Pediatric Acute lymphoblastic leukemia accounts for 25% of pediatric cancer patients, with approximately 85% expressing a B-cell immunophenotype (e.g., CD19, cCD79a, CD22, PAX5; B-ALL) and 15% expressing a T-cell immunophenotype (e.g., CD3 (cCD3), CD5, CD7; T-ALL). Advances in our understanding of the underlying oncogenesis, the association of genomic profiles with prognosis, and the stratified therapy based on risk classification have led to a high cure rate for most ALL cases. B-ALL can be grouped into at least 23 subgroups, each with class-defining genes or chromosome aberrations.¹ The genomic profile of T-ALL is also extensively characterized, with TCR rearrangements detected in most T-ALL, leading to overexpression of a variety of oncogenes. However, a clear relationship between genomic changes and risk stratification remains unclear in T-ALL.² Currently, a combination of laboratory tests are utilized to determine the genomic profile of ALL. While karyotyping remains a standard technology in clinical laboratories, it has limitations as a substantial number of ALL cases do not divide in vitro, leading to failed or normal karyotyping results (from normal bone marrow cells). Moreover, several fusions, including the chromosomally cryptic t(12;21)(p13;q22)ETV6::RUNX1 found in 25% of pediatric B-ALL, cannot be detected by karyotyping. Furthermore, karyotyping may fail to distinguish true hyperdiploid B-ALL (with a favorable prognosis) from the masked hypodiploid B-ALL (with a poor prognosis). FISH is also a standard assay, typically including probes for BCR::ABL1, KMT2A (MLL), ETV6::RUNX1, CEP4, CEP10 for pediatric B-ALL, and TCR for pediatric T-ALL. NGS is increasingly used in clinical laboratories for genome profiling. Targeted RNA NGS or whole transcriptome sequencing can detect gene fusions, which are common dominant drivers in ALL. Targeted DNA NGS is helpful in identifying drivers such as PAX5 alt, PAX5 P80R, IKZF1 indel, or IG or TCR-related rearrangements.³

MRD testing is an essential component of ALL management and can be used to adjust therapy. For instance, patients with favorable genomic changes but slow MRD clearance can be cured by increasing the intensity of their post-remission chemotherapy.^4,5 MRD technologies include next-generation flow cytometry, which can be used for almost all ALL patients, PCR assay for fusion-driving ALL, and patient-specific V(D)J sequencing for approximately 90% of ALL cases. Among these technologies, V(D)J NGS is the most sensitive, capable of detecting one cell in one million. Additionally, immunophenotyping drifting, which can occur during disease progression or be induced by immunotherapy, does not affect MRD testing by V(D)J sequencing but can impact MRD detection by flow cytometry.