression (including HER2 IHC 1+ and ultra-low) belong to two different types of the tumor and the disease with HER2 IHC 1+ and HER2 ultra-low expression may be the same. It is necessary to further test the performance of HER2 mRNA detection for stratifying the HER2 weak expression subgroup and to determine the threshold.

Abstract PO2-14-05: COMPARISON OF HER2 mRNA PCR LEVELS AND IMMUNOHISTOCHEMISTRY (IHC) IN HORMONE RECEPTOR POSITIVE (HR+)/HER2 NEGATIVE (HER2-) EARLY BREAST CANCER

Abstract BACKGROUND IHC assays were developed to identify HER2-positive breast cancers deemed to be treated with trastuzumab-based therapies. However, these assays lack sensitivity to reliably identify tumors with low levels of HER2 protein on the cell surface. HR+/HER2-low may benefit from Trastuzumab Deruxtecan (TDxd) and therefore accurate quantification of HER2 expression levels becomes a critical issue. As a result, additional technologies are needed to achieve a quantitative and reproducible detection of HER2 in the low range expression compared with the standard IHC testing. METHODS We conducted a retrospective study of all incident stage I-II HR +/HER2- breast cancer patient whose genomic risk was assessed with the Oncotype 21 gene assay from 2009 to 2022 in Hospital Clínico San Carlos (Spain). Local HER2 expression (IHC w/wo cerbB2 FISH) was performed in all patients according to ASCO/CAP guidelines available during the different periods. HER2 mRNA expression levels by RT-PCR were obtained from OncotypeDx patient reports. The primary objective was to determine the distribution of HER2 mRNA levels across the different HER2 expression groups. Other objectives were to compare the clinicopathological characteristics, management and OncotypeDX Recurrence Score (RS) in the different HER2 expression groups. Qualitative variables are presented with their frequency distribution. Quantitative variables are summarized in their mean and standard deviation (SD). Welch’s F-test ANOVA was used to measure association between the two HER2 measuring methods. RESULTS 500 early breast cancer patients with HER2 IHC 0, 1+ and 2+ (FISH negative) and HER2 mRNA expression by RT-PCR tumors were included. Among them, 169 (34%) were IHC 0, 211(42%) were IHC 1+ and 120 (24%) were IHC 2+ (FISH negative). The distribution of clinicopathological characteristics was similar between different IHC HER2 expression groups, and the distribution of chemotherapy treatment was also similar among them (Table 1). All patients had undergone surgery, and most of them had received hormone therapy (487, 97%) and radiotherapy when indicated (375, 77%). The mean OncotypeDx® recurrence score across HER2 IHC 0, 1, 2 showed not significant difference between groups (16,85, 17,36 and 17,89 respectively). The mean OncotypeDx® HER2 gene score expression across the different levels of HER2 (0, 1+, 2+) determined by IHC were 9.07, 9.25 and 9.51, respectively. One-way ANOVA and Games-Howell post-hoc test revealed that differences between all the groups were significant. The ranges of HER2 gene score data for the 3 IHC groups were widely overlapped, with IQR of [8,5-9,5], [8,83-9,7] and [9,07-10] respectively. CONCLUSIONS Our data show statistically significant differences between HER2 mRNA expression levels and IHC groups. This suggests that RT-qPCR could complement the data obtained by IHC. However, the wide range and dispersion of mRNA expression within groups limits its clinical utility. Clinicopathological characteristics and management between different IHC HER2 expression groups. Citation Format: Julia Tejerina-Peces, Marta Amann-Arévalo, Pablo Ballestín, Beatriz González-Diez, Mateo Paz-Cabezas, Alejandro Pascual, Alicia de Luna, Vanesa García-Barberán, Alfonso López de Sá, Jose Angel García-Sáenz, Fernando Moreno. COMPARISON OF HER2 mRNA PCR LEVELS AND IMMUNOHISTOCHEMISTRY (IHC) IN HORMONE RECEPTOR POSITIVE (HR+)/HER2 NEGATIVE (HER2-) EARLY BREAST CANCER [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-14-05.

Molecular Characterization of HER2-Low Invasive Breast Carcinoma by Quantitative RT-PCR Using Oncotype DX Assay.

HER2 immunohistochemistry (IHC) reproducibility is suboptimal for HER-low cases (IHC 1+ or 2+). The Yale cohort included 214 stages I-II estrogen receptor positive breast cancers with IHC scores 0, 1+, and 2+ and routine Oncotype DX Recurrence Score (RS) results. The Exact Sciences (ES) cohort included 9 57 624 patients who had an Oncotype DX RS assay that assigns HER2-negative, equivocal, or positive status based on HER2 mRNA levels. HER2 mRNA levels varied across IHC categories but with increasing medians of 9.10 (n = 89), 9.20 (n = 71), and 9.45 (n = 54) in IHC 0, 1+, and 2+, respectively. 22.4% of HER2-low (1+/2+) cancer had RS > 25. Over 98% of HER-low cancers were HER2-negative by Oncotype DX assignment. Cancers with higher mRNA levels exist within IHC 0 and low categories, most of the HER2-low patients by IHC have low RS indicating no benefit from current adjuvant chemotherapies.

