A high-throughput sequencing approach for minimal residual disease detection in acute lymphoblastic leukemia
Session type: Proffered paper sessions
Minimal residual disease (MRD) analysis is the standard of care in childhood acute lymphoblastic leukaemia (ALL) and is integral to the personalised risk stratification onto appropriate intensity chemotherapy. MRD assessment by real time quantitative PCR (qPCR) is the current gold standard method due to its increased sensitivity and ease of standardisation compared to the alternative flow cytometric approach. Current techniques for identifying biomarkers for quantitation of MRD levels involves; multiple PCRs, gel electrophoresis and Sanger sequencing. These methods are expensive, time consuming and not applicable to all patients. Newer techniques, such as high-throughput sequencing (HTS) are now able to be used instead of the current standard methods. We have developed and applied an approach in a number of real-life clinical scenarios.
HTS techniques are a promising alternative in driving changes to MRD methodologies. HTS has high-throughput, rapid turnaround times, relatively low costs and increased sensitivity. We used BIOMED- 2 primers with standard PCR reagents to generate libraries, and utilised Promega Pronex size selection beads for dual clean up of the PCR products. Tapestation and Qubit platforms were used to assess library quantity and quality.
Markers identified by HTS were used to track MRD using standard real-time quantitative PCR. In our study, we show the clinical benefits of utilising HTS to screen diagnostic samples and its necessity, especially where traditional screening techniques fail. We have provided practical evidence that current MRD diagnostic marker screening should be replaced by an HTS approach.
In our study, we show the clinical benefits of utilising HTS to screen diagnostic samples and its necessity, especially where traditional screening techniques fail. We have provided practical evidence that current MRD diagnostic marker screening should be replaced by an HTS approach. Our study demonstrates that HTS can identify suitable diagnostic markers, including in cases where traditional screening has been unsuccessful. We now routinely use HTS to screen diagnostic samples for clonal rearrangements and qPCR for follow up MRD.