Targeted Microarray Analyses Augment the Clinical Cytogenetic Diagnosis of Acute Lymphoblastic Leukemia (ALL): Submicroscopic Genetic Events Improve Diagnosis, Contribute to Risk Stratification, and Provide Genetic Markers for Minimal Residual Disease (MRD) Testing

Abstract 2690

Cytogenetic aberrations are key diagnostic and prognostic markers in ALL; however, suboptimal chromosome morphology, low lymphoblast mitotic activity in cell culture and the inability to detect subtle (≤ 5∼10 Mb) abnormalities often preclude a rapid and accurate ALL clinical cytogenetic workup. Recent genome-wide microarray studies have reported submicroscopic DNA copy number alterations (CNAs) in ALL that target key genes involved in B-cell and T-cell cellular processes (cell cycle regulation, differentiation, proliferation and survival) and alterations of these genes are rapidly being associated with clinical treatment and outcome (e.g., IKZF1, NUP214/ABL1, NUP214/SET, PTEN, PTPN2). To improve the cytogenetic diagnosis of ALL, we analyzed DNA samples from 22 newly-diagnosed, pediatric T-cell and 22 B-cell (16 newly diagnosed and 6 relapse) ALL patients (pts) using a 133K targeted oligo-based microarray. The aCGH results were compared to their cytogenetic, FISH and clinic-pathological findings. When sufficient material was available, locus-specific FISH studies were performed to confirm the submicroscopic CNAs. The 22 pediatric T-cell samples, obtained from the COG Cell Bank, showed the following karyotypes: del(6q) alone (n=10), del(6q) with one or two additional abnormalities (n=8), or normal karyotypes (n=4). The B-cell ALL karyotypes showed one to three abnormalities (n=10), ≥5 abnormalities (n=10), including hypodiploidy/hyperdiploidy/near-triploid/near tetraploidy, or normal karyotypes (n=2). Twelve B-cell ALL pts showed prognostically-significant translocations. By aCGH, CNAs were observed in all 44 cases, ranging from five to14 (median, 8) CNAs in T-cell and three to 35 (median, 10) CNAs in B-cell ALL. aCGH detected del(6q) in all 18 known T-cell pts (size range, 16.8 kb to 106 Mb) and in six B-cell pts. Submicroscopic aberrations detected in T-cell ALL included: CDKN2A (mono or bi-allelic) deletions (n=19), ranging in size from 24 kb to 6.76 Mb, including 6 focal deletions under 200 kb, a ∼77 Kb deletion in 1p33, resulting in a STIL/TAL1 fusion (n=8), other TAL1 or STIL deletions (n=3), PTEN deletions (n=6) ranging from 15 kb to 1 Mb (n=6), 49 kb biallelic GSTT1 deletions (n=4), and TLX3 rearrangements (n=2) including a case with a ABL1/NUP214 fusion and a focal biallelic PTPN2 deletion. Cryptic deletions in 4q31.3/FBXW7 and in 9q34 resulting in a SET/NUP214 fusion and duplication of MYB were observed in a single case. In B-cell ALL, recurring “cryptic” deletions were of IKZF1 (n=6), TOX (n=3), PAX5 (n= 5) CDKN2A (mono- or bi-allelic) (n=10), ETV6 (n=6), BTG1 (n=4), C20orf94 (n=3), EBF1 (n=2), TP53 (n=2), and miR650 (n=2). Single CNAs observed in B-cell ALL included: CDKN2C, LCK, BTLA, MECOM, TBL1XR1, AFF1, LEF1, HEF, RAG1, ATF7IP, JAK2, PTEN, ID4, CASC3, COMMD1, and the drug receptor gene NRCC1. Gains observed in residual or relapsed B-cell ALL were MYC, MLL, miR657, and at 13q31.3, which includes GPC5 and multiple miRNAs (latter also seen in three cases of T-cell), and PPP2R5A in a t(4;11) pt. Imbalances in TRG (7p14), TRB (7q34), and TRA/TRD (14q11.2) were noted in T-cell pts whereas IGH, IGK and/or IGL were clearly seen in B-cell pts. Three translocations, t(9;22), t(4;11) and t(12;21), were also detected by microarray using linear amplification followed by aCGH. These findings demonstrated that aCGH can improve the cytogenetic diagnosis of ALL, contribute to risk stratification, and provide genetic markers for MRD testing. Moreover, these results provide a rationale for the integration of targeted microarray technology in the clinical evaluation of ALL.