3MO HER2 expression and early response to patritumab deruxtecan (HER3-DXd) in early-stage hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC): A correlative analysis from SOLTI-TOT-HER3 trial

HER3-DXd and trastuzumab deruxtecan (T-DXd) are antibody-drug-conjugates (ADCs) directed against HER3 and HER2, respectively. Both drugs have shown efficacy in patients (pt) with HR+/HER2- BC. Baseline HER3 protein or mRNA levels do not predict efficacy from HER3-DXd. Here, we aimed to evaluate the association of early response to HER3-DXd with HER2 levels. TOT-HER3 (NCT04610528), a window-of-opportunity trial, evaluated a single dose of HER3-DXd in 77 pts with untreated HR+/HER2- early BC in Part A (6.4 mg/kg) and 17 in Part B (5.6 mg/kg). Primary objective was CelTIL score [a surrogate of response that combines tumor cellularity and tumor-infiltrating lymphocytes] variation between baseline and day 21 tumor samples. Here, CelTIL response was defined as an absolute increase of 20 at day 21. HER2 expression was determined by immunohistochemistry (IHC). RNA and DNA were purified from baseline FFPE tumor samples and analyzed using the nCounter and the VHIO-300 NGS platforms, respectively. Logistic regression models and area under the ROC Curve (AUC) estimated the performance of HER2 IHC, mRNA or copy-number (CN) levels with CelTIL response. In TOT-HER3 Part A, HER2 IHC status was 32% HER2 0, 38% HER2 1+ and 30% HER2 2+. Distribution of high CelTIL responders was 40% in HER2 0, 45% in HER2 1+ and 13% in HER2 2+ (p=0.034). High ERBB2 mRNA and RNA-based HER2 amplicon signatures were significantly associated with low CelTIL response (ERBB2: Odds Ratio [OR]=0.34, p<0.001, AUC=0.71; and HER2 amplicon: OR=0.20, p=0.005, AUC=0.74). The association of ERBB2 mRNA with CelTIL response was independent of HER2 IHC expression (p=0.002), PAM50 subtype (p=0.001) or proliferation (p=0.003). High HER2 CN signal was found associated with low CelTIL response (p=0.005). The association of HER2 IHC and ERBB2 mRNA with CelTIL response will be independently validated in TOT-HER3 Part B. Low HER2 IHC, mRNA or CN levels in early-stage HR+/HER2- BC are associated with high response to HER3-DXd. HER3-DXd might be highly active in HR+ BC with very low HER2 levels, thereby offering a new therapeutic paradigm to this subset of patients.

56P Association between HER2 expression level and prognosis using RT-PCR in HER2-low breast cancer

The variation and heterogeneity of conventional immunohistochemistry (IHC) or in situ hybridization (ISH) techniques for measuring HER2 has long been pointed out, and the method of testing for HER2 has been discussed for the selection of appropriate treatment. Therefore, we compared HER2 expression in RT-PCR using OncotypeDX have correlation with conventional IHC, and evaluated HER2 expression in RT-PCR as a prognostic factor in HER2-negative early breast cancer. ER+/HER2- patients who underwent breast cancer surgery between 2004 and 2016 and underwent OncotypeDX testing were included. Data from 632 patients were collected and analyzed by retrospective chart review at 9 centers to determine if there was a correlation between HER2 expression by IHC and RT-PCR. HER2 expression was classified as negative, HER2-low, and positive when HER2 mRNA levels were ≤ 9.1, 9.2-11.4, and11.5≤, respectively, based on the median mRNA level in HER2 negative in IHC. Survival curves were estimated using the Kaplan-Meier method with a log-rank test between the three groups. The median age of the 632 patients was 51 years. Of the 632 patients, 311(49.2%) were pStage I, the 309 (48.9%) were pStage II, and 11(1.7%) were pStage III. 127 (20%) patients received postoperative chemotherapy, and 54 (8.5%) patients had relapse. HER2 IHC rates were HER2 0: 166 (26.3%), 1+: 304 (48.1%), and 2+: 162 (25.6%). There was a weak correlation between the IHC and RT-PCR methods for HER2 expression (Spearman rank correlation P<0.001, r=0.33). The recurrence rates of IHC 0, 1+, and 2+ were 1.8%, 3.9%, 2.6%, respectively. There was no statistically significant difference in DFS between the three groups of HER2 expression by the IHC method (P=0.59). In addition, in the study of HER2 expression and prognosis by RT-PCR, the patients were divided into HER2 negative: 269, HER2-low: 357, and HER2 positive: 1. There was no statistically significant difference between the three groups (P=0.832). The recurrence rates were 3.9%, 4.4%, and 0%, respectively. In the analysis of HER2 expression, there was a weak correlation between IHC and RT-PCR mRNA expression levels; in the HER2-negative population, RT-PCR mRNA expression of HER2 was not a prognostic factor in early breast cancer.

Incidence, Clinicopathologic Features, HER2 Fluorescence In Situ Hybridization Profile, and Oncotype DX Results of Human Epidermal Growth Factor Receptor 2-Low Breast Cancers: Experience From a Single Academic Center

Human epidermal growth factor receptor 2 (HER2)-low breast cancer, defined by an immunohistochemical (IHC) score of 1+ or 2+ with negative in situ hybridization, is emerging as a predictive marker for the use of the antibody-drug conjugate. To understand how this category differs from HER2-zero cases, we investigated clinicopathological characteristics and HER2 fluorescence in situ hybridization results in a large cohort of 1309 continuous HER2-negative invasive breast carcinomas from 2018 to 2021 evaluated by the Food and Drug Administration-approved HER2 IHC test. Additionally, we compared Oncotype DX recurrence scores and HER2 mRNA expression between HER-low and HER2-zero cases in a separate cohort of 438 estrogen receptor-positive (ER+) early-stage breast carcinoma cases from 2014 to 2016. Based on the cohort from 2018 to 2021, the incidence of HER2-low breast cancers was approximately 54%. HER2-low cases had less frequent grade 3 morphology, less frequent triple-negative results, ER and progesterone receptor negativity, and a higher mean HER2 copy number and HER2/CEP17 ratio than HER2-zero cases (P < .0001). Among ER+ cases, HER2-low cases showed significantly less frequent Nottingham grade 3 tumors. In the cohort from 2014 to 2016, HER2-low cases showed significantly higher ER+ percentages, fewer progesterone receptor-negative cases, lower Oncotype DX recurrence scores, and higher HER2 mRNA expression scores than HER2-zero cases. In summary, this is the first study, to our knowledge, using a large cohort of continuous cases evaluated by the Food and Drug Administration-approved HER2 IHC companion diagnostic test for HER2-low expression and HER2 fluorescence in situ hybridization profile in a real-world setting. Although HER2-low cases showed a higher HER2 copy number, ratio, and mRNA level than HER2-zero cases statistically, such small differences are unlikely to be biologically or clinically meaningful. However, our study suggests that HER2-low/ER+ early-stage breast carcinoma may represent a less aggressive group of breast carcinoma, given its association with a lower Nottingham grade and Oncotype DX recurrence score.

