Improving risk stratification for pediatric patients with rhabdomyosarcoma by molecular detection of disseminated disease

Background Survival of children with rhabdomyosarcoma that suffer from recurrent or progressive disease is poor. Identifying these patients upfront remains challenging, indicating a need for improvement of risk stratification. Detection of tumor-derived mRNA in bone marrow (BM) and peripheral blood (PB) using reverse-transcriptase quantitative PCR (RT-qPCR) is a more sensitive method to detect disseminated disease. We identified a panel of genes to optimize risk stratification by RT-qPCR. Methods Candidate genes were selected using gene expression data from rhabdomyosarcoma and healthy hematological tissues, and a multiplexed RT-qPCR was developed. Significance of molecular disease was determined in a cohort of 99 Dutch patients with rhabdomyosarcoma (72 localized and 27 metastasized) treated according to the EpSSG RMS2005 protocol. Findings We identified the following 11 rhabdomyosarcoma markers: ZIC1, ACTC1, MEGF10, PDLIM3, SNAI2, CDH11, TMEM47, MYOD1, MYOG, PAX3/7-FOXO1. RT-qPCR was performed for this 11-marker panel on BM and PB samples from the patient cohort. Five-year EFS was 35.5% (95%CI 17.5-53.5%) for the 33/99 RNA-positive patients, versus 88.0% (95%CI 78.9-97.2%) for the 66/99 RNA-negative patients (p<0.0001). Five-year OS was 54.8% (95%CI 36.2-73.4%) and 93.7% (95%CI 86.6-100.0%), respectively (p<0.0001). RNA panel-positivity was negatively associated with EFS (Hazard Ratio 9.52 95%CI (3.23-28.02), while the RMS2005 risk group stratification was not, in the multivariate Cox regression model. Interpretation This study shows a strong association between PCR-based detection of disseminated disease at diagnosis with clinical outcome in pediatric patients with rhabdomyosarcoma, also compared to conventional risk stratification. This warrants further validation in prospective trials as additional technique for risk stratification