Abstract
Recurrent clonal genetic alterations are the hallmark of Acute Lymphoblastic Leukemia (ALL) and govern the risk stratification, response to treatment and clinical outcome. In this retrospective study conducted on ALL patient samples, the purpose was to estimate the copy number alterations (CNAs) in ALL by digitalMLPA (dMLPA), validation of the dMLPA data by conventional MLPA and RT-PCR, and correlation of CNAs with Minimal Residual Disease (MRD) status. The ALL patient samples (n = 151; B-ALL, n = 124 cases and T-ALL, n = 27 cases) were assessed for CNAs by dMLPA for detection of sub-microscopic CNAs and ploidy status. This assay allowed detection of ploidy changes and CNAs by multiplexing of karyotyping probes and probes covering 54 key gene targets implicated in ALL. Using the dMLPA assay, CNAs were detected in ~89% (n = 131) of the cases with 66% of the cases harboring ≥3 CNAs. Deletions in CDKN2A/B, IKZF1, and PAX5 genes were detectable in a quarter of these cases. Heterozygous and homozygous gene deletions, and duplications were observed in genes involved in cell cycle control, tumor suppression, lineage differentiation, lymphoid signaling, and transcriptional regulators with implications in treatment response and survival outcome. Distinct CNAs profiles were evident in B-ALL and T-ALL cases. Additionally, the dMLPA assay could reliably identify ploidy status and copy number-based gene fusions (SIL-TAL1, NUP214-ABL, EBF1-PDGFRB). Cases of B-ALL with no detectable recurrent genetic abnormalities could potentially be risk stratified based on the CNA profile. In addition to the commonly used gene deletions for risk assessment (IKZF1, EBF1, CDKN2A/B), we identified a broader spectrum of gene alterations (gains of- RUNX1, LEF1, NR3C2, PAR1, PHF6; deletions of- NF1, SUZ12, MTAP) that significantly correlated with the status of MRD clearance. The CNAs detected by dMLPA were validated by conventional MLPA and showed high concordance (r = 0.99). Our results demonstrated dMLPA to be a robust and reliable alternative for rapid detection of key CNAs in newly diagnosed ALL patients. Integration of ploidy status and CNAs detected by dMLPA with cytogenetic and clinical risk factors holds great potential in further refinement of patient risk stratification and response to treatment in ALL.
Highlights
Acute lymphoblastic leukemia (ALL), the most common childhood cancer manifests as a clinically and genetically heterogenous malignancy
The frequency distribution of copy number alterations (CNAs) detected in B ALL T-cell acute lymphoblastic leukemia (T-ALL) ETP-ALL KARYOTYPE (B-ALL) (n = 121) were predominantly heterozygous or homozygous deletions in IKZF1 (22%) and CDKN2A/B (22%) followed by PAX5 (19%) (Figure 2A, Supplementary Table S1)
In the 27 T-ALL cases (18% of total ALL cases), deletions were noted in 59% (n = 16) cases primarily in the CDKN2A/B gene locus followed by MTAP (∼33%), 15% in MLLT3/ PTEN/ JAK2, and
Summary
Acute lymphoblastic leukemia (ALL), the most common childhood cancer manifests as a clinically and genetically heterogenous malignancy Both B-cell and T-cell ALL are primarily initiated by recurrent chromosomal translocations and driven by accumulation of distinct constellations of gross and sub-microscopic somatic genetic alterations including copy number alterations (CNAs), aneuploidy, structural variants, and DNA sequence mutations [1,2,3,4]. Several of these lesions are important determinants of the risk of treatment failure and disease relapse but a universal agreement for inclusion of these genetic alterations in clinical risk stratification is still lacking. In addition to gross chromosomal rearrangements, partial, or complete deletions of the IKZF1, CDKN2A/B, EBF1, and RB1 genes have emerged as independent predictors of poor outcome in B-ALL [10,11,12]
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