Abstract P2-23-14: Molecular characterization of HER2-low invasive breast carcinoma by quantitative RT-PCR using Oncotype DX®

Abstract Background: The DESTINY-Breast04 clinical trial demonstrated the superiority of trastuzumab deruxtecan (T-DXd) versus physician’s choice chemotherapy in HER2-low [immunohistochemistry (IHC) staining score 1+ or 2+ with no gene amplification by in-situ hybridization] metastatic breast cancer. The reproducibility of HER2 IHC category 0 versus 1+ scoring is poor in community practice. 89% of the DESTINY-04 participants were hormone receptor positive (HR+). In HR+ breast cancers, the Oncotype DX® (ODX) assay is widely used to estimate risk of recurrence and guide adjuvant chemotherapy selection. It also provides standardized quantification of HER2 mRNA expression by RT-PCR. Prior studies have demonstrated a high degree of overall concordance between central IHC and RT-PCR using ODX in HER2+ and HER2- cases. Here, we (i) compare HER2 mRNA levels quantified by ODX in HER2 IHC 0, 1+ and 2+ invasive breast carcinomas (IBC), (ii) compare the Recurrence Score® (RS) distribution across these three IHC categories and (iii) describe RS distribution and proliferation score in HER2-low IBC. Methods: 212 patients with HER2 IHC 0, 1+ and 2+ who were negative for HER2 gene amplification by FISH and had RS results were identified in Yale Department of Pathology archives. All US samples submitted for IBC ODX testing between 2005 to 2021 were reviewed in the Exact Sciences database. RS, quantitative HER2 mRNA expression, and proliferation scores were examined. IHC results were not available. Based on quantitative RT-PCR measures of HER2 expression, cases were assigned to: HER2 positive ≥11.5, equivocal ≥10.7 to &amp;lt; 11.5, and negative &amp;lt; 10.7 (1 unit increment is equivalent to approximately 2-fold change). Results: In the Yale cohort, 42%, 33%, and 25% of cases were IHC 0, 1+ and 2+, respectively. There was no difference in age or tumor grade by IHC category. HER2 mRNA levels increased across IHC categories (means 9.05, 9.16, 9.39, respectively), but in group-wise comparisons, only the IHC 0 compared to IHC 2+ reached statistical significance (Mann-Whitney test, p=0.0014). The RS scores were also modestly, but significantly higher in IHC 2+ compared to IHC 0 cases (mean 19.5 vs 14.51, Mann-Whitney p=0.034). Among IHC 0 and 1+ cases, 14% had RS &amp;gt;25, and among IHC 2+ cases, 32% had RS &amp;gt;25. All IHC 0 and 1+ cases were HER2 negative by RT-PCR. Of the HER2 2+ cases, there was one HER2 positive and one HER2 equivocal by RT-PCR. There was substantial variation in HER2 expression by RT-PCR in all IHC groups. In the Exact Sciences cohort, a total of 957,624 samples were analyzed. 0.8% of samples were HER2 positive, 1.2% were equivocal, and 98% were negative by RT-PCR. There was a wide range of RS results. Of the HER2 positive cases, 94.7% had RS &amp;gt;25. Among the HER2 equivocal cases 39.1% had RS &amp;gt;25, and among the HER2 negative cases, 15.5% had RS &amp;gt;25. Conclusions: HER2 IHC 0 and HER2 low IBC have a broad and overlapping range of HER2 expression by RT-PCR. Whether RT-PCR based HER2 expression predicts benefit from T-DXd is yet to be determined. Most HER2 low cases have RS ≤25, indicating no or limited benefit from adjuvant chemotherapy. Further studies are required to determine if patients with HER2 low IBC and RS ≤25, particularly with high anatomical risk, could benefit from adjuvant T-DXd. Table 1. Clinicopathologic and molecular characteristics by HER2 IHC group Table 2. Quantitative gene expression by HER2 IHC group Citation Format: Mariya Rozenblit, Hao-Kuen Lin, Nhu Thuy Can, Cynthia A. Flannery, Jess Hoag, Alekhya Akkunuri, Helen Bailey, Frederick Baehner, Lajos Pusztai. Molecular characterization of HER2-low invasive breast carcinoma by quantitative RT-PCR using Oncotype DX® [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 P2-23-14.

Abstract P5-02-33: Deep learning-based assessment of HER2-low expression on breast cancer H&amp;E digital whole slide images

