PF188 NGS COMBINED WITH ASO REAL‐TIME PCR BASED MRD DETECTION ASSAY FOR PEDIATRIC ALL PATIENTS
Authors: K. Nicolaou, J. Chi, L. Koumas, L. Loizou, E. Karaoli, E. Papachristodoulou, A. Loizos, P. Costeas
Journal: HemaSphere (2019)

Background: Monitoring of minimal residual disease (MRD) has become routine clinical practice in frontline treatment of pediatric acute lymphoblastic leukemia (ALL) patients. MRD diagnostics has proven to be the strongest prognostic factor, allowing for risk group assignment into different treatment categories, ranging from significant treatment reduction to mild or strong intensification. MRD diagnostics is also guiding treatment decisions in relapsed ALL patients and patients undergoing stem cell transplantation. Aims: Our aim is to develop and apply a sensitive (≤ 10 –4 ), universal, accurate, reliable and fast assay for MRD detection in ALL patients. To date, flow cytometry and polymerase chain reaction (PCR) analysis of rearranged immunoglobulin and T‐cell receptor genes (allele‐specific oligonucleotide [ASO]‐PCR) are claimed to meet these criteria, but classical flow cytometry does not reach a solid 10 –4 , whereas classical ASO‐PCR is time‐consuming and labor intensive. Methods: LymphoTrack ® IGH + TRG Assay – MiSeq is applied on diagnosis sample using the Illumina MiSeq platform. This is used for the identification of clonal IGH VH‐JH and TRG V‐J rearrangements, DNA sequences and the distribution frequency of V and J segment utilization. The clone sequence is then compared against the germline sequences contained in the IMGT V‐QUEST databases for the identification of patient‐specific junctional regions following by the design of six different D‐N‐J specific ASOs primers for each clone according to UKALL protocol. Five point standard curve is used with triplicates at 5x10^‐5 and 1x10^‐5 for more reliable results at the critical lower level and six pooled negative controls for confirmation of the specificity of the assay. Results: Forty pediatric ALL patients have been tested at several time points. All informative patient sample dilutions reached 5x10^‐5, and most of the patients reached 1x10^‐5. The R 2 of standard curves were within the expected range (≥0.98), and all slopes within range (3.1–3.9) with an average of 3.21 +/‐ 0.16. In most negative control samples no amplification was detected. In 8 assays amplification was detected in two negative control samples. In these cases the CT value was more than 3 CT from the Quantitative Range (QR). Our coupled NGS‐ASO‐PCR method sensitivity was compared to Flow cytometry and Fusion Transcript Real Time PCR methods. NGS‐ASO based MRD method has detected MRD populations in several samples were Flow cytometry or real time PCR failed. None of the patient tested were positive by other methods and was negative by ASO based MRD method. Summary/Conclusion: In the ALL field, MRD has become part of diagnostic patient care. Consequently, standardized MRD diagnostics should be available for assessment of treatment response in each individual ALL patient, to be used for personalized medicine such as accurate risk group assignment with risk‐adapted treatment. The coupled NGS‐ASO‐PCR method seams to fulfill a series of requirements for acceptance in the field, such as broad availability, easy implementation, applicability in the vast majority of patients, sufficient sensitivity (quantitative range of 5x10^‐5) and fast (short turn‐around time, particularly for follow‐up samples).