Replication of genetic associations of chemotherapy-related cardiotoxicity in the adjuvant NSABP B-31 clinical trial.

Background: The cardiotoxic effects of doxorubicin, trastuzumab, and other anticancer agents are well known, but molecular genetic testing is lacking for the early identification of patients at risk for therapy-related cardiac toxicity. Methods: Using the Agena Bioscience MassARRAY system, we genotyped TRPC6 rs77679196, BRINP1 rs62568637, LDB2 rs55756123, RAB22A rs707557, intergenic rs4305714, LINC01060 rs7698718, and CBR3 rs1056892 (V244M) (previously associated with either doxorubicin or trastuzumab-related cardiotoxicity in the NCCTG N9831 trial of anthracycline-based chemotherapy ± trastuzumab) in 993 patients with HER2+ early breast cancer from the NSABP B-31 trial of adjuvant anthracycline-based chemotherapy ± trastuzumab. Association analyses were performed with outcomes of congestive heart failure (N = 29) and maximum decline in left ventricular ejection fraction (LVEF) using logistic and linear regression models, respectively, under an additive model with age, baseline LVEF, and previous use of hypertensive medications as covariates. Results: Associations of maximum decline in LVEF in the NCCTG N9831 patients did not replicate in the NSABP B-31 patients. However, TRPC6 rs77679196 and CBR3 rs1056892 were significantly associated with congestive heart failure, p < 0.05, with stronger associations observed in patients treated with chemotherapy only (no trastuzumab) or in the combined analysis of all patients relative to those patients treated with chemotherapy + trastuzumab. Conclusions: TRPC6 rs77679196 and CBR3 rs1056892 (V244M) are associated with doxorubicin-induced cardiac events in both NCCTG N9831 and NSABP B-31. Other variants previously associated with trastuzumab-related decline in LVEF failed to replicate between these studies.

TRPC6 N338S is a gain-of-function mutant identified in patient with doxorubicin-induced cardiotoxicity.

The canonical transient receptor potential 6 gene, TRPC6, has been implicated as a putative risk gene for chemotherapy-induced congestive heart failure, but knowledge of specific risk variants is lacking. Following our genome-wide association study and subsequent fine-mapping, a rare missense mutant of TRPC6 N338S, was identified in a breast cancer patient who received anthracycline-containing chemotherapy regiments and developed congestive heart failure. However, the function of N338S mutant has not been examined. Using intracellular Ca2+ imaging, patch clamp recording and molecular docking techniques, we assessed the function of N338S mutant heterologously expressed in HEK293 cells and HL-1 cardiac cells. We found that expression of TRPC6 N338S significantly increased intracellular Ca2+ levels ([Ca2+]i) and current densities in response to 50 µM 1-oleoyl 2-acetyl-sn-glycerol (OAG), an activator of TRPC6 channels, compared to those of TRPC6 WT. A 24-h pretreatment with 0.5 µM doxorubicin (DOX) further potentiated the OAG effects on TRPC6 N338S current densities and [Ca2+]i, and these effects were abolished by 1 µM BI-749327, a highly selective TRPC6 inhibitor. Moreover, DOX treatment significantly upregulated the mRNA and protein expressions of TRPC6 N338S, compared to those of TRPC6 WT. Molecular docking and dynamics simulation showed that OAG binds to the pocket constituted by the pore-helix, S5 and S6 domains of TRPC6. However, the N338S mutation strengthened the interaction with OAG, therefore stabilizing the OAG-TRPC6 N338S complex and enhancing OAG binding affinity. Our results indicate that TRPC6 N338S is a gain-of-function mutant that may contribute to DOX-induced cardiotoxicity by increasing Ca2+ influx and [Ca2+]i in cardiomyocytes.

Adaptive immune signature in HER2-positive breast cancer in NCCTG (Alliance) N9831 and NeoALTTO trials.

