Kachergus, Jennifer M.

Abstract Introduction: Stromal tumor-infiltrating lymphocytes (sTILs) have established prognostic and predictive significance in triple-negative breast cancer (TNBC). However, the roles of other immune cells in TNBC are less well-established. We performed high-plex quantitative spatial profiling in a cohort of early-stage TNBC to 1) apply spatial context to tumoral immune landscapes and 2) identify immune proteins associated with clinical outcomes, independently of TILs and other established prognostic clinicopathologic variables, in patients (pts) treated with or without adjuvant chemotherapy (CTX). Methods: The Mayo TNBC cohort comprises pts with centrally-verified, CTX-naive tumors resected from 1985-2012. Using a cohort-based TMA, with Nanostring GeoMX DSP, we quantitated 58 proteins within spatially-distinct intra-epithelial, cytokeratin-positive tumor segments and adjacent cytokeratin-negative/nuclei-positive stromal segments. Differentially-expressed (DE) proteins were identified using a negative binomial generalized linear model (SNR>2, p< 0.05) and a target DE protein set was dichotomized (80th percentile). After adjusting for prognostic clinicopathologic variables, proteins associated with recurrence-free survival (RFS, defined as time from surgery to either local, regional, and distant recurrence, or death by any cause) were identified by performing variable selection using the Akaike Information Criterion (AIC) obtained from fitting all possible Cox proportional hazards regression models (performed separately for intra-epithelial/stromal segments, and in groups +/- adjuvant CTX. Results: From the TNBC TMA, DSP data (N=250 tumors) included 169 pts who received adjuvant CTX+ and 81 who did not (CTX-). Overall, 85/250 developed recurrent disease. In the CTX+ group, intra-epithelial tumor segments from pts without recurrent disease were enriched in 10 immune proteins, including CD8, markers involved in antigen presentation/dendritic cells (CD11c, CD40, HLA-DR) or NK cells (CD56) (FC: 1.4-2.1, p<0.05); CD14 was increased in stroma (FC: 1.5, p<0.05). In contrast, in the CTX- group, both the intra-epithelial tumor and stromal segments from pts without recurrences were enriched in immune proteins (N= 12 and 15 respectively; FC 1.6-5.5, p< 0.05) most markedly CD40, IDO1 and HLA-DR (FC: 3.2-5.5, p< 0.05). Overall, CD3, CD4, CD27, CD44, and ICOS among others were enriched only in the CTX- group; CD14 and CD56 were enriched only in the CTX+ group. Based on these spatial data, biologic function and DSP data from another set of TNBC (FinXX trial), CD11c, CD14, CD27, CD40, CD56, and IDO1 were selected for RFS analysis. After applying our model selection criterion and adjusting for pt age at surgery, tumor size, lymph node status, and sTILs, intra-epithelial CD56 was independently associated with improved RFS in the CTX+ group (HR: 0.31[0.12, 0.81]). In the CTX- group, intra-epithelial CD11c was independently associated with improved RFS (0.10 [0.01, 0.81]). Conclusion: In this early-stage TNBC cohort, spatially-distinct tumor immune landscapes were associated with RFS but differed according to receipt of CTX after surgical resection. In the patients who received CTX, the intra-epithelial compartment, rather than stromal compartment, was immune-enriched in pts without recurrences. Among a targeted protein set, intra-epithelial CD56 remained associated with improved outcomes, independent of sTILs and other clinicopathologic features. In the CTX- group, spatial landscapes were more balanced, and intra-epithelial CD11c was independently associated with improved outcomes. These data provide insight into the spatial context of intrinsic immune landscapes in TNBC, and identify candidate prognostic immune biomarkers which may inform therapeutic strategies. Citation Format: Jodi M Carter, Saranya Chumsri, David W Hillman, David M Zahrieh, Yaohua Ma, Xue Wang, Jennifer M Kachergus, Judy C Boughey, Minetta C Liu, Krishna R Kalari, JC Villasboas, Roberto A Leon Ferre, Fergus J Couch, Matthew P Goetz, E. Aubrey Thompson. Intra-epithelial tumor immune landscapes are associated with clinical outcomes in early-stage triple-negative breast cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS16-01.