Abstract Background: Antibody drug conjugates (ADCs) against HER2 have shown meaningful clinical activity in HER2 low breast cancers, defined as 1+ or 2+ staining on immunohistochemistry (IHC) without gene amplification by in situ hybridization (ISH) techniques. Given that these methods were originally developed for an accurate detection of HER2 3+, their sensitivity and robustness for the detection of low and ultra-low levels of HER2 are questionable. We have recently described a deep learning algorithm that can detect signatures of HER2 expression based on training utilizing scanned H&amp;E whole slide images (WSI) of breast cancers for which IHC and mRNA expression levels of HER2 were available. Here, we report the application of our algorithm to two independent breast cancer cohorts. Methods: A model was developed based on recognition of invasive breast cancer in whole slide images of H&amp;E staining, and then trained via computational neural network with multiple instance learning for binary classification of cases as HER2 “negative” and HER2 “expressed” (low). For training, true negatives were defined as having HER2 IHC-0 and mRNA level &amp;lt; 7.6. HER2-low cases were defined as IHC-1+/2+ and mRNA &amp;gt;9. IHC-0 cases with mRNA &amp;gt;7.6 were excluded from the training cohorts. The resulting model (HER2Complete) was able to distinguish HER2-negatives from HER2-low cases with an AUC of 0.91 (+/- 0.08). Here we use Her2Complete to assess HER2 in two additional cohorts that include 901 ER+/HER2 IHC-0 and 52 HER2 IHC 0+ breast cancers from MSK and TCGA cohorts, respectively. For the TCGA cohort, concomitant transcriptomics data (RNASeq) as a reference for HER2 mRNA expression were retrieved and “HER2 expressed” defined as RNASeq expression of HER2 greater than the 90th percentile of the geometric mean of expression of three reference genes not expressed in breast tissues (TTN, MUC13, OR10A6). Values less than this reference cut-off in the TCGA cohort were considered “HER2 not expressed.” Results: Among the 901 IHC-0 test cases from the MSK cohort, the model identified 82 as ‘negative’, whereas 819 were found to have features of HER2 expression (HER2-Low). Of the 82 negative cases in the MSK cohort, all except 13 cases expressed mRNA levels &amp;lt; 9, and 786/819 of the HER2-low cases expressed mRNA levels &amp;gt;8. Of the 52 IHC 0+ cases in the TCGA cohort, 33 also had “HER2 not expressed” by our reference based RNASeq expression cut-off. Our model identified 15 of these 33 as ‘negative’, while 15 of the 19 TCGA cases with IHC 0+ and HER2 ‘expressed’ by our cut-off were identified as ‘HER2-Low’ by our model. Conclusions: AI tools based on the analysis of WSIs of routinely prepared H&amp;E sections may predict HER2 status in breast cancer. This work requires further investigation using treatment response data to demonstrate that cases with morphologic features of low level HER2 expression will respond to ADCs. Citation Format: Gerard Oakley III, Jorge Reis-Filho, David Klimstra, Marc Goldfinger, Yikan Wang, Antonio Marra. Deep learning-based assessment of HER2-low expression on breast cancer H&amp;E digital whole slide images [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 P5-02-33.

HER2 mRNA Levels, Estrogen Receptor Activity and Susceptibility to Trastuzumab in Primary Breast Cancer.

While the results thus far demonstrate the clinical benefit of trastuzumab in breast cancer (BC), some patients do not respond to this drug. HER2 mRNA, alone or combined with other genes/biomarkers, has been proven to be a powerful predictive marker in several studies. Here, we provide evidence of the association between HER2 mRNA levels and the response to anti-HER2 treatment in HER2-positive BC patients treated with adjuvant trastuzumab and show that this association is independent of estrogen receptor (ER) tumor positivity. While HER2 mRNA expression was significantly correlated with HER2 protein levels in ER-negative tumors, no correlation was found in ER-positive tumors, and HER2 protein expression was not associated with relapse risk. Correlation analyses in the ER-positive subset identified ER activity as the pathway inversely associated with HER2 mRNA. Associations between HER2 levels and oncogene addiction, as well as between HER2 activation and trastuzumab sensitivity, were also observed in vitro in HER2-positive BC cell lines. In ER-positive but not ER-negative BC cells, HER2 transcription was increased by reducing ligand-dependent ER activity or inducing ER degradation. Accordingly, HER2 mRNA levels in patients were found to be inversely correlated with blood levels of estradiol, the natural ligand of ER that induces ER activation. Moreover, low estradiol levels were associated with a lower risk of relapse in HER2-positive BC patients treated with adjuvant trastuzumab. Overall, we found that HER2 mRNA levels, but not protein levels, indicate the HER2 dependency of tumor cells and low estrogen-dependent ER activity in HER2-positive tumors.

Can HER2 1+ Breast Cancer Be Considered as HER2-Low Tumor? A Comparison of Clinicopathological Features, Quantitative HER2 mRNA Levels, and Prognosis among HER2-Negative Breast Cancer.

Simple SummaryHER2-low breast cancer is a new entity featuring low HER2 expression (HER2 1+ or HER2 2+/FISH−) and can potentially benefit from novel antibody-drug conjugates. A precise estimation of HER2-low expression is warranted. Here, we found that HER2 1+ and HER2 0 tumors shared similar clinicopathologic features and HER2 mRNA expression, significantly different from HER2 2+/FISH− tumors. A poor concordance rate was found between IHC/FISH and qRT-PCR for the estimation of HER2 expression in HER2-negative tumors. This study suggests that the current definition of HER2-low expression with the lower boundary of HER2 IHC 1+ may be inaccurate. IHC and qRT-PCR were not optimal to quantify HER2-low expression, especially for HER2 1+ patients.Background: Human epidermal growth factor receptor 2 (HER2)-low tumor is a new entity defined as HER2 immunohistochemistry (IHC) 1+ or 2+/fluorescence in situ hybridization (FISH)-negative. We aimed to evaluate whether HER2 mRNA levels tested by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) could better define HER2-low tumors. Patients and methods: Consecutive breast cancer patients with hormonal receptor-positive, HER2-negative diseases, and HER2 mRNA results were included. Clinicopathologic features, HER2 mRNA expression level, and prognosis were compared among HER2 0, 1+ and 2+/FISH− groups. Concordance of the HER2 category between qRT-PCR and IHC/FISH was analyzed for each group. Results: 2296 patients were included: 368 (16.0%) HER2 0, 911 (39.7%) 1+, and 1017 (44.3%) 2+/FISH− tumors. HER2 1+ cases shared similarities with HER2 0 tumors in terms of clinicopathologic features (all p > 0.05), whereas IHC 2+/FISH− cases were less often non-IDC (p = 0.045), node-negative (p = 0.044), and Ki-67 < 14% (p <0.001). The mRNA expression was similar between HER2 0 and 1+ cases (p = 0.063), and both were lower than 2+/FISH− cases (p < 0.001). A poor concordance rate was found between IHC/FISH and qRT-PCR for HER2 0 and HER2-low cases (Cohen’s kappa 0.126, p < 0.001). No survival difference was observed among these groups, whether stratified by HER2 IHC/FISH status or mRNA level (all p > 0.05). Conclusions: HER2 1+ cases had similar clinicopathological features to HER2 0 breast cancers, and both were different from HER2 2+/FISH− cases. HER2 mRNA levels were comparable between HER2 0 and 1+ tumors, and both were significantly lower than IHC 2+/FISH− tumors. Neither IHC nor qRT-PCR may be optimal to quantify HER2-low expression, especially for HER2 1+ patients.

