MINIMAL RESIDUAL DISEASE ASSESSMENT BY DIGITAL DROPLET PCR USING PATIENT- MINIMAL RESIDUAL DISEASE ASSESSMENT BY DIGITAL DROPLET PCR USING PATIENT- SPECIFIC FUSION GENES IN PHILADELPHIA-LIKE ACUTE LYMPHOBLASTIC LEUKEMIA

Background Background Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), included in the 2022 WHO classification1 , displays a gene expression profile resembling that of Ph+ ALL. Most cases carry individual fusion genes involving tyrosine kinases or cytokine receptor rearrangements. Ph-like ALL is associated with a lower response to induction treatment, a higher relapse rate and an unfavorable outcome. Minimal residual disease (MRD) could be important to guide management also in this ALL subgroup. Currently, real-time quantitative PCR (RQ-PCR) of clonal immunoglobulin/T-cell receptor (IG/TR) gene rearrangements is mostly used for MRD assessment. Unlike in other ALLs, IG/TR MRD monitoring seems to correlate less strictly with response to treatment and outcome2 . Aims Aims Given the frequent presence of gene rearrangements in Ph-like ALL, we aimed at assessing MRD by digital droplet PCR (ddPCR) based on patient-specific fusion genes to investigate their predictive role on clinical outcome. Methods Methods The Ph-like signature was assessed using the B C R / A B L 1 -like predictor tool3 . At diagnosis, targeted-RNA sequencing (RNA-seq) was carried out to identify patient-specific lesions. Whenever feasible, we designed primers and probes starting from the fusion transcript sequence documented by RNA-seq, to perform MRD analysis by ddPCR. The probe (FAM-labeled) was designed for each fusion gene and the A B L 1 gene (HEX-labeled) was used to calculate the ratio between the target and control gene. DdPCR was performed as described4 . MRD was also monitored by IG/TR RQ-PCR. Results Results RNA-seq led to the identification of patient-specific fusion genes in 48/87 cases (55%, 17 ABL-class genes rearranged). So far, in 2 cases, we performed MRD analysis by ddPCR, using the fusion transcript (A B L 2 : : R C S D 1 and B C R : : F G F R 1 respectively), at available timepoints and documented a positive signal. Interestingly, while 1 case was positive also by IG/TR RQ-PCR with at least a 1-log difference, the second case was negative by IG/TR. Summary/Conclusion These preliminary results suggest that in Ph-like ALL cases carrying a fusion gene, and possibly also in other settings, the specific lesion can be used for MRD monitoring, in addition to the IG/TR marker, in an attempt to increase MRD sensitivity and specificity, and to further improve patients’ management. Additional patients are undergoing this dual monitoring strategy.