Abstract Introduction: The immune microenvironment of high-risk HR+ breast cancer (BC) is poorly understood. BEAUTY is a prospective neoadjuvant chemotherapy (NAC) study of stage I-III BC patients treated with neoadjuvant weekly taxane followed by anthracycline-based chemotherapy. Among clinical luminal B BC (St. Gallen criteria) from BEAUTY, we used high-plex digital spatial profiling (DSP) to characterize the intra-epithelial tumor and stromal immune landscapes to 1) assess the impact of NAC on these landscapes and 2) identify immune biomarkers predictive of response to NAC. Methods: The tissue set included FFPE sections of resected tumors from 35 patients (median 51y; range: 21-71y) with clinically-defined luminal B BC (ER > 10%/grade 2/Ki67 ≥ 15% or ER > 10%/grade 3), and 16 paired pre-NAC biopsies. Nanostring GeoMX DSP was used to quantitate 58 immune and BC biomarker proteins in intra-epithelial, cytokeratin-positive tumor segments and adjacent stromal (cytokeratin-negative/SYTO13 (nuclear stain)-positive) segments. Data were normalized using the geometric mean of two negative controls (RbIgG and MsIgG1). A general linear model with negative binomial identified differentially-expressed (DE) proteins in pre/post-NAC tumors. Based on DE protein data and biologic function, a targeted protein subset (N=19) was evaluated in pre-NAC biopsies for associations between protein abundance and residual cancer burden (RCB) class (0-II vs. III) (Wilcoxon-rank sum test: p-value < 0.025 considered significant) or RCB ‘breast only’ index (Spearman correlation). Results: Comparing tumor segments in pre-NAC specimens, intra-epithelial segments were predictably enriched in cytokeratin, ER, PR and Ki-67, but also CD127 and NY-ESO-1; whereas stromal segments were enriched in proteins associated with T cell subsets (e.g. CD3, CD4, CD8), macrophages (CD68 and CD163), antigen presentation/dendritic cells (CD11c, HLA-DR) and immune checkpoint proteins (PD-L1, PD-L2, B7-H3, TIM-3, VISTA) among others. While NAC did not alter the spatial distribution of proteins (intra-epithelial vs. stromal segments), it markedly attenuated the immune landscape, with decreased abundance of functionally-diverse immune proteins (e.g. CD45, CD3, CD4, CD127, granzyme B, CTLA4; STING, B7-H3, CD11c, and CD68, log2FC: 0.27-1.52 p < 0.05), including low abundance proteins PD-1, PD-L1, PD-L2, CD20, and OX40L (log 2FC: 0.15-1.16, p < 0.05). Both PR and Ki-67 decreased in post-NAC tumors whereas ER was not significantly altered (p < 0.05). CD8, CD14, CD163, HLA-DR, IDO-1 and TIM-3 were not significantly altered by NAC. In the pre-NAC biopsies (which had subsequent residual tumor burdens of RCB class 0 or I (n=1 each), II (n=6), and III (n=8), and median RCB ‘breast only’ index of 3.39 (range: 0.00-38.02), 19 proteins were used for RCB analysis (CD3, CD4, CD8, CD14, CD34, CD44, CD45, CD68, CD127, CD163, CTLA4, granzyme B, STING, B7-H3, fibronectin, Ki-67, HLA-DR, SMA and TIM3). Among them, stromal CD127 was significantly higher in RCB class III than RCB class 0-II (p=0.021) and RCB ‘breast only’ index was positively correlated with intra-epithelial granzyme B (rho = 0.61; p=0.012), and negatively correlated with intra-epithelial Ki-67 (rho= -0.71; p=0.0022). Conclusion: In this series of clinical luminal B BC, NAC markedly attenuated the tumoral immune landscapes with a small set of “NAC-resistant” immune proteins. Among a targeted set, stromal CD127 was significantly higher in RCB III, and RCB breast-only index was positively correlated with intra-epithelial granzyme B. These data provide insight into the impact of NAC on HR+ BC, and identify potential immune biomarkers to predict response to neoadjuvant chemotherapy. Citation Format: Jodi M Carter, Judy C Boughey, Jun He, Vera J Suman, Xue Wang, Jennifer M Kachergus, Krishna R Kalari, Liewei Wang, Richard Weinshilboum, Ann M Moyer, Sarah A McLaughlin, Alvaro Moreno-Aspitia, Donald W Northfelt, Richard J Gray, James N Ingle, E. Aubrey Thompson, Matthew P Goetz. Neoadjuvant chemotherapy selectively alters spatially-defined immune landscapes in clinical luminal B HR+/HER2- breast cancers: Analysis of the breast cancer genome guided therapy study (BEAUTY) [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD7-05.