Abstract LB088: Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd) in tamoxifen-resistant ER+ breast cancer cells by reprogramming cell cycle progression

Abstract Background: HER3, a member of the ErbB family of receptor tyrosine kinases that activate multiple oncogenic signaling pathways, is overexpressed in 50-70% of breast cancers (BC). HER3 mRNA levels are higher in ER+ breast tumors than in other molecular subtypes. Inhibition of ER activity using an antagonist increased HER3 protein expression and activation, which was essential for growth and survival of ER+ BC cells resistant to the ER antagonist. Therefore, therapeutically targeting HER3 with HER3-DXd, an antibody-drug conjugate (ADC) composed of a fully human anti-HER3 monoclonal antibody (patritumab) conjugated to a topoisomerase I inhibitor payload (an exatecan derivative) via a tetrapeptide-based cleavable linker, can be an effective treatment for tamoxifen-resistant (TMR) ER+ BC. After first assessing HER3-DXd as a single agent, we sought to identify a synergistic partner to maximize HER3-DXd’s antitumor activity in HER3+/ER+ TMR BC. Methods: Whole-genome high-throughput siRNA screening was performed to identify targets whose inhibition enhances HER3-DXd’s antitumor efficacy. The antitumor effects of HER3-DXd plus the synergistic partners were assessed using a soft agar colony formation assay and a clonogenic assay in TMR HER3+/ER+ MCF7 and T47D BC cells. Treatment effects on cell cycle distribution, apoptosis, and expression of proteins that regulate cell cycle progression were assessed by flow cytometry, annexin V-PE and 7-AAD staining, and Western blotting, respectively. Results: HER3-DXd inhibited the anchorage-independent growth of HER3+/ER+ cells by &amp;gt;50% at 5 nM and their colony formation at 5-25 nM (P&amp;lt;0.05). To maximize HER3-DXd’s antitumor efficacy, we performed high-throughput siRNA screening and identified ATR, CD247, RAB7A, UPK3A, ROCK2, SLC29A1, and WNT7A as potential synergistic targets. Among these targets, inhibiting ATR with siRNA or BAY1895344 showed the most synergistic effect with HER3-DXd in HER3+/ER+ BC cells. In contrast, no synergistic effect was observed with the combination of BAY1895344 plus patritumab or control ADC (IgG-DXd), suggesting its dependence on HER3-DXd-mediated delivery of DXd. The combination of HER3-DXd plus BAY1895344 reprogrammed cell cycle progression from G2/M arrest to G1 arrest by inhibiting both ATR/Chk1/cyclin A2/CDK2 and ATR/Chk1/cyclin E/CDK2 signaling. The combination also reduced expression of H2AX, an ATR substrate that contributes to DNA repair, but increased that of γH2AX, indicating the induction of DNA damage. HER3-DXd and BAY1895344 synergistically inhibited growth of HER3+/ER+ BC cells by inducing apoptosis. Conclusion: The combination of HER3-DXd plus ATR inhibitors has therapeutic potential for overcoming tamoxifen resistance in HER3+/ER+ BC. We are currently validating the synergy in endocrine-resistant ER+ BC xenograft models. Citation Format: Xuemei Xie, Jangsoon Lee, Jon A. Fuson, Huey Liu, Young Jin Gi, Pang-Dian Fan, Kumiko Koyama, Debu Tripathy, Naoto T. Ueno. Targeting ATR enhances the antitumor efficacy of patritumab deruxtecan (HER3-DXd) in tamoxifen-resistant ER+ breast cancer cells by reprogramming cell cycle progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB088.

Abstract 5418: Comprehensive genomic profiling of HER2low breast carcinomas unveils unique genomic features and actionable genetic alterations

Abstract Introduction &amp; Purpose: “HER2low breast cancer” is an operational term identifying breast carcinomas (BCs) displaying a HER2 score 1+ or score 2+ in immunohistochemistry (IHC) and lacking HER2 amplification. This paradigm shift in HER2 assessment in BC stems from substantial antitumor activity of potent antibody-drug conjugates (ADCs) targeting the HER2 receptor on the tumor cell membrane. Whether these tumors constitute a distinct pathological entity is a matter of debate. Experimental procedures: To dissect the biology of HER2low BCs we performed a high throughput mutational analysis coupled with gene expression profiling on 98 HER2low BCs [34 cases score 1+ (1), 15 cases score 2+ HER2 not amplified (2N), 49 cases score 2+ with HER2 copy numbers in the equivocal range (2E)], of which 89 were estrogen receptor (ER)-positive, and 9 ER-negative. DNA and RNA were extracted and subjected to targeted DNA sequencing (Illumina TSO500 panel) and targeted gene expression analysis (NanoString BC360 panel). Mutation rates were compared with in silico data from 1317 samples of the MSK Breast Cancer cohort (PMID: 30205045). Results: The most frequently mutated genes were PIK3CA (33/98, 33%), GATA3 (18/98, 18%), TP53 (17/98, 17%), HER2 (8/98, 8%, private to 2E cases). Tumor mutational burden mean values were significantly higher in score 1+ compared to score 2+ cases (p= 0.04), with a single score 1+ tumor harboring microsatellite instability (MSI). Mutational signatures 1, 5 and 13 (APOBEC) were the most prevalent, with signature 30 being private to HER2 score 1+ cases. Comparative statistics revealed mutational drift of HER2low BCs from both HER2 negative (19 differentially mutated genes) and HER2 positive BCs (11 differentially mutated genes). Potentially actionable genetic alterations (OncoKB classes) accounted for 43 SNVs (predominantly HER2 and PIK3CA mutations) and 17 CNVs (e.g. MET amplification and PTEN homozygous deletions), including a MSI tumor, distributed over 47 independent patients.Subgroup stratifications based on gene expression were observed when considering genes involved in angiogenesis, stromal marker and cytokines signalling. HER2 mRNA levels were significantly higher in 2E versus 2N and in 2E versus 1. Differential gene expression Kegg pathway analysis identified RTK activation restricted to 2E cases. Conclusions: HER2low BCs harbour a different mutational landscape from ER- and grade- matched HER2 positive and HER2 negative BCs. Differences across IHC score classes within HER2low carcinomas were demonstrated, with the 2E category showing distinct features (private HER2 mutations, HER2 mRNA levels significantly higher compared to the other groups, RTK pathway activation). Of note, besides the option of ADCs we demonstrate that up to 48% of cases may be targeted with different approaches exploiting actionable alterations. Citation Format: Enrico Berrino, Laura Annaratone, Ivana Sarotto, Maria Cecilia Zecchillo, Laura Casorzo, Riccardo Ponzone, Filippo Montemurro, Anna Sapino, Caterina Marchio. Comprehensive genomic profiling of HER2low breast carcinomas unveils unique genomic features and actionable genetic alterations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5418.

