Genomic Analysis of the Clonal Origins of Relapsed Acute Lymphoblastic Leukemia
Authors: Mullighan Charles G , Phillips Letha A , Su Xiaoping , Ma Jing , Miller Christopher B , Downing James R. 
Published: April 2020

Abstract
Relapsed acute lymphoblastic leukemia (ALL) is the fourth most common pediatric malignancy and carries a dismal prognosis. To gain insights into the genetic alterations responsible for relapse, we performed genome-wide analysis of matched diagnosis and relapse samples from 61 pediatric ALL patients, including 47 B-progenitor and 14 T-ALL cases. All samples were flow sorted as required to ensure at least 80% tumor cell purity prior to DNA extraction. DNA copy number abnormality (CNA) and loss-of-heterozygosity (LOH) data was generated using Affymetrix SNP 6.0 arrays (1.87 million markers) for 47 diagnosis-relapse pairs, and 500k arrays for the remainder. Remission marrow samples were also examined for 48 cases. Analysis of diagnostic samples identified 10.8 CNAs per B-ALL case and 7.4 per T-ALL case. The most frequent target of CNAs at diagnosis were deletions of genes involved in B-lymphoid development (49% of B-ALL cases, involving PAX5 and IKZF1 in 12 cases each), CDKN2A/B in 36% of B-ALL and 71% of T-ALL, and ETV6 (11 B-ALL cases). At relapse, we observed a striking degree of change in the number, extent, and nature of CNAs. In B-ALL the average number of CNAs increased from 10.8/case at diagnosis to 14.0/case at relapse (P=0.0005), with the majority of this change due to an increase in deletions. By contrast, no difference in the number of CNAs was observed between diagnosis and relapse in T-ALL. Most relapse samples (54 of 61) harbored some of the CNAs present at diagnosis, and had identical or similar antigen receptor locus deletions, indicating a common clonal origin. Nevertheless, 92% of relapse cases exhibited significant changes in CNAs, including the acquisition of new lesions (34%), loss or alteration of lesions present at diagnosis (12%), or both acquisition of new lesions and loss of diagnosis lesions (46%). In the cases where a clear clonal relationship existed, almost half of the relapse clones were derived from a pre-leukemic cell and not from the clone predominating at diagnosis. The most frequent targets of relapse-acquired CNAs were CDKN2A/B, ETV6, and regulators of B-lymphoid development. 18 cases developed new CDKN2A/B deletions, with 70% showing bi-allelic loss; 11 developed new ETV6 lesions; and 15 developed new or more extensive CNAs involving B-lymphoid regulators. In contrast to diagnosis where PAX5 is most frequently involved, at relapse Ikaros and related gene family members were most common (IKZF1, 8 cases; IKZF2, 2 cases; IKZF3, 1 case). Other CNAs previously identified at diagnosis were also detected as new lesions at relapse, including deletions of ADD3, ATM, BTG1, FHIT, KRAS, NF1, PTCH, TBL1XR1, TOX and WT1, suggesting that these lesions contribute to treatment resistance. To further define the biological pathways most frequently altered by relapse–acquired CNAs, the genes within altered regions were categorized into 148 different biological pathways and each cases was then assessed for overrepresentation of these pathways. This analysis identified cell cycle and B-cell transcriptional regulatory pathways as the most significantly targeted pathways at relapse. To determine whether CNAs identified at relapse were present at low levels at diagnosis or were acquired during therapy, we developed qualitative genomic PCR assays for deletions involving ADD3, C20orf94, DMD, ETV6, IKZF2 and IKZF3, and tested corresponding diagnosis and relapse samples. This analysis detected evidence of the relapse clone at diagnosis in 7 of 10 cases tested, indicating that in the majority of cases, CNAs that emerge in the predominant clone at relapse are present at low levels at diagnosis and are selected for during treatment. These results provide critical insights into the spectrum of genetic lesions that underlie ALL relapse. Although our data are limited to a single class of mutations (CNAs), they demonstrate that no single genetic lesion or alteration of a single pathway is responsible for relapse. Instead, a diversity of mutations appear to contribute to relapse with the most common alterations targeting key regulators of tumor suppression, cell cycle control, and lymphoid/B cell development. Notably, few lesions involved genes with roles in drug import, metabolism, export and/or response, (an exception being the glucocorticoid receptor gene NR3C1) suggesting that that the mechanism of relapse is more complex than simple “drug resistance”.