Trastuzumab acts in part through the adaptive immune system. Previous studies showed that enrichment of immune-related gene expression was associated with improved outcomes in HER2-positive (HER2+) breast cancer. However, the role of the immune system in response to lapatinib is not fully understood. Gene expression analysis was performed in 1,268 samples from the North Central Cancer Treatment Group (NCCTG) N9831 and 244 samples from the NeoALTTO trial. In N9831, enrichment of CD45 and immune-subset signatures were significantly associated with improved outcomes. We identified a novel 17-gene adaptive immune signature (AIS), which was found to be significantly associated with improved RFS among patients who received adjuvant trastuzumab (HR 0.66, 95% CI 0.49-0.90, Cox regression model p = 0.01) but not in patients who received chemotherapy alone (HR 0.96, 95% CI 0.67-1.40, Cox regression model p = 0.97). This result was validated in NeoALTTO. Overall, AIS-low patients had a significantly lower pathologic complete response (pCR) rate compared with AIS-high patients (χ2 p < 0.0001). Among patients who received trastuzumab alone, pCR was observed in 41.7% of AIS-high patients compared with 9.8% in AIS-low patients (OR of 6.61, 95% CI 2.09-25.59, logistic regression model p = 0.003). More importantly, AIS-low patients had a higher pCR rate with an addition of lapatinib (51.1% vs. 9.8%, OR 9.65, 95% CI 3.24-36.09, logistic regression model p < 0.001). AIS-low patients had poor outcomes, despite receiving adjuvant trastuzumab. However, these patients appear to benefit from an addition of lapatinib. Further studies are needed to validate the significance of this signature to identify patients who are more likely to benefit from dual anti-HER2 therapy. ClinicalTrials.gov Identifiers: NCT00005970 (NCCTG N9831) and NCT00553358 (NeoALTTO).

Inter-Individual Variation and Cardioprotection in Anthracycline-Induced Heart Failure.

Anthracyclines are one of the most widely used and effective chemotherapies in oncology, but their most important side effect is the cumulative, dose-related cardiotoxicity leading to congestive heart failure in ~5% of individuals. Methodology and pharmacogenetic studies for predicting which individuals are at high risk and subsequently the development of targeted and individualized cardioprotective plans are beginning to make progress. Here, we review current putative risk genes and variants, the strength of evidence for each genetic association and the interaction between risk genes, in the context of known clinical risk factors and potential novel cardioprotective strategies.

Disruption of MAP7D1 Gene Function Increases the Risk of Doxorubicin-Induced Cardiomyopathy and Heart Failure.

Doxorubicin is a cornerstone chemotherapeutic drug widely used to treat various cancers; its dose-dependent cardiomyopathy, however, is one of the leading causes of treatment-associated mortality in cancer survivors. Patients' threshold doses leading to doxorubicin-induced cardiomyopathy (DIC) and heart failure are highly variable, mostly due to genetic variations in individuals' genomes. However, genetic susceptibility to DIC remains largely unidentified. Here, we combined a genetic approach in the zebrafish (Danio rerio) animal model with a genome-wide association study (GWAS) in humans to identify genetic susceptibility to DIC and heart failure. We firstly reported the cardiac and skeletal muscle-specific expression and sarcomeric localization of the microtubule-associated protein 7 domain-containing protein 1b (Map7d1b) in zebrafish, followed by expression validation in mice. We then revealed that disruption of the map7d1b gene function exaggerated DIC effects in adult zebrafish. Mechanistically, the exacerbated DIC are likely conveyed by impaired autophagic degradation and elevated protein aggregation. Lastly, we identified 2 MAP7D1 gene variants associated with cardiac functional decline and heart failure in cancer patients who received doxorubicin therapy. Together, this study identifies MAP7D1 as a clinically relevant susceptibility gene to DIC and heart failure, providing useful information to stratify cancer patients with a high risk of incurring severe cardiomyopathy and heart failure after receiving chemotherapy.

Sex Differences, Genetic and Environmental Influences on Dilated Cardiomyopathy.

Dilated cardiomyopathy (DCM) is characterized by dilatation of the left ventricle and impaired systolic function and is the second most common cause of heart failure after coronary heart disease. The etiology of DCM is diverse including genetic pathogenic variants, infection, inflammation, autoimmune diseases, exposure to chemicals/toxins as well as endocrine and neuromuscular causes. DCM is inherited in 20-50% of cases where more than 30 genes have been implicated in the development of DCM with pathogenic variants in TTN (Titin) most frequently associated with disease. Even though male sex is a risk factor for heart failure, few studies have examined sex differences in the pathogenesis of DCM. We searched the literature for studies examining idiopathic or familial/genetic DCM that reported data by sex in order to determine the sex ratio of disease. We found 31 studies that reported data by sex for non-genetic DCM with an average overall sex ratio of 2.5:1 male to female and 7 studies for familial/genetic DCM with an overall average sex ratio of 1.7:1 male to female. No manuscripts that we found had more females than males in their studies. We describe basic and clinical research findings that may explain the increase in DCM in males over females based on sex differences in basic physiology and the immune and fibrotic response to damage caused by mutations, infections, chemotherapy agents and autoimmune responses.

