Single Center Experience Combining Multiple Tools for Genetic Diagnosis of B-Cell Acute Lymphoblastic Leukemia

Introduction: Pediatric B-cell acute lymphoblastic leukemia (B-ALL) is characterized by distinct genetic profiles essential for prognosis and treatment. About 70% of B-ALL cases can be classified into well-known genetic subtypes, while 30% remain under the "B-other" category. Accurate genetic diagnosis requires multiple techniques; however, their accessibility varies significantly among laboratories.

This study investigates an integrated approach to classify B-ALL into genetic subtypes, elucidate their frequency and clinical significance, and identify the most effective diagnostic methods.

Methods: This study included 175 pediatric patients with B-ALL treated at Vall d’Hebron University Hospital from 2014 to 2023. Diagnostic techniques included karyotyping, FISH, RT-PCR, MLPA, targeted NGS (t-NGS), and Optical Genome Mapping (OGM).

Results: Utilizing the combined diagnostic approach, we classified 83% (n=145) of B-ALL patients into 17 genetic subtypes. Karyotyping and FISH identified abnormalities in 73% (n=128) of cases, with t-NGS and OGM contributing an additional 10% (n=17) to the classification rate. Among the 145 patients with a defined genetic diagnosis, 71% harbored classical abnormalities: high hyperdiploidy (HeH) (n=46), ETV6::RUNX1 (n=36), BCR::ABL1-like (n=17), KMT2A rearrangements (n=8), TCF3::PBX1 (n=6), BCR::ABL1 (n=4), iAMP21 (n=4), and hypodiploidy (n=3). The remaining 29% (n=51) were classified as B-other, with specific genetic subtypes identified 21 cases, including ZNF384 rearrangements (n=6), PAX5 alterations (PAX5alt) (n=4), PAX5 P80R (n=3), DUX4 rearrangements (n=3), IGH rearrangements (n=3), NUTM1 rearrangements (n=1), and IKZF1 N159Y (n=1).

Copy number alterations (CNA) were detected in 60% of patients (median: 1 deletion), with the most frequent deletions observed in ETV6, CDKN2A/B, PAX5, and IKZF1 genes. CNA profiles were classified as good-risk (CNA-GR; n=69) or poor-risk (CNA-PR; n=51) according to Hamadeh et al.(1). ETV6::RUNX1 cases showed frequent deletions of ETV6 and PAR1 (p<0.01). BCR::ABL1-like were associated with a CNA-PR, with significant deletions in IKZF1 and PAR1 region (p<0.001). The B-other subgroup displayed a high deletion frequency, particularly in PAX5, CDKN2A/B, IKZF1 and RB1. Specifically, PAX5alt and ZNF384r showed a higher number of deletions (median 3 and 2.5, respectively).

Pathogenic mutations were found in 71% of patients (median: 1.5 mutations), with KRAS, NRAS, and PAX5 being the genes most commonly affected. BCR::ABL1-like patients had a higher mutation frequency, and all JAK2 mutations occurred in CRLF2r patients. The B-other subtype had prevalent KRAS, NRAS, PAX5 and IKZF1 mutations. Concretely, PAX5alt group had the highest median number of mutations (3.5), and all ZNF384r patients had mutations in EGFR and EZH2.

A total of 144 B-ALL patients were treated according to SEHOP-PETHEMA 2013 guidelines, which use risk-adapted treatment regimens. The entire B-ALL cohort showed high OS and EFS rates of 88.9% and 78.9%, respectively, with a relapse rate of 17.4%. Genetic subtypes significantly influenced outcomes: HeH and ETV6::RUNX1 had the best outcomes (5-years 98.5% OS, 94.9% EFS; p<0.001), while B-other patients showed intermediate-risk profiles (5-years 87.8% OS, 66.8% EFS); specific ZNF384r and PAX5 P80R subtypes exhibited favorable outcomes. BCR::ABL1-like, concretely CRLF2r, had poor outcomes (5-years 52.5% OS, 26.5% EFS), similar to KMT2Ar and hypodiploidy subtypes (5-years 50% OS).

Discussion: An integrated diagnostic approach classified 83% of B-ALL cases into 17 subtypes, the distribution of genetic subtypes aligned with previous studies. Although 58% of B-other cases (17% of all B-ALL) in our study remained unclassified, this is comparable to other large studies like Schwab et al.(2), where even using WGS, 53% were left unclassified.

The study highlights the importance of combining multiple diagnostic tools for accurate genetic classification and risk stratification in pediatric B-ALL. Our study confirms the utility of conventional techniques combined with t-NGS and OGM for comprehensive genetic diagnosis. Karyotyping and FISH remain robust, cost-effective, and widely accessible for rapid chromosomal abnormality assessment, while NGS and OGM significantly improve diagnostic yield, especially in cases with incomplete cytogenetic results.