Pediatric T-cell lymphoblastic leukemia evolves into relapse by clonal selection, acquisition of mutations and promoter hypomethylation

Abstract

Relapsed precursor T-cell acute lymphoblastic leukemia is characterized by resistance against chemotherapy and is frequently fatal. We aimed at understanding the molecular mechanisms resulting in relapse of T-cell acute lymphoblastic leukemia and analyzed 13 patients at first diagnosis, remission and relapse by whole exome sequencing, targeted ultra-deep sequencing, multiplex ligation dependent probe amplification and DNA methylation array. Compared to primary T-cell acute lymphoblastic leukemia, in relapse the number of single nucleotide variants and small insertions and deletions approximately doubled from 11.5 to 26. Targeted ultra-deep sequencing sensitively detected subclones that were selected for in relapse. The mutational pattern defined two types of relapses. While both are characterized by selection of subclones and acquisition of novel mutations, 'type 1' relapse derives from the primary leukemia whereas 'type 2' relapse originates from a common pre-leukemic ancestor. Relapse-specific changes included activation of the nucleotidase NT5C2 resulting in resistance to chemotherapy and mutations of epigenetic modulators, exemplified by SUZ12, WHSC1 and SMARCA4. While mutations present in primary leukemia and in relapse were enriched for known drivers of leukemia, relapse-specific changes revealed an association with general cancer-promoting mechanisms. This study thus identifies mechanisms that drive progression of pediatric T-cell acute lymphoblastic leukemia to relapse and may explain the characteristic treatment resistance of this condition.

Figure 1.

Ultra-deep sequence analysis of primary and relapsed T-ALL distinguishes two types of relapse. (A, C) Variant allele frequencies in relapse were plotted over the variant allele frequency in the corresponding primary leukemia sample. Allele frequencies were determined by HaloPlex sequencing, only in the rare case that a certain allele was not covered by HaloPlex, allele frequencies from WES were used. (B, D) The simplified models show each mutation as a single symbol (circle, square, pentagon, ellipse). Blue symbols denote mutations that were detected in the major clone of primary leukemia, red symbols denote mutations that were specific for relapse. (A, B) Type 1 relapse: all mutations present in the major clone from primary disease were also present in relapse. The clone giving rise to relapse carried all mutations that were detected in the major clone of primary leukemia. (C, D) Type 2 relapse: the major clone from primary leukemia was lost in relapse, as indicated by the mutations that were present in the major clone in primary leukemia but absent in relapse. The clone giving rise to relapse shared some but not all mutations with the major clone of primary leukemia and was derived from a common ancestor, but evolved independently already before the initial diagnosis.

Figure 2.

Methylation analysis identifies promoter specific hypomethylation in relapsed T-ALL. β values in relapse were plotted over β values in primary leukemia in scatter plots. (A) Average β values (single sites) of all patients analyzed. (B) The β value for each promoter in relapse was compared to that in primary leukemia. Promoters with a β value in relapse that was at least 0.2 higher than in primary leukemia were considered to be hypermethylated. Promoters with a β value in relapse that was at least 0.2 lower than in primary leukemia were considered to be hypomethylated. Only promoters of those genes for which at least one gene symbol is assigned and which are represented by at least three probes on the 450k array are represented here.