Abstract Background: The intrinsic immune response in triple negative breast cancer (TNBC) has both prognostic and predictive utility, and immune-related biomarkers are now actionable targets in TNBC. Current modalities for immune profiling are limited by lack of spatial context and precise quantitation. We used quantitative digital spatial profiling (DSP) to 1) characterize the immune architecture of tumoral and stromal regions in TNBC and 2) identify prognostic immune signatures. Methods: From the FinXX trial of adjuvant capecitabine therapy, tumor sections (FFPE) matched for patient characteristics, treatment arm and RFS-based outcome (N=44) were assessed with Nanostring GeoMxTM DSP. Briefly, with 3-plex immunofluorescence (pan-cytokeratin, CD45, CD68 with SYTO-13 dye for nuclei), spatially-defined CD45-enriched or CD68-enriched stromal segments and adjacent tumor segments (N=950) were selected for digital quantitation (NanoString nCounter) of 39 immune proteins (e.g. immune cell profiling proteins: CD3, CD4, CD8, CD11c, CD20, CD56, CD68, HLA-DR, PD-1, PD-L1, Granzyme B), immune drug targets (e.g. VISTA, STING, IDO1), and activation status-related proteins (e.g. ICOS, PD-L2, CD40, CD40L, CD44). Normalized, differentially-expressed (DE) proteins were evaluated, and weighted gene co-expression network analysis (WGCNA) was performed to identify modules of highly co-expressing immune proteins, and association with RFS as a categorical variable was determined. Results: 20 DE proteins in tumor segments were associated with RFS. Among them, those with the highest RFS-associated fold changes (FC) included CD20, CD40, CD56, HLA-DR, Granzyme B, and PD-L2 (FC): 2.1-4.4) (p < 0.05). In contrast, only 4 DE proteins were associated with RFS in stromal segments (Granzyme B, IDO-1, PD-L1, and PD-L2 (FC: 1.5-3.3) (p < 0.05). By WGCNA, 2 tumor-based immune modules [IM1 (lymphocyte infiltration) and IM2 (lymphocyte activation)] were identified based on their co-expression. The top three co-expressed proteins in IM1 were CD4, CD40, and CD45 (r >0.9). The three top co-expressed proteins in IM2 were PD-L1, PD-L2, and Granzyme B (r > 0.85). Together with CD56 expression, IM1 and IM2 comprised a set of 3 intratumoral immune features that associated with RFS. For recurring tumors, all 3 immune features clustered with low scores . Among non-recurrent tumors, two distinct, non-overlapping clusters with inverse characteristics were identified: high IM2, low IM1, low CD56 vs. low IM2, high IM1, and high CD56. Overall, across all 3 immune features, PDL2, CD40 and CD56 expression most strongly associated with RFS. Conclusion: With spatially-defined, precise quantification of immune proteins, we identified 3 immune features, including two distinct immune modules, IM1 and IM2 that correlate with durable RFS. These immune modules of highly co-expressing proteins were associated with RFS only when present in the tumor compartment but not tumor-adjacent, immune cell-rich stroma. Citation Format: Jodi M. Carter, Saranya Chumsri, Yaohua Ma, Xue Wang, Jennifer M. Kachergus, Douglas Hinerfeld, Heather Ann Brauer, Sarah Warren, Heikki Joensuu, Edith A. Perez, Aubrey Thompson. Quantitative digital spatial profiling reveals novel prognostic intratumoral immune signatures in triple-negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1004.

