Building a Targeted Approach to Assess Risk of Anthracycline Induced Cardiomyopathy Amongst Paediatric Cancer Patients

Introduction

Anthracyclines are chemotherapeutic agents used in the treatment of over 70% of childhood and adolescent malignancies and their use has significantly improved cancer survival rates (Lipshultz, Alvarez et al. 2008). However, a major side effect of this drug class is the potential to develop subclinical, irreversible and fatal cardiomyopathy with the risk of sudden cardiac death. Despite the high incidence of cardiotoxicity in paediatric and adolescent cancer survivors as well as the emerging evidence that alteration in the genes that control drug absorption, distribution, metabolism and excretion (ADME) may be predictive of a patient's sensitivity to this complication, there are no current standard approaches to identify the 'at risk' group (Visscher, Amstutz et al. 2011). Consequently, there is no way to tailor therapy according to a patient's individual risk.

This study aims to create a functional assay that will assess patient sensitivity to anthracyclines using a cohort of paediatric cancer survivors, with and without, evidence of anthracycline cardiotoxicity (ACT). A novel, exciting approach will be used through the creation of patient specific cardiomyocytes from induced pluripotent stem cells (iPSC). Through the exposure of iPSC derived cardiomyocytes to anthracyclines, a series of functional assays will be performed to help determine the patient's sensitivity to this drug toxicity. The cardiomyocytes in both cases and controls will be assessed for variations in the genes involved in drug ADME. The genetic analysis will help to validate the results of our collaborators at the British Columbia (BC) Children's Hospital (Vancouver) and the Canada Pharmacogenomic Network for Drug Sensitivity (CPNDS).

Methods: An audit of paediatric patients diagnosed across two tertiary institutes between 2000-2015 was performed. From this patients were deemed to be either cases (no evidence of cardiac damage) or controls (evidence of cardiac damage). The degree of cardiac damage was defined as severe (ejection fraction < 24 %), intermediate (ejection fraction 24 - 27 % or a decline of > 10 % ejection fraction from baseline). Patients were then enrolled to the study upon routine presentation to clinic. Upon enrolment peripheral blood mononuclear cells (PBMC) were collected for DNA/RNA storage and a cell pellet was cryopreserved for iPSC development. Following storage, the aims of the project are to 1) Establish a biobank from anthracycline treated childhood cancer survivors, including both sensitive and resistant patients (cases and controls). 2) To confirm the capacity of patient iPSC derived cardiomyocytes to predict ACT and to explore the mechanistic basis and functional responses of human cardiomyocytes to anthracyclines. 3) To perform detailed genomic profiling of the cohort and assess if genetic variation in ADME genes is predictive of ACT in the Australian paediatric population. 4) To document the diastolic changes evident on echocardiography and correlate these findings with long-term anthracycline cardiotoxicity in all enrolled patients.

Results: To date 56 cases and 199 controls have enrolled onto the study and had their blood processed accordingly. The median age of the cases is 11.5 years and controls 8.0 years. Of the 56 cases, 52 % (n = 29) are female and 48 % (n = 27) are male. The predominant anthracycline exposure in the cases was doxorubicin alone (47.3 %), daunorubicin and doxorubicin (29.1 %) and daunorubicin alone (14.1 %). Overall 63 % of cases and controls had a diagnosis of Acute Lymphoblastic leukaemia. The median anthracycline dose exposure in the cases was 172 mg/m² (range 22 - 438) versus 96 mg/m² (range 18 - 389) in the controls. To date we have successfully been able to derive cardiomyocytes from 8 cases and 8 controls and have begun a range of viability assays to assess susceptibility to anthracycline exposure.

Conclusions: We hope over the next 12 months to complete whole exome sequencing and microarray SNP analysis to further characterise the genetic basis of anthracycline cardiomyopathy. Furthermore, using human induced pluripotent stem cell derived cardiomyocytes as a functional platform we hope to further characterise the molecular basis of the disease and create a novel functional assay to stratify patients according to their risk of anthracycline induced cardiomyopathy.