Trpc6 Promotes Doxorubicin-Induced Cardiomyopathy in Male Mice With Pleiotropic Differences Between Males and Females.

Background: Doxorubicin is a widely used and effective chemotherapy, but the major limiting side effect is cardiomyopathy which in some patients leads to congestive heart failure. Genetic variants in TRPC6 have been associated with the development of doxorubicin-induced cardiotoxicity, suggesting that TRPC6 may be a therapeutic target for cardioprotection in cancer patients. Methods: Assessment of Trpc6 deficiency to prevent doxorubicin-induced cardiac damage and function was conducted in male and female B6.129 and Trpc6 knock-out mice. Mice were treated with doxorubicin intraperitoneally every other day for a total of 6 injections (4 mg/kg/dose, cumulative dose 24 mg/kg). Cardiac damage was measured in heart sections by quantification of vacuolation and fibrosis, and in heart tissue by gene expression of Tnni3 and Myh7. Cardiac function was determined by echocardiography. Results: When treated with doxorubicin, male Trpc6-deficient mice showed improvement in markers of cardiac damage with significantly reduced vacuolation, fibrosis and Myh7 expression and increased Tnni3 expression in the heart compared to wild-type controls. Similarly, male Trpc6-deficient mice treated with doxorubicin had improved LVEF, fractional shortening, cardiac output and stroke volume. Female mice were less susceptible to doxorubicin-induced cardiac damage and functional changes than males, but Trpc6-deficient females had improved vacuolation with doxorubicin treatment. Sex differences were observed in wild-type and Trpc6-deficient mice in body-weight and expression of Trpc1, Trpc3 and Rcan1 in response to doxorubicin. Conclusions: Trpc6 promotes cardiac damage following treatment with doxorubicin resulting in cardiomyopathy in male mice. Female mice are less susceptible to cardiotoxicity with more robust ability to modulate other Trpc channels and Rcan1 expression.

Association of Genetic Variants at TRPC6 With Chemotherapy-Related Heart Failure.

Background: Our previous GWAS identified genetic variants at six novel loci that were associated with a decline in left ventricular ejection fraction (LVEF), p < 1 × 10-5 in 1,191 early breast cancer patients from the N9831 clinical trial of chemotherapy plus trastuzumab. In this study we sought replication of these loci. Methods: We tested the top loci from the GWAS for association with chemotherapy-related heart failure (CRHF) using 26 CRHF cases from N9831 and 984 patients from the Mayo Clinic Biobank which included CRHF cases (N = 12) and control groups of patients treated with anthracycline +/- trastuzumab without HF (N = 282) and patients with HF that were never treated with anthracycline or trastuzumab (N = 690). We further examined associated loci in the context of gene expression and rare coding variants using a TWAS approach in heart left ventricle and Sanger sequencing, respectively. Doxorubicin-induced apoptosis and cardiomyopathy was modeled in human iPSC-derived cardiomyocytes and endothelial cells and a mouse model, respectively, that were pre-treated with GsMTx-4, an inhibitor of TRPC6. Results: TRPC6 5' flanking variant rs57242572-T was significantly more frequent in cases compared to controls, p = 0.031, and rs61918162-T showed a trend for association, p = 0.065. The rs61918162 T-allele was associated with higher TRPC6 expression in the heart left ventricle. We identified a single TRPC6 rare missense variant (rs767086724, N338S, prevalence 0.0025% in GnomAD) in one of 38 patients (2.6%) with CRHF. Pre-treatment of cardiomyocytes and endothelial cells with GsMTx4 significantly reduced doxorubicin-induced apoptosis. Similarly, mice treated with GsMTx4 had significantly improved doxorubicin-induced cardiac dysfunction. Conclusions: Genetic variants that are associated with increased TRPC6 expression in the heart and rare TRPC6 missense variants may be clinically useful as risk factors for CRHF. GsMTx-4 may be a cardioprotective agent in patients with TRPC6 risk variants. Replication of the genetic associations in larger well-characterized samples and functional studies are required.