Abstract Background: Growing evidence supports the critical role of preexisting immune response in triple negative breast cancer (TNBC). However, there are limitations with current evaluation approaches: inability to functionally assess the type of immune infiltration with traditional pathologic evaluation and loss of spatial distribution in conventional high-plex gene or protein expression analyses from the whole tumor section. Here, we report the initial analysis of immune protein expression as a function of spatial distribution and clinical outcomes in TNBC samples. Methods: NanoString GeoMxTM Digital Spatial Profiling (DSP) was used to quantify 39 immune-related proteins in stromal and tumor segments from 44 TNBC samples from the FinXX trial. Samples were matched for patient characteristics, treatment arm (capecitabine vs. 5-fluorouracil), and outcome based on recurrence-free survival (RFS) with 22 samples from patients who recurred and 22 samples from patients with durable RFS. Regions of interest (ROIs) were selected based on expression of cytokeratin (tumor), CD45 (leukocytes), or CD68 (macrophages). Each ROI was segmented into tumor (pancytokeratin-positive area) and stroma (pancytokeratin-negative/nuclear SYTO13-positive area). The general linear model was used for statistical analysis of differential expression with RFS as a categorical variable (recur yes or no). Results: A total of 950 tumor and stroma segments were included in this initial analysis. In both tumor and stroma segments, over-expression of T cell activation markers (CD137, GITR) was associated with better outcome, whereas T cell markers (CD3, CD4, CD8) were not significantly associated with outcome. In tumor segments alone, improved outcome was significantly associated with increased protein expression [> 2-fold change (FC) at p<0.001] of CD56, PD-L2, HLA-DR, CD137, GITR, and CD40. In CD45-enriched stroma, improved outcome was associated with elevated expression (FC>2.0, p<0.001) of PD-L2; whereas durable RFS was associated with elevated PD-L2 and IDO1 expression in CD68-enriched stroma. In contrast, macrophage/dendritic cell markers CD68 and CD11c were not associated with outcome. In tumor cells adjacent to CD45-enriched stroma, durable RFS was associated with increased abundance (FC>2.0, p<0.001) of PD-L2, CD56, CD27, GITR, CD20, HLA-DR, and IDO1. Similarly, in tumor segment associated with CD68-enriched stroma, proteins associated with RFS included PD-L2, HLA-DR, CD56, GITR, and CD137. Among 39 immune function proteins, only elevated CTLA4 expression in CD68-enriched stroma was associated with recurrence (FC=0.55, p<0.001), whereas tumor-segment CTLA4 was associated with RFS. Conclusions: Using an in-depth analysis to precisely quantify the abundance of multiple immune function proteins in a spatially defined manner, we observed that PD-L2, IDO1, and T cell activation markers were robustly associated with durable RFS in both tumor and stromal segments. In contrast, MHC components (HLA-DR, beta-2-microglobulin), B cell markers (CD20), and NK cell markers (CD56) were strongly associated with favorable outcome in tumor but not in stromal segments. Our study highlights the conclusion that the immune landscape of TNBC is far too complex to be encompassed by any single molecular marker, and more detailed analyses of the DSP data reported here are ongoing with a view towards using quantitative multiplex analyses to refine our understanding of how therapeutic outcome is influenced by interactions among immune cells and between immune and tumor cells. Acknowledgements: Supported by the Breast Cancer Research Foundation (BCRF18-161), Bankhead Coley (6BC05 Florida Department of Health), 26.2 with Donna Foundation, US National Cancer Institute (CA15083), and the Canadian Cancer Center Citation Format: Saranya Chumsri, Douglas Hinerfeld, Jennifer M. Kachergus, Yaohua Ma, Heather A Brauer, Sarah Warren, Xue Wang, Torsten O. Nielsen, Karama Asleh, Heikki Joensuu, Edith A. Perez, E. A. Thompson. Digital spatial mapping of the immune landscape of triple negative breast cancer reveals novel features of immune-tumor cell interaction [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr PD5-06.