Abstract 5661: HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers

Abstract HER2 is amplified in about 20% of breast cancers. HER3 is as essential as HER2 for maintaining cell viability in HER2+ breast cancer cells. It is known that inhibition of HER2 tyrosine kinase activity results in upregulation of HER3 transcription and phosphorylation. We sought to identify HER3 binding partners upon pharmacological inhibition of HER2 using neratinib. We immunoprecipitated HER3 using a HER3 antibody from BT474 cells treated ± neratinib. Fmass spectrometry experiments identified non-muscle myosin IIA (NMIIA) increased upon inhibition of HER2 with neratinib and decreased under DMSO control treatment from HER3 immunoprecipitates. To validate the presence of NMIIA, we performed immunoprecipitation experiments in BT474 and MDA-MB-453 cells using a HER3 antibody. Immunoblots showed increased NMIIA levels upon treatment with 200nM neratinib for 24 hours in both cell lines. Myosin heavy chain 9 (MYH9) gene encodes a protein called non-muscle myosin of class II, isoform A (NMIIA). It localizes to actin stress fibers and has been implicated in many cell functions. To confirm the interaction between HER3 and NMIIA we immunoprecipitated NMIIA from BT474 and MDA-MB-453 cells using a NMIIA antibody. Immunoblots indicated that HER3 levels were increased upon inhibition of HER2 with 200 nM neratinib for 24 hours. We next examined overall survival of primary breast cancer patients who have high gene expression for MYH9 from the METABRIC cohort. We observed that patients with high levels of MYH9 have a statistically significant worse overall survival versus patients with low levels of MYH9. Furthermore, we evaluated the overall levels of HER3 and MYH9 mRNA and protein upon treatment with neratinib in BT474 and MDA-MB-453 whole cell lysates. RT-qPCR and immunoblots showed increased HER3 and NM-IIA mRNA and protein levels upon neratinib treatment for 24 hours We examined the affect NMIIA loss has on HER3 signaling. Transduced MDA-MB-453 and BT474 cells with shMYH9 and doxycycline induction demonstrate a reduction in HER3 protein levels compared to cells transduced with a control sequence and doxycycline induction. We observed a concomitant reduction in P-HER3 (Y1289), downstream P-Akt (T308), and P-Erk1/2. In addition, NM-IIA knockdown suppresses cells growth, proliferation, migration, and invasion. In conclusion, there is a bidirectional relationship between NMIIA and HER signaling in HER2+ breast cancer cells. HER2 inhibition increases NMIIA and NMIIA promotes HER3 expression. Loss of NMIIA reduces HER3 protein and concomitant reductions in PI3K/Akt and MAPK signaling. Loss of NMIIA in combination with HER2 inhibition results in reduction in HER2+ breast cancer cell proliferation, growth on matrigel, migration, and invasion. Studies are ongoing to decipher the mechanisms for the bidirectional relationship between NMIIA and HER signaling. Citation Format: Samar M. Alanazi, Rosalin Mishra, Hima Patel, Mary K. Kilroy, Joan T. Garrett. HER2 inhibition increases non-muscle myosin IIa to promote tumorigenesis in HER2+ breast cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5661.

Assessing the use of tumor-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex before autotransplantation

To assess the use of tumor-specific designed ankyrin repeat proteins (DARPins) fused to a domain of Pseudomonas aeruginosa exotoxin A for purging of cancer metastases from the ovarian cortex. Experimental study. University medical center. Human ovarian cortex. Ovarian cortex harboring artificially induced breast cancer metastases was treated with DARPins targeted to epithelial cell adhesion molecule (EpCAM) and human epidermal growth factor receptor 2 (HER2). The presence of any remaining cancer cells after purging was analyzed by (immuno)histochemistry and reverse transcriptase polymerase chain reaction. Effects on the viability of the ovarian cortex were determined by (immuno)histology, a follicular viability assay, and an assay to determine the invitro growth capacity of small follicles. After purging with EpCAM-targeted DARPin, all EpCAM-positive breast cancer cells were eradicated from the ovarian cortex. Although treatment had no effect on the morphology or viability of small follicles, a sharp decrease in oocyte viability during invitro growth was observed, presumably due to low-level expression of EpCAM on oocytes. The HER2-targeted DARPins had no detrimental effects on the morphology, viability, or invitro growth of small follicles. HER2-positive breast cancer foci were fully eliminated from the ovarian cortex, and the reverse transcriptase polymerase chain reaction showed a decrease to basal levels of HER2 mRNA after purging. Purging cancer metastases from ovarian cortex without impairing ovarian tissue integrity is possible by targeting tumor cell surface proteins with exotoxin A-fused DARPins. By adapting the target specificity of the cytotoxic DARPin fusions, it should be possible to eradicate metastases from all types of malignancies.

