Whole genome sequencing facilitates patient-specific quantitative PCR-based minimal residual disease monitoring in acute lymphoblastic leukaemia, neuroblastoma and Ewing sarcoma.
In this study we use whole genome sequencing (WGS) data to identify tumour-specific DNA breakpoints for each patient, and demonstrate that these breakpoints provide reliable targets for Minimal Residual Disease (MRD) detection in multiple paediatric tumours.
Recent advances in genomics and bioinformatics, linked with fast throughput and cost reductions for whole genome sequencing (WGS) have facilitated the development of precision medicine programs aimed at improving patient survival. Whilst the primary focus of precision medicine is the rapid identification of targetable genetic alterations and new therapy recommendations, we hypothesised that WGS data would identify individualised, tumour-specific DNA breakpoints that were reliable targets for Minimal Residual Disease (MRD) analysis in patients regardless of their tumour diagnosis. Our study leveraged the WGS data collected through clinical trials run by the Zero Childhood Cancer personalised medicine program (ZERO) led jointly by Children’s Cancer Institute and the Kids Cancer Centre at Sydney Children’s Hospital. The study was driven by the need to provide advanced diagnostics for children with high-risk solid tumours, neuroblastoma (NB) and Ewing sarcoma (EWS), using a robust and non-invasive detection technology with utmost accuracy and sensitivity. We sought to enable monitoring of treatment response with greater sensitivity, to detect impending relapse earlier than currently possible and to more accurately determine when a patient is disease free.
Molecular MRD testing started as a prognostic tool in acute lymphoblastic leukemia (ALL) because slow responses to induction therapy are associated with high risk of relapse later. MRD testing has enabled the early identification of high-risk ALL patients and early treatment intensification to prevent relapse. More recently, MRD testing has been used to monitor ALL patients after haematopoietic stem-cell transplant or to rapidly evaluate the effectiveness of personalised treatment. Unlike ALL, developing patient-speciﬁc, DNA based MRD assays in NB and EWS has been limited by the absence of readily identiﬁable, tumour-speciﬁc targets and standardisation guidelines. Instead, MRD detection in these cancers has focused on detection of tumour-specific mRNA and gene fusion transcripts. However, expression of mRNA markers often shows high transcript level variability between primary and disseminated sites, and it is unclear whether these transcripts undergo treatment-related changes in expression.
Transitioning to DNA based MRD measurement in solid tumours was a challenge, which we addressed by drawing on the expertise of our Molecular Diagnostics team, who perform MRD testing nationally for children with ALL according to international guidelines and with quality assurance. By targeting DNA break points identified from WGS data, we have developed a framework for quantitative analysis and interpretation of patient-specific MRD tests that has potential applicability across multiple cancers and treatment protocols. The Computational Biology and Personalised Medicine teams designed analysis pipelines to ensure that a WGS-guided MRD assay could be developed and validated within 6 weeks of receipt of a patient’s diagnostic sample. The MRD in bone marrow or peripheral blood samples collected after therapy could then be measured with a turnaround of less than 3 days. A major achievement of this study is the implementation of robust DNA-based MRD detection in solid paediatric cancers based on the multi-laboratory standardization guidelines established by the Euro-MRD consortium for ALL.
In summary, in this study we used WGS data to develop MRD assays that can be applied to multiple paediatric tumour types. Using patient-specific assays on qPCR and ddPCR platforms, we were able to reproducibly measure the burden of disseminated disease. The patient-specific DNA markers were stable through the course of treatment and were able to track disease course. This provides the rationale for complementing or even substituting the current approaches for MRD detection in solid paediatric tumours with more sensitive and patient-specific DNA-based assays.
This work was made possible by funding from Children’s Cancer Foundation, Australia and the funding partners of the Zero Childhood Cancer Program.