1037 Background: Folate receptor alpha (FRA the product of the FOLR1 gene) has been identified as a potential prognostic and therapeutic target in a number of cancers. A correlation has been shown between intense expression of FRA in breast tumors and poor prognosis, yet little is known about FOLR1/FRA expression across clinically relevant breast cancer subtypes. Methods: 131 breast cancer tumors including 4 benign, 33 ER+, 26 HER2+, and 68 triple negative (TN) were constructed into tissue microarrays (TMAs). FRA expression was analyzed by immunohistochemistry (IHC) using a high affinity FRA antibody. Tumor membrane staining intensity was scored by a pathologist as negative (0), weak (1+), moderate (2+) and strong (3+). The percent of cells within each tissue core stained at each intensity was recorded to calculate an H-score. The H-score is a weighted score that captured both the proportion of positive staining and intensity for each tumor. H-score values can range from zero (no membrane staining) to a maximum of 300 (100% membrane staining at 3+). H-scores for each patient sample were averaged over 3 TMA cores. The mean H-scores for each tumor subtype and the percentage of 3+ staining in >30% of tumor cells were compared by a Mann-Whitney test. The distribution of FOLR1 mRNA was completed using a TCGA RNA-seq dataset from 691 breast tumors classified as ER+, HER2+ and TN. FOLR1 levels of TN versus ER+ and HER2+ were compared by a Mann-Whitney test. Results: The mean H-score for the benign tumors was 0, ER+ (13.31), HER2+ (39.36), TN (119.02). The median H-score for the benign tumors was 0, ER+ (0), HER2+ (7.5), TN (127.5). The TN tumors mean and median H scores were significantly higher than benign, ER+ or HER2+ (p<0.001). The largest percentage of 3+ staining in >30% of tumor cells was observed in TN tumors (36.7%) and lowest in ER+ tumors (0%) (p<0.001). TN tumors had significantly higher levels of FOLR1 mRNA compared to ER+ and HER2+ subtypes (p<0.0001). Conclusions: Our data indicate that expression of FRA is highly prevalent in TN tumors and is supported by FOLR1 mRNA levels. Anti-FRA therapy may represent an important therapeutic intervention in TNBC who to this point have no active targeted treatment options.

3142 Background: Recent studies have demonstrated a benefit of adjuvant capecitabine, particularly in triple negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy. However, biomarkers to predict which patients are more likely to benefit from capecitabine are needed. Methods: The nanoString Breast Cancer 360 (BC360) and PanCancer Immunoncology (IO360) panels were used to quantify mRNA expression in TNBC samples in the FinXX trial. FinXX is a phase III trial which randomized high risk patients to receive either 3 cycles of docetaxel followed by 3 cycles of cyclophosphamide, epirubicin, and fluorouracil (Arm A: T+CEF) vs. 3 cycles of docetaxel plus capecitabine followed by 3 cycles of cyclophosphamide, epirubicin, and capecitabine (Arm B: TX+CEX). Gene signature scores were analyzed using prespecified algorithms developed by nanoString. Digital Spatial Profiling was carried out using GeoMX platform. Results: A total of 111 TNBC patients in FinXX trial were included (57 in Arm A and 54 in Arm B) with 10.2 years median follow up. There were 7 cancer- and immune-related gene signatures identified by BC360 and IO360 panels that were significantly associated with improved recurrent free survival favoring an addition of capecitabine. These include cytotoxic cell signature (HR 0.37, 95%CI 0.15-0.92, p 0.03), endothelial signature (HR 0.18, 95%CI 0.04-0.83, p 0.03), mast cell signature (HR 0.43, 95%CI 0.21-0.88, p 0.02), PDL2 gene (HR 0.29, 95%CI 0.09-0.99, p 0.05), immunoproteasome (HR 0.34, 95%CI 0.13-0.89, p 0.02), exhausted CD8 (HR 0.29, 95%CI 0.09-0.97, p 0.04), and PD1 (HR 0.44, 95%CI 0.20-1.02, p 0.05). Conclusions: Analysis of RNA abundance signatures strongly suggests that there are important immune features that are associated with benefit from capecitabine in TNBC. However, analysis of RNA extracted from whole tumor sections lacks spatial discrimination. We anticipate that a more detailed, spatially-defined analysis of protein abundance, using the novel NanoString GeoMX platform, will provide more insights and define specific immune features associated with improved outcome. Additional results of GeoMX will be reported at the meeting. Clinical trial information: NCT00114816.