O-191 Assessing the use of tumour-specific DARPin-toxin fusion proteins for ex vivo purging of cancer metastases from human ovarian cortex tissue fragments before autotransplantation

Abstract Study question Is it possible to eradicate cancer cells from ovarian cortex by using tumour-specific designed ankyrin repeat protein (DARPin)-toxin fusion proteins, without compromising the ovarian tissue? Summary answer Purging ovarian cortex ex vivo from experimentally induced breast cancer tumour foci is possible by tumour-targeted DARPin-toxin fusion proteins trough inhibition of protein synthesis. What is known already Ovarian tissue cryopreservation and autotransplantation is a successful technique for fertility restoration in cancer patients. The procedure is not without risk since malignant cells may still be present in the graft. Procedures to detect cancer cells render the tissue fragment useless for autotransplantation. Strategies to circumvent this problem such as in vitro maturation of follicles or the construction of artificial ovaries are pursued but are still experimental. Alternatively, we have shown ex vivo purging of ovarian cortex is possible by elimination of rhabdomyosarcoma after treatment with verteporfin. This allows treatment of cortex fragments before autotransplantation without compromising ovarian tissue integrity. Study design, size, duration Human ovarian cortex fragments harbouring breast cancer tumour foci were exposed for 24 h to DARPins fused to the translocation and catalytic domain of Pseudomonas aeruginosa exotoxin A (DARPin-toxin fusion proteins) targeting EpCAM or HER2. After treatment with the DARPin-toxin fusion proteins the tissue was cultured for an additional 6 days to allow any remaining tumour cells to form foci. In addition, the functional integrity of the ovarian tissue was analysed after purging. Participants/materials, setting, methods Breast cancer cell lines expressing different levels of EpCAM and HER2 were introduced in human ovarian tissue to form tumour foci. After purging with DARPin-toxin fusion proteins, the presence of any remaining cancer cells in the tissue was analysed with (immuno)histochemistry and RT-qPCR. Possible detrimental effects on the viability of ovarian cortex and follicles were determined by (immuno)histology, a follicular viability assay and an assay to determine the in vitro growth capacity of small follicles. Main results and the role of chance Ovarian cortex harbouring EpCAM-positive breast cancer cells showed a significant decrease in the number of tumour foci after treatment with the EpCAM-targeted DARPin-toxin fusion proteins. Although exposure to the EpCAM-specific DARPin had no effect on morphology or viability of follicles, a decrease in oocyte viability after in vitro growth experiments was observed, presumably due to low level expression of EpCAM on oocytes. In contrast to the EpCAM-specific DARPin-toxin fusion protein, the DARPin-toxin fusion protein targeting HER2 had no detrimental effects on morphology, viability or in vitro growth of follicles while foci of HER2-positive breast cancer cells were severely affected as indicated by the presence of apoptotic bodies, tumour cell remnants and the absence of viable tumour cells. The histological results after purging with the HER2-specific DARPin-toxin fusions proteins were confirmed by RT-qPCR, showing a decrease to basal levels of HER2 mRNA in the ovarian cortex tissue. Limitations, reasons for caution The effect of DARPin-toxin fusion proteins depends heavily on the expression of their target on the cancer cell. The target protein should not be expressed by ovarian cortex as this may lead to tissue damage. The functional integrity of ovarian cortex after the treatment requires further investigation in vivo. Wider implications of the findings Purging metastases from ovarian cortex without harming ovarian tissue is possible by targeting tumour specific surface expressed antigens with DARPin-toxin fusion proteins. Purging ovarian cortex tissue with DARPin-toxin fusion proteins provides a feasible therapeutic strategy to prevent reintroduction of cancer by autotransplantation in case of malignancies expressing tumour-specific surface markers. Trial registration number not applicable

Quantitative assessments and clinical outcomes in HER2 equivocal 2018 ASCO/CAP ISH group 4 breast cancer

We quantified human epidermal growth factor receptor 2 (HER2) RNA and protein expression in 2018 American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) in situ hybridization (ISH) group 4 (HER2/centromeric probe 17 (CEP17) ratio <2.0, average HER2 copy number ≥4.0 and <6.0, and 2013 ASCO/CAP ISH equivocal) breast cancers. Breast cancers in 2018 ASCO/CAP ISH group 4 between 2014 and 2017 were identified from the Yale archives. Sixty-three patients (34 with HER2 immunohistochemistry (IHC) 0/1+ and 29 with HER2 IHC 2+) were included. We compared patient characteristics, systemic treatments, and outcomes. We assessed HER2 by real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and quantitative immunofluorescence (QIF). Among ISH group 4 cancers, higher HER2 mRNA (P < 0.0001) but similar HER2 protein levels were observed in IHC 2+ compared to IHC 0/1+ cancers. The distribution of RT-qPCR and QIF scores were independent of fluorescence in situ hybridization (FISH) ratio/copy number. Concordance between HER2 RT-qPCR and QIF was 69.8% (r = 0.52). Among 29 patients with IHC2+ results, 16 were HER2 positive by RT-qPCR and 12 were HER2 positive by QIF. Systemic treatment, recurrence, and survival outcomes were comparable among ISH group 4 cancers regardless of IHC 0/1+ or 2+ results. ISH group 4 cancers appear to form a distinct group with intermediate levels of RNA/protein expression, close to positive/negative cut points. Therefore, adjudication into positive or negative categories may not be meaningful. Our results support the 2018 ASCO/CAP recommendation to refrain from routine additional testing of these samples. Additional outcome information after trastuzumab treatment for patients in this special group might help to guide treatment decisions in these patients.