Folate receptor alpha expression associates with improved disease-free survival in triple negative breast cancer patients.

Triple negative breast cancer (TNBC) comprises 15-20% of all invasive breast cancer and is associated with a poor prognosis. As therapy options are limited for this subtype, there is a significant need to identify new targeted approaches for TNBC patient management. The expression of the folate receptor alpha (FRα) is significantly increased in patients with TNBC and is therefore a potential biomarker and therapeutic target. We optimized and validated a FRα immunohistochemistry method, specific to TNBC, to measure FRα expression in a centrally confirmed cohort of 384 patients with TNBC in order to determine if expression of the protein is associated with invasive disease-free survival (IDFS) and overall survival (OS). The FRα IHC demonstrated exceptional performance characteristics with low intra- and interassay variability as well as minimal lot-to-lot variation. FRα expression, which varied widely from sample to sample, was detected in 274 (71%) of the TNBC lesions. In a multivariable model adjusted for baseline characteristics, FRα expression was associated with improved IDFS (HR = 0.63, p = 0.01) but not with OS. The results demonstrate the potential of targeting the FRα in the majority of TNBC patients and suggest that variable expression may point to a need to stratify on FRα expression in clinical studies.

Retinoid X receptor alpha is a spatiotemporally predominant therapeutic target for anthracycline-induced cardiotoxicity.

To uncover the genetic basis of anthracycline-induced cardiotoxicity (AIC), we recently established a genetic suppressor screening strategy in zebrafish. Here, we report the molecular and cellular nature of GBT0419, a salutary modifier mutant that affects retinoid x receptor alpha a (rxraa). We showed that endothelial, but not myocardial or epicardial, RXRA activation confers AIC protection. We then identified isotretinoin and bexarotene, two FDA-approved RXRA agonists, which exert cardioprotective effects. The therapeutic effects of these drugs only occur when administered during early, but not late, phase of AIC or as pretreatment. Mechanistically, these spatially- and temporally-predominant benefits of RXRA activation can be ascribed to repair of damaged endothelial cell-barrier via regulating tight-junction protein Zonula occludens-1. Together, our study provides the first in vivo genetic evidence supporting RXRA as the therapeutic target for AIC, and uncovers a previously unrecognized spatiotemporally-predominant mechanism that shall inform future translational efforts.

Rapid Generation of Sustainable HER2-specific T-cell Immunity in Patients with HER2 Breast Cancer using a Degenerate HLA Class II Epitope Vaccine.

PURPOSE: Patients with HER2+ breast cancer benefit from trastuzumab-containing regimens with improved survival. Adaptive immunity, including cytotoxic T-cell and antibody immunity, is critical to clinical efficacy of trastuzumab. Because Th cells are central to the activation of these antitumor effectors, we reason that HER2 patients treated with trastuzumab may benefit by administering vaccines that are designed to stimulate Th-cell immunity. PATIENTS AND METHODS: We developed a degenerate HER2 epitope-based vaccine consisting of four HLA class II-restricted epitopes mixed with GM-CSF that should immunize most (≥84%) patients. The vaccine was tested in a phase I trial. Eligible women had resectable HER2+ breast cancer and had completed standard treatment prior to enrollment and were disease free. Patients were vaccinated monthly for six doses and monitored for safety and immunogenicity. RESULTS: Twenty-two subjects were enrolled and 20 completed all six vaccines. The vaccine was well tolerated. All patients were alive at analysis with a median follow-up of 2.3 years and only two experienced disease recurrence. The percent of patients that responded with augmented T-cell immunity was high for each peptide ranging from 68% to 88%, which led to 90% of the patients generating T cells that recognized naturally processed HER2 antigen. The vaccine also augmented HER2-specific antibody. Immunity was sustained in patients with little sign of diminishing at 2 years following the vaccination. CONCLUSIONS: Degenerate HLA-DR-based HER2 vaccines induce sustainable HER2-specific T cells and antibodies. Future studies, could evaluate whether vaccination during adjuvant treatment with trastuzumab-containing regimens improves patient outcomes.

Generation of HER2-specific antibody immunity during trastuzumab adjuvant therapy associates with reduced relapse in resected HER2 breast cancer.

