Diagnostic Utility of Multimodal Genomic Profiling for Molecular Classification and MRD Assessment in Adult B-Cell Acute Lymphoblastic Leukemia

Genomic markers define molecular subtypes and measurable residual disease (MRD) targets in B-cell acute lymphoblastic leukemia/lymphoma (B-ALL) and are essential determinants of treatment. Current diagnostic approaches typically involve serial multi-step testing utilizing conventional cytogenetics (CC)/FISH and molecular genetic (RT-qPCR, MLPA, clonality PCR, NGS panel) techniques which are time and sample consuming and ultimately may not adequately identify genomically complex B-ALL subtypes. In contrast, single-step comprehensive genomic profiling by whole genome and whole transcriptome sequencing (WGS/WTS) may be more efficient for the molecular classification of established and newly described entities which are of increasing therapeutic relevance. We have instituted a multimodal platform for molecular testing in B-ALL performing WGS/WTS in parallel with deep NGS-based immunoglobulin (IG) rearrangement MRD and exploratory DNA-breakpoint based MRD assays. We aimed to determine the utility of this approach for subtype classification compared to a standard-of-care diagnostic approach of CC/FISH testing.

Forty-two consecutive adult patients underwent both standard-of-care diagnostic testing and WGS/WTS. 20/42 (48%) patients had an abnormal CC/FISH result supporting classification into recognized molecular subgroups. WGS/WTS assessment incorporating somatic coding and non-coding mutations, structural variants, fusions, copy number abnormalities and gene expression subtype prediction (ALLSorts, https://github.com/Oshlack/ALLSorts) was performed with concordant results in all 20 patients. 16/22 patients that were unclassified by CC/FISH were successfully reclassified by WGS/WTS including subtypes enriched for cryptic rearrangements (Ph-like, DUX4, MEF2D) and groups characterized by heterogeneous genomic alterations or a distinctive gene expression signature (PAX5alt, ZEB2/CEBP). A low hypodiploid karyotype was observed in two cases with an apparently normal karyotype by CC. The six patients who remained without a subtype defining driver genetic alteration after comprehensive testing frequently harbored novel IGH translocations or a Ph-like expression signature but without a described fusion.

In order to understand the relative contribution from WGS versus WTS, analysis of 36 patients was performed using a truth classification. WGS and WTS produced equivalent classifications for 22 cases. Two cases were based solely on WGS findings (iAMP21 and ZEB2/CEBP) and three cases were based solely on WTS findings (DUX4). Importantly the combination of both WGS and WTS was critical to correctly classify nine cases (Ph-like and PAX5alt).

MRD was assessed by a sensitive NGS assay to IG rearrangements (Adaptive Biotechnologies) and by quantitative probe-based droplet digital PCR (ddPCR) assays designed to structural rearrangement DNA breakpoints from genome data (analytical sensitivity 10 ^-4). Patient-specific ddPCR assays were designed to eight structural variants (KMT2A and IGH translocations, and IKZF1 deletions) in seven patients and assessed in 36 remission samples with parallel testing by multiparametric flow cytometry (MFC). Concordant MFC and ddPCR was observed in 30/36 samples (19 MRD ^pos, 11 MRD ^neg). Discordances included two MRD ^pos by MFC-only and four MRD ^pos by ddPCR-only; the latter often occurring in the setting of antigen directed therapy or in ALL with a less informative immunophenotype, demonstrating the additional utility of non-MFC based MRD assessment in specific clinical settings. 27/42 patients in our cohort had ≥1 genomic structural rearrangement identified by WGS that could be used for patient-specific MRD monitoring to complement existing MRD assessment.

In conclusion WGS/WTS provided a molecular subtype classification in 86% of our cohort compared to 48% by standard-of-care diagnostic testing highlighting that CC/FISH alone is inadequate for contemporary molecular classification of B-ALL, which may have implications for treatment decisions. Importantly, the combination of WGS and WTS was superior to WGS-only or WTS-only for correct molecular subtype assignment. This approach has the potential to improve risk assessment in adult B-ALL and the routine feasibility, improvement in clinical outcomes and health economic impact warrant further assessment.