Abstract 316: Parallel activation of MEK as a mechanism of resistance to ALK inhibitor therapy

Abstract Chromosomal rearrangements of ALK can result in a fusion protein that serves as an oncogenic driver in non-small cell lung cancer (NSCLC). Although patients with ALK-positive (ALK+) lung cancer have significant response rates when treated with tyrosine kinase inhibitors (TKIs), resistance against ALK targeted therapy consistently develops. Here, we modeled acquired resistance to ALK TKI therapy using 5 different FDA-approved ALK TKIs, including crizotinib, ceritinib, alectinib, brigatinib, and lorlatinib, using two distinct ALK+ lung cancer cell lines, H3122 and STE-1 (10 resistant cell lines total). Both of these cell lines harbor an EML4-ALK E13;A20 fusion and were derived from patients prior to ALK directed therapy. An ALK kinase domain mutation (F1174L) was detected in the H3122 ceritinib resistant cells, but none of the other resistant cell lines harbored mutations in the ALK kinase domain known to confer TKI resistance. For each of the cell lines tested, we found that ALK TKI treatment in TKI-resistant cells failed to inhibit MAP kinase pathway activity. We hypothesized that inhibition of the MAP kinase pathway, via MEK inhibition, could restore drug sensitivity. The allosteric MEK inhibitors, trametinib and binimetinib, did not effectively reduce cell viability as single agents. However, ALK TKI co-treatment with a MEK inhibitor resensitized these cells by reducing cell viability in a synergistic manner across all ALK TKI-resistant cell lines tested. Whole transcriptomic analysis of ALK TKI-resistant cell lines also revealed an upregulation in EGFR and HER2 mRNA levels. These results provide rationale for combined ALK+MEK inhibitor therapy in patients who have relapsed on first line ALK TKI therapy. Citation Format: Vincent Huang, Yingjun Yan, Huan Qiao, Yunkai Zhang, Christine M. Lovly. Parallel activation of MEK as a mechanism of resistance to ALK inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 316.

BioPATH: A Biomarker Study in Asian Patients with HER2+ Advanced Breast Cancer Treated with Lapatinib and Other Anti-HER2 Therapy

PurposeBioPATH is a non-interventional study evaluating the relationship of molecular biomarkers (PTEN deletion/downregulation, PIK3CA mutation, truncated HER2 receptor [p95HER2], and tumor HER2 mRNA levels) to treatment responses in Asian patients with HER2+ advanced breast cancer treated with lapatinib and other HER2-targeted agents. Materials and MethodsFemale Asian HER2+ breast cancer patients (n=154) who were candidates for lapatinib-based treatment following metastasis and having an available primary tumor biopsy specimen were included. The primary endpoint was progression-free survival (PFS). Secondary endpoints were response rate, overall survival on lapatinib, correlation between biomarker status and PFS for any previous trastuzumab-based treatment, and conversion/conservation rates of the biomarker status between tissue samples collected at primary diagnosis and at recurrence/metastasis. Potential relationships between tumor mRNA levels of HER2 and response to lapatinib-based therapy were also explored.Resultsp95HER2, PTEN deletion/downregulation, and PIK3CA mutation did not demonstrate any significant co-occurrence pattern and were not predictive of clinical outcomes on either lapatinib-based treatment or any previous trastuzumab-based therapy in the metastatic setting. Proportions of tumors positive for p95HER2 expression, PIK3CA mutation, and PTEN deletion/down-regulation at primary diagnosis were 32%, 31.2%, and 56.2%, respectively. Despite limited availability of paired samples, biomarker status patterns were conserved in most samples. HER2 mRNA levels were not predictive of PFS on lapatinib.ConclusionThe prevalence of p95HER2 expression, PIK3CA mutation, and PTEN deletion/downregulation at primary diagnosis were similar to previous reports. Importantly, no difference was observed in clinical outcome based on the status of these biomarkers, consistent with reports from other studies.

Relationship between tumor biomarkers and efficacy in MARIANNE, a phase III study of trastuzumab emtansine \xb1 pertuzumab versus trastuzumab plus taxane in HER2-positive advanced breast cancer

BackgroundThe phase III EMILIA and TH3RESA trials demonstrated clinical benefits of trastuzumab emtansine (T-DM1) therapy in patients with previously treated HER2-positive metastatic breast cancer (MBC). Data from these and other trials showed that T-DM1–associated survival benefits were observed across biomarker subgroups tested in these trials. Prespecified, exploratory analyses of the phase III MARIANNE study examined the effects of HER2-related biomarkers on PFS in patients administered T-DM1 in the first-line MBC setting.MethodsIn MARIANNE, patients with previously untreated HER2-positive MBC were randomized (1:1:1) to trastuzumab plus taxane, T-DM1 plus placebo, or T-DM1 plus pertuzumab. Biomarker subgroups included HER2 and HER3 mRNA expression levels (≤median vs. >median), HER2 staining intensity (IHC 3+ vs. 2+ vs. 0/1+), PIK3CA status (mutated vs. non-mutated), PTEN H-score (≤median vs. >median), and PTEN protein expression level (0 vs. 1+ vs. 2+ vs. 3+ vs. 4+). PFS was analyzed descriptively for each subgroup using Kaplan–Meier methodology. Additional exploratory post-hoc analyses evaluated the effects of HER2 heterogeneity. Multivariate analyses were also performed.ResultsMedian PFS was numerically longer for patients with HER2 mRNA levels >median versus ≤median across treatment arms. In general, there were no predictive biomarkers of benefit for either T-DM1 treatment arm; most hazard ratios were close to 1 with wide confidence intervals that included the value 1. Focal HER2 expression (IHC 3+ or IHC 2+) was present in 3.8% of patients and was associated with numerically shorter PFS in the T-DM1–containing treatment arms versus trastuzumab plus taxane. Compared with non-mutated PIK3CA, mutated PIK3CA was associated with numerically shorter median PFS across treatment groups. Post-hoc multivariate analysis showed HER2 mRNA expression and mutated PIK3CA were prognostic for PFS (P ≤ 0.001 for both biomarkers).ConclusionsIn MARIANNE, biomarkers related to the HER2 pathway did not have predictive value for PFS when comparing T-DM1 (with or without pertuzumab) with trastuzumab plus taxane. However, HER2 mRNA level and PIK3CA mutation status showed prognostic value. Evaluation of other potential biomarkers, including immune markers, is ongoing.Trial registrationRegistration number: NCT01120184. Date of registration: April 28, 2010 (registered prospectively).