BACKGROUND: Resected HER2 breast cancer patients treated with adjuvant trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induce HER2-specific antibodies which correlate with improved survival in HER2 metastatic breast cancer patients. It remains unclear whether the generation of immunity required trastuzumab and whether endogenous antibody immunity is associated with improved disease-free survival in the adjuvant setting. In this study, we addressed this question by analyzing serum anti-HER2 antibodies from a subset of patients enrolled in the NCCTG trial N9831, which includes an arm (Arm A) in which trastuzumab was not used. Arms B and C received trastuzumab sequentially or concurrently to chemotherapy, respectively. METHODS: Pre-and post-treatment initiation sera were obtained from 50 women enrolled in N9831. Lambda IgG antibodies (to avoid detection of trastuzumab) to HER2 were measured and compared between arms and with disease-free survival. RESULTS: Prior to therapy, across all three arms, N9831 patients had similar mean anti-HER2 IgG levels. Following treatment, the mean levels of antibodies increased in the trastuzumab arms but not the chemotherapy-only arm. The proportion of patients who demonstrated antibodies increased by 4% in Arm A and by 43% in the Arms B and C combined (p = 0.003). Cox modeling demonstrated that larger increases in antibodies were associated with improved disease-free survival in all patients (HR = 0.23; p = 0.04). CONCLUSIONS: These results show that the increased endogenous antibody immunity observed in adjuvant patients treated with combination trastuzumab and chemotherapy is clinically significant, in view of its correlation with improved disease-free survival. The findings may have important implications for predicting treatment outcomes in patients treated with trastuzumab in the adjuvant setting. TRIAL REGISTRATION: ClinicalTrials.gov, NCT00005970 . Registered on July 5, 2000.

Determining the genetic basis of anthracycline-cardiotoxicity by molecular response QTL mapping in induced cardiomyocytes.

Anthracycline-induced cardiotoxicity (ACT) is a key limiting factor in setting optimal chemotherapy regimes, with almost half of patients expected to develop congestive heart failure given high doses. However, the genetic basis of sensitivity to anthracyclines remains unclear. We created a panel of iPSC-derived cardiomyocytes from 45 individuals and performed RNA-seq after 24 hr exposure to varying doxorubicin dosages. The transcriptomic response is substantial: the majority of genes are differentially expressed and over 6000 genes show evidence of differential splicing, the later driven by reduced splicing fidelity in the presence of doxorubicin. We show that inter-individual variation in transcriptional response is predictive of in vitro cell damage, which in turn is associated with in vivo ACT risk. We detect 447 response-expression quantitative trait loci (QTLs) and 42 response-splicing QTLs, which are enriched in lower ACT GWAS [Formula: see text]-values, supporting the in vivo relevance of our map of genetic regulation of cellular response to anthracyclines.

Genome-wide association study of cardiotoxicity in the NCCTG N9831 (Alliance) adjuvant trastuzumab trial.

OBJECTIVES: The major clinical side effect of the ERBB2-targeted breast cancer therapy, trastuzumab, is a decline in the left ventricular ejection fraction (LVEF). Improved markers are needed to better identify patients susceptible to cardiotoxicity. METHODS: The NCCTG N9831 trial compared adjuvant doxorubicin and cyclophosphamide followed by either weekly paclitaxel (arm A); paclitaxel then trastuzumab (arm B); or concurrent paclitaxel and trastuzumab (arm C) in patients with HER2-positive breast cancer. A genome-wide association study was performed on all patients with available DNA (N=1446). We used linear regression to identify single nucleotide polymorphisms (SNPs) associated with decline in LVEF, adjusting for age, baseline LVEF, antihypertensive medications, and the first two principle components. RESULTS: In total, 618 863 SNPs passed quality control and DNA from 1191 patients passed genotyping quality control and were identified as Whites of non-Hispanic origin. SNPs at six loci were associated with a decline in LVEF (P=7.73×10 to 8.93×10), LDB2, BRINP1, chr6 intergenic, RAB22A, TRPC6, and LINC01060, in patients who received chemotherapy plus trastuzumab (arms BC, N=800). None of these loci were significant in patients who received chemotherapy only (arm A, N=391) and did not increase in significance in the combined analysis of all patients. We did not observe association, P<0.05, with SNPs previously associated with trastuzumab-induced cardiotoxicity at ERBB2, I655V, and P1170A. We replicated association, P<0.05, with SNPs previously associated with anthracycline-induced cardiotoxicity at CBR3 and ABCB1. CONCLUSION: Our study identified six putative novel cardiotoxicity loci in patients treated with combination chemotherapy and trastuzumab that require further investigation and confirmed known associations of anthracycline-induced cardiotoxicity.

