Pediatric ALL relapses after allo-SCT show high individuality, clonal dynamics, selective pressure, and druggable targets

Abstract

Survival of patients with pediatric acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-SCT) is mainly compromised by leukemia relapse, carrying dismal prognosis. As novel individualized therapeutic approaches are urgently needed, we performed whole-exome sequencing of leukemic blasts of 10 children with post-allo-SCT relapses with the aim of thoroughly characterizing the mutational landscape and identifying druggable mutations. We found that post-allo-SCT ALL relapses display highly diverse and mostly patient-individual genetic lesions. Moreover, mutational cluster analysis showed substantial clonal dynamics during leukemia progression from initial diagnosis to relapse after allo-SCT. Only very few alterations stayed constant over time. This dynamic clonality was exemplified by the detection of thiopurine resistance-mediating mutations in the nucleotidase NT5C2 in 3 patients' first relapses, which disappeared in the post-allo-SCT relapses on relief of selective pressure of maintenance chemotherapy. Moreover, we identified TP53 mutations in 4 of 10 patients after allo-SCT, reflecting acquired chemoresistance associated with selective pressure of prior antineoplastic treatment. Finally, in 9 of 10 children's post-allo-SCT relapse, we found alterations in genes for which targeted therapies with novel agents are readily available. We could show efficient targeting of leukemic blasts by APR-246 in 2 patients carrying TP53 mutations. Our findings shed light on the genetic basis of post-allo-SCT relapse and may pave the way for unraveling novel therapeutic strategies in this challenging situation.

Graphical abstract

Figure 1.

Large oncogenomes are characterized by acquired mutations in DNA repair genes. (A) Experimental setup including oncogenomes (OG). For patients 202 and 735, no material of the initial leukemia (INIT) was available; patient 107 did not relapse before allo-SCT. (B) Sizes of the individual oncogenomes. (C) Mutational loads across the oncogenomes. The increase from OG1 vs OG2/OG3 was statistically significant (P = .005). (D) Mutations in DNA polymerase/repair genes explaining the large oncogenomes. n/a, not available; REMI, first remission; RLPS, first relapse; TREMI, remission after allo-SCT; TRLPS, relapse after allