Mutational Landscape and Temporal Evolution during Treatment of Relapsed Acute Lymphoblastic Leukemia

Introduction

Relapsed childhood acute lymphoblastic leukemia (ALL) is a leading cause of cancer-related death in children and has poor prognosis due to acquired drug resistance. However, the clonal evolution leading to drug resistance at ALL relapse is incompletely understood.

Methods

We performed whole-genome sequencing (WGS) of samples at diagnosis and relapse from 103 Chinese patients, most of whom were enrolled on the Shanghai Children's Medical Center (SCMC) ALL2005 frontline treatment protocol. We also performed ultra-deep sequencing at 5,000-50,000X coverage of 211 serial bone-marrow samples from 17 of these patients (3-23 per case) collected during ALL therapy. Fifteen ALL subtypes were identified based on copy number variation, structural re-arrangement and gene fusion by WGS and RNA-Seq analysis. Cases were selected based on sample availability with no bias on age, gender, cytogenetics, or immunophenotype compared to the characteristics of all relapsed cases at SCMC; however only one CRLF2-rearranged case was observed suggesting that CRLF2-rearrangement could be a rare event in Asian patients. Functional impact of novel mutations was assessed by ectopic expression in ALL cell lines.

Results

Relapse-specific somatic alterations were significantly enriched in 12 genes (NT5C2, NR3C1/2, PRPS1/2, TP53, CREBBP, MSH2/6, PMS2, WHSC1, and FPGS) predominantly involved in drug metabolism and response pathways. These somatic alterations were present in over 50% (56/103) of relapsed ALLs and were enriched in patients with early relapse (9-36 months from diagnosis) compared to very early (<9 months) or late relapse (>36 months) patients. NR3C1 mutations resulted in loss of glucocorticoid receptor transcriptional activity and severely impaired glucocorticoid response in vitro, while FPGS mutations led to reduced enzymatic activation of methotrexate. Genome-wide analysis identified 9 mutational signatures, including two novel ones exclusive to relapse. Novel signature 1 was characterized by C>G mutations and was present in 15% of the relapsed cases with an enrichment for hyperdiploid ALL, while novel signature 2, present in 14% of the relapsed cases, was characterized by C mutations (C>T in particular) followed by a G. These novel signatures gave rise to relapse-specific drug resistance mutations, including PRPS1, TP53, NT5C2, and KRAS mutations.

The majority (59%) of the cases underwent a selective sweep as the relapse clone arose from a subclone detectable at diagnosis; however, very early relapse cases were less likely to experience a clonal sweep and had multiple lineages present at relapse. In patients tracked serially through multiple relapses, the clonal composition of drug resistance variants evolved substantially in response to chemotherapy. For example, in one patient, two independent NT5C2-mutant clones appeared at relapse, manifesting convergent evolution (Fig. 1a). One of the NT5C2-mutant clones expanded after subsequent treatment while the other diminished in prevalence. Sequential acquisition of multiple drug resistance mutations was also evident, with one patient sequentially acquiring KRAS, FBXW7, NR3C1 and FPGS mutations over a period of 5 years through multiple relapses (Fig. 1b). Indeed, 17% (18/103) of patients acquired multiple drug resistance mutations at relapse. The median time from detecting resistant clones to relapse was 41 days, suggesting that ultra-deep sequencing offers a means for early detection of genetic lesions that could serve as an indicator of relapse to enable earlier therapeutic intervention.

Conclusions

Very early relapses likely have different resistance mechanisms than early or late relapses, perhaps due to increased intrinsic resistance or the presence of multiple drug-resistant clones. At least a subset of relapse-specific drug resistance mutations may be acquired de novo, rather than pre-existing, as evidenced by resistance mutations bearing the novel relapse-specific mutational signatures which are likely to be therapy-induced. Relapsed ALL can acquire multiple drug resistance mutations targeting different drug classes. Frequent monitoring of disease, such as via cell-free DNA sequencing, may enable earlier detection of relapse and facilitate timely treatment to avoid selection for drug-resistant clones.

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