The antineoplastic drug, trastuzumab, dysregulates metabolism in iPSC-derived cardiomyocytes.

BACKGROUND: The 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. RESULTS: Transcriptome 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). CONCLUSIONS: Our study suggests dysregulation of cardiac gene expression and metabolism as key elements of ERBB2 signaling that could potentially be early biomarkers of cardiotoxicity.

Intrinsic Subtype and Therapeutic Response Among HER2-Positive Breaty st Tumors from the NCCTG (Alliance) N9831 Trial.

Background: Genomic data from human epidermal growth factor receptor 2-positive (HER2+) tumors were analyzed to assess the association between intrinsic subtype and clinical outcome in a large, well-annotated patient cohort. Methods: Samples from the NCCTG (Alliance) N9831 trial were analyzed using the Prosigna algorithm on the NanoString platform to define intrinsic subtype, risk of recurrence scores, and risk categories for 1392 HER2+ tumors. Subtypes were evaluated for recurrence-free survival (RFS) using Kaplan-Meier and Cox model analysis following adjuvant chemotherapy (n = 484) or chemotherapy plus trastuzumab (n = 908). All statistical tests were two-sided. Results: Patients with HER2+ tumors from N9831 were primarily scored as HER2-enriched (72.1%). These individuals received statistically significant benefit from trastuzumab (hazard ratio [HR] = 0.68, 95% confidence interval [CI] = 0.52 to 0.89, P = .005), as did the patients (291 of 1392) with luminal-type tumors (HR = 0.52, 95% CI = 0.32 to 0.85, P = .01). Patients with basal-like tumors (97 of 1392) did not have statistically significantly better RFS when treated with trastuzumab and chemotherapy compared with chemotherapy alone (HR = 1.06, 95% CI = 0.53 to 2.13, P = .87). Conclusions: The majority of clinically defined HER2-positive tumors were classified as HER2-enriched or luminal using the Prosigna algorithm. Intrinsic subtype alone cannot replace conventional histopathological evaluation of HER2 status because many tumors that are classified as luminal A or luminal B will benefit from adjuvant trastuzumab if that subtype is accompanied by HER2 overexpression. However, among tumors that overexpress HER2, we speculate that assessment of intrinsic subtype may influence treatment, particularly with respect to evaluating alternative therapeutic approaches for that subset of HER2-positive tumors of the basal-like subtype.

Breast Cancer Clinical Trial of Chemotherapy and Trastuzumab: Potential Tool to Identify Cardiac Modifying Variants of Dilated Cardiomyopathy.

Doxorubicin and the ERBB2 targeted therapy, trastuzumab, are routinely used in the treatment of HER2+ breast cancer. In mouse models, doxorubicin is known to cause cardiomyopathy and conditional cardiac knock out of Erbb2 results in dilated cardiomyopathy and increased sensitivity to doxorubicin-induced cell death. In humans, these drugs also result in cardiac phenotypes, but severity and reversibility is highly variable. We examined the association of decline in left ventricular ejection fraction (LVEF) at 15,204 single nucleotide polymorphisms (SNPs) spanning 72 cardiomyopathy genes, in 800 breast cancer patients who received doxorubicin and trastuzumab. For 7033 common SNPs (minor allele frequency (MAF) > 0.01) we performed single marker linear regression. For all SNPs, we performed gene-based testing with SNP-set (Sequence) Kernel Association Tests: SKAT, SKAT-O and SKAT-common/rare under rare variant non-burden; rare variant optimized burden and non-burden tests; and a combination of rare and common variants respectively. Single marker analyses identified seven missense variants in OBSCN (p = 0.0045-0.0009, MAF = 0.18-0.50) and two in TTN (both p = 0.04, MAF = 0.22). Gene-based rare variant analyses, SKAT and SKAT-O, performed very similarly (ILK, TCAP, DSC2, VCL, FXN, DSP and KCNQ1, p = 0.042-0.006). Gene-based tests of rare/common variants were significant at the nominal 5% level for OBSCN as well as TCAP, DSC2, VCL, NEXN, KCNJ2 and DMD (p = 0.044-0.008). Our results suggest that rare and common variants in OBSCN, as well as in other genes, could have modifying effects in cardiomyopathy.

