Abstract 5931: Unraveling the genetic complexity of Ph-like ALL: development of a cost-effective diagnostic qPCR panel

Abstract

Background: Advancements in high-throughput sequencing have significantly expanded our understanding of B-cell Acute Lymphoblastic Leukemia (B-ALL). In particular, whole-transcriptome sequencing (WTS) has facilitated the discovery of the Ph-like ALL subtype. However, clinical integration of WTS remains challenging due to high costs and lack of standardized workflows. Ph-like ALL is marked by genetic heterogeneity, including diverse fusions and mutations, which complicates its diagnosis and limits targeted therapeutic options. This genetic complexity correlates with poor prognosis, emphasizing the need for accessible and effective diagnostic approaches. Aim: To develop a cost-effective, qPCR-based diagnostic panel for its detection and classification in clinical settings. Methods: A cohort of 394 newly diagnosed B-ALL cases were analyzed using RT-PCR and FISH for initial genetic screening. WTS was employed for comprehensive fusion detection, mutation analysis, and subtype classification. Kaplan-Meier survival analysis was used to evaluate patient outcomes, and a qPCR-based likelihood scoring model was developed based on differential gene expression (DEGs) to identify Ph-like ALL. Results: Whole transcriptome sequencing revealed that 13.1% of the B-ALL cases have Ph-like phenotype, found in different age groups: 9.7% in pediatric patients (n=15), 20.5% in adolescents and young adults (AYA, n=8), and 50% in adults (n=3). Ph-like ALL cases predominantly exhibited fusions involving JAK-STAT pathway genes (n=10), ABL-class genes (n=9), or NTRK1 (n=1), in addition to a novel RUNX1::FGF13 fusion (n=2). Differentially expressed genes were tested for diagnostic potential, with 9 genes (FZH2, PON2, CA6, OLFML2A, BAALC, PRKCZ, ZFPM2, TCFL5 and ENPP2) demonstrating significant ROC (p<0.0001 and sensitivity and specificity >80%) curves in a discovery cohort (n=79). This likelihood model also effectively distinguished Ph-like ALL in a validation cohort (n=150). Additionally, survival analysis revealed significantly worse event-free survival (EFS) and overall survival (OS) in Ph-like ALL patients compared to non-Ph-like patients (EFS: 0% vs. 43.3%, HR 3.40, p=0.006; OS: 0% vs. 45.4%, HR 3.57, p=0.005). Conclusion: This study underscores the genetic diversity of Ph-like ALL. The poor prognosis of Ph-like ALL, marked by significantly reduced EFS and OS, highlights the need for targeted therapeutic approaches. The cost-effective qPCR-based diagnostic panel provides a promising tool for the early detection of Ph-like ALL, offering potential for improved patient outcomes through timely intervention.