Abstract Background: Understanding heterogeneity within individual breast tumors is key to the ability to predict therapeutic outcome. Molecular heterogeneity is commonly evaluated based on genomic features, including mRNA abundance, gene copy number events, and somatic mutations. The expression profile and activation state of key proteins is widely recognized as another key element in defining tumor heterogeneity. We have taken advantage of NanoString 3D Biology™ technology (for research use only) and curated nCounter Vantage 3DTM Solid Tumor Assay to interrogate a survey panel of HER2-positive breast tumors with the ultimate goal of determining key relationships between multiple genomic and proteomic profiles in individual tumors. Methods: We analyzed samples from 24 HER2+ breast cancer patients using NanoString technology to quantify the expression profile for over 25 total and phospho signaling proteins, including PI3K/MAPK/EGFR/HER2, 770 mRNA corresponding to 13 canonical cancer pathways, and 104 somatic mutations and small INDELS that are commonly associated with cancer, including 8 known PIK3CA mutations. These analyses were carried out in a matched fresh frozen and FFPE samples on the nCounter paltform. Data were analyzed by nSolver to identify genotype specific expression profiles across the 24 samples. Results: In our proof-of-concept data set, we successfully demonstrate that NanoString’s 3D biology Technology shows concordance across both FFPE and fresh frozen sample types for DNA, RNA, and protein. NanoString analysis also showed high concordance to gold-standard techniques used to assess genotype and RNA expression profiles. The combination of digital DNA, RNA, and protein data from our HER2+ breast cancer samples yielded potentially actionable data based on mapping of mutational status as the driver of key differences in protein expression and mRNA abundance of the signaling targets profiled. This work sheds new light on HER2+ breast cancer biology and the interplay between genomic and proteomic profiles while setting the stage for future studies that further probe the differences observed in this sample set. Conclusions: Simultaneous analysis of mutational status (SNV) and expression at the level of both mRNA and protein promises to enable a more detailed view of the relationship between genotype and the biological and clinical behavior of key tumor types. The NanoString Vantage 3DTM Solid Tumor platform provides a rapid, reliable, and economic means of assessing these analytes simultaneously. The application of these analytes to models that make clinically actionable predictions will require additional analyses of large sample cohorts, but such analysis is quite feasible using a variety of sample types. Acknowledgements: Supported in part by grants from the Breast Cancer Research Foundation and the 26.2 with Donna Foundation. Citation Format: Sarayna Chumsri, Daniel J. Serie, Brian M. Necela, Jennifer M. Kachergus, Bianca C. Axenfeld, Gokhan Demirkan, Gavin Meredith, P. Martin Ross, Anisha Kharkia, Erin Piazza, Afshin Mashadi-Hossein, Sarah Warren, Sarah A. McLaughlin, Joseph Beechem, Gary Geiss, E. Aubrey Thompson. Simultaneous analysis of the mutational landscape and RNA and protein expression profile of HER2-positive breast cancer using 3D BiologyTM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3377. doi:10.1158/1538-7445.AM2017-3377

AbstractBackgroundThe targeted ERBB2 therapy, trastuzumab, has had a tremendous impact on management of patients with HER2+ breast cancer, leading to development and increased use of further HER2 targeted therapies. The major clinical side effect is cardiotoxicity but the mechanism is largely unknown. On the basis that gene expression is known to be altered in multiple models of heart failure, we examined differential gene expression of iPSC‐derived cardiomyocytes treated at day 11 with the ERBB2 targeted monoclonal antibody, trastuzumab for 48 h and the small molecule tyrosine kinase inhibitor of EGFR and ERBB2.ResultsTranscriptome sequencing was performed on four replicates from each group (48 h untreated, 48 h trastuzumab and 48 h lapatinib) and differential gene expression analyses were performed on each treatment group relative to untreated cardiomyocytes. 517 and 1358 genes were differentially expressed, p < 0.05, respectively in cardiomyocytes treated with trastuzumab and lapatinib. Gene ontology analyses revealed in cardiomyocytes treated with trastuzumab, significant down‐regulation of genes involved in small molecule metabolism (p = 3.22 × 10−9) and cholesterol (p = 0.01) and sterol (p = 0.03) processing. We next measured glucose uptake and lactate production in iPSC‐derived cardiomyocytes 13 days post‐plating, treated with trastuzumab up to 96 h. We observed significantly decreased glucose uptake from the media of iPSC‐derived cardiomyocytes treated with trastuzumab as early as 24 h (p = 0.001) and consistently up to 96 h (p = 0.03).ConclusionsOur study suggests dysregulation of cardiac gene expression and metabolism as key elements of ERBB2 signaling that could potentially be early biomarkers of cardiotoxicity.