Improved Survival of HER2+ Breast Cancer Patients Treated with Trastuzumab and Chemotherapy Is Associated with Host Antibody Immunity against the HER2 Intracellular Domain.

The addition of trastuzumab to chemotherapy extends survival among patients with HER2(+) breast cancer. Prior work showed that trastuzumab and chemotherapy augments HER2 extracellular domain (ECD)-specific antibodies. The current study investigated whether combination therapy induced immune responses beyond HER2-ECD and, importantly, whether those immune responses were associated with survival. Pretreatment and posttreatment sera were obtained from 48 women with metastatic HER2(+) breast cancer on NCCTG (now Alliance for Clinical Trials in Oncology) studies, N0337 and N983252. IgG to HER2 intracellular domain (ICD), HER2-ECD, p53, IGFBP2, CEA, and tetanus toxoid were examined. Sera from 25 age-matched controls and 26 surgically resected HER2(+) patients were also examined. Prior to therapy, some patients with metastatic disease had elevated antibodies to IGFBP2, p53, HER2-ICD, HER2-ECD, and CEA, but not to tetanus toxin, relative to controls and surgically resected patients. Treatment augmented antibody responses to HER2-ICD in 69% of metastatic patients, which was highly associated with improved progression-free survival (PFS; HR = 0.5, P = 0.0042) and overall survival (OS; HR = 0.7, P = 0.038). Augmented antibody responses to HER2-ICD also correlated (P = 0.03) with increased antibody responses to CEA, IGFBP2, and p53, indicating that treatment induces epitope spreading. Paradoxically, patients who already had high preexisting immunity to HER2-ICD did not respond to therapy with increased antibodies to HER2-ICD and demonstrated poorer PFS (HR = 1.6, P < 0.0001) and OS (HR = 1.4, P = 0.0006). Overall, the findings further demonstrate the importance of the adaptive immune system in the efficacy of trastuzumab-containing regimens. Cancer Res; 76(13); 3702-10. ©2016 AACR.

Assessment of Tumor Heterogeneity, as Evidenced by Gene Expression Profiles, Pathway Activation, and Gene Copy Number, in Patients with Multifocal Invasive Lobular Breast Tumors.

BACKGROUND: Invasive lobular carcinoma (ILC) comprises approximately ~10-20% of breast cancers. In general, multifocal/multicentric (MF/MC) breast cancer has been associated with an increased rate of regional lymph node metastases. Tumor heterogeneity between foci represents a largely unstudied source of genomic variation in those rare patients with MF/MC ILC. METHODS: We characterized gene expression and copy number in 2 or more foci from 11 patients with MF/MC ILC (all ER+, HER2-) and adjacent normal tissue. RNA and DNA were extracted from 3x1.5 mm cores from all foci. Gene expression (730 genes) and copy number (80 genes) were measured using Nanostring PanCancer and Cancer CNV panels. Linear mixed models were employed to compare expression in tumor versus normal samples from the same patient, and to assess heterogeneity (variability) in expression among multiple ILC within an individual. RESULTS: 35 and 34 genes were upregulated (FC>2) and down-regulated (FC<0.5) respectively in ILC tumor relative to adjacent normal tissue, q<0.05. 9/34 down-regulated genes (FIGF, RELN, PROM1, SFRP1, MMP7, NTRK2, LAMB3, SPRY2, KIT) had changes larger than CDH1, a hallmark of ILC. Copy number changes in these patients were relatively few but consistent across foci within each patient. Amplification of three genes (CCND1, FADD, ORAOV1) at 11q13.3 was present in 2/11 patients in both foci. We observed significant evidence of within-patient between-foci variability (heterogeneity) in gene expression for 466 genes (p<0.05 with FDR 8%), including CDH1, FIGF, RELN, SFRP1, MMP7, NTRK2, LAMB3, SPRY2 and KIT. CONCLUSIONS: There was substantial variation in gene expression between ILC foci within patients, including known markers of ILC, suggesting an additional level of complexity that should be addressed.