Norton, Nadine

Abstract Introduction Doxorubicin is an anthracycline used as a chemotherapeutic drug for the treatment of a wide range of adult and pediatric cancers. Doxorubicin is associated with an increased risk of cardiomyopathy and heart failure with up to 10% of patients developing cardiac complications. A GWAS conducted of 1,191 patients from the N9831 clinical trial identified that cardiac gene expression and genetic variants of TRPC6 were associated with a decline in left ventricular ejection fraction (LVEF) (p=0.005 and p=1.6x10–6, respectively). TRPC6 is a non-selective cation channel expressed in heart and vascular tissue. TRPC6 participates in the pathogenesis of cardiac hypertrophy as a pathological response to chronic mechanical stress. Chronic activation has been found to promote cardiac fibrosis leading to heart failure. Purpose TRPC6 variants could be associated with increased risk of doxorubicin-induced cardiotoxicity. Data/tests to determine which patients may progress to cardiomyopathy and heart failure are currently lacking and there are no targeted treatments to prevent cardiomyopathy in these patients. Methods In preliminary in vivo data, B6.129 wild-type mice or TRPC6 knockout mice were treated with either 6x intraperitoneal saline or 4mg/kg doxorubicin injections (cumulative dose of 24mg/kg). Results We found doxorubicin increased cardiac vacuolation (male, p≤0.001 and female, p≤0.05) in WT mice compared to controls. Higher HW/BW ratio was observed in male TRPC6 knock out mice compared to wild-type mice when both were treated with doxorubicin (males, p=0.005 and females, p=0.19). Additionally, we found that doxorubicin-induced cardiac injury was significantly reduced in TRPC6 knock-out mice compared to wild-type mice based on reduced vacuolation (p=0.0004 males, p=0.03 females), with the effect being greater in male mice than female mice. Furthermore, a significant decrease in stroke volume (p=0.007), diastolic volume (p=0.01) and cardiac output (p=0.004) in wild-type male mice treated with doxorubicin compared to control and TRPC6 knock-out mice. Our in vitro preliminary data show that inhibition of TRPC6 using the TRPC6 inhibitor GsMTx-4 in human iPSC-derived cardiomyocytes significantly reduced doxorubicin-induced apoptosis (p<0.0001). Further we treated male mice with the TRPC6 inhibitor, GsMTx4, and found that they had less global longitudinal cardiac strain (p=0.04) and higher ejection fraction (p=0.01) compared to mice who were only treated with doxorubicin. Histologically we found that mice given the TRPC6 inhibitor had less fibrosis as measured by Trichrome stained heart sections (p=0.004) which could account for improvements in cardiac function. Conclusion TRPC6 could be a novel therapeutic target in the prevention of chemotherapy-induced cardiomyopathy and heart failure. Additionally genetic mapping of TRCP6 functional variants may provide a new screening tool to determine increased risk of developing heart failure. Funding Acknowledgement Type of funding sources: Private hospital(s). Main funding source(s): Mayo Clinic

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.

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.

522 Background: In the adjuvant setting, patients with HER2 breast cancer treated with trastuzumab and chemotherapy have superior survival compared to patients treated with chemotherapy alone. We previously showed that trastuzumab and chemotherapy induces HER2-specific antibodies which correlate with response to therapy in patients with HER2+ metastatic breast cancer. It remained unclear from those studies, however, whether the HER2-specific immunity played a role and if antibody immunity was associated with improved disease free survival in the adjuvant setting. In the present study, we addressed these questions by analyzing sera samples from a subset of patients enrolled in the NCCTG adjuvant 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 (22 Arm A; 14 Arm B, and 14 Arm C). Lambda IgG antibodies (to avoid detection of trastuzumab) to HER2 were measured and presented as an index (>0.2 was considered a positive response). Results: Prior to therapy, across all three arms, N9831 patients had similar mean HER2 IgG levels (0.19 units in Arm A, 0.14 in Arm B, and 0.23 in Arm C, P=0.85). Following treatment, the mean levels of antibodies increased in Arm B to 0.35 units and in Arm C to 0.56 units and were higher (p<0.001) than in Arm A where levels did not increase. The proportion of patients who demonstrated antibody immunity increased by 9% in Arm A, 50% in Arm B and 28% in Arm C (P=0.026). Although the event rate was low in this cohort, Cox modeling suggested that larger increases in antibodies were associated with improved disease free survival (HR=0.23; p=0.04). Conclusions: These results show that the increased antibody immunity observed in adjuvant patients treated with combination trastuzumab and chemotherapy is clinically significant and results from the inclusion of trastuzumab. The findings may have important implications for improving treatment outcomes in patients treated with trastuzumab.

521 Background: The addition of trastuzumab to chemotherapy improves responses to therapy and extends survival among patients with metastatic HER2 breast cancer. Several mechanisms have been proposed for the activity of this combination therapy. Trastuzumab, specifically, is thought to activate NK cells and blunt HER2 signaling. Prior work from us has shown that combination trastuzumab and chemotherapy induces HER2-specific antibodies which correlate with response to therapy. Despite that, it remains unclear whether the immunity that was induced was due to complexing of non-tumor derived HER2 or antigen derived from the tumor site. In the present work, we addressed this question by assessing if combination therapy induced epitope spreading to tumor antigens other than HER2. Methods: Pre-and post-treatment sera were obtained from 56 women enrolled in 2 NCCTG clinical trials, N0337 and 98-32-52. IgG antibodies to HER2 intracellular domain (HER2), p53, IGFBP2, CEA and tetanus toxoid (TT) were examined using ELISAs. Sera from an age-matched group (N=56) of controls and 12 patients treated in the adjuvant setting were also examined. Results: Prior to therapy, metastatic patients had higher IgG levels (≥ 2-fold) to p53 and HER2 but not CEA, IGFBP2 and TT, relative to the controls. Similarly, adjuvant patients had elevated IgGs to multiple tumor antigens prior to therapy, relative to controls. Following therapy, levels of IgG to IGFBP2, HER2, and p53 increased in 81% of metastatic patients, with mean increases of 3.2 (±0.6 sem), 6.2 (±2.7) and 2.7 (±0.7) fold, respectively (p<0.05). Levels of antibodies to TT and CEA were not elevated by treatment. In contrast, IgGs were not increased in adjuvant patients; consistent with the idea that immunity depends on the presence of threshold levels of antigens. Conclusions: These results show that combination treatment induces adaptive immunity to antigens released by tumor and that metastatic patients remain capable of responding immunologically to their cancer. Thus, in metastatic breast cancer patients, combination trastuzumab and chemotherapy may behave as a vaccine.

RXRA is an endothelial- and early stage–predominant therapeutic target for treating anthracycline-induced cardiotoxicity.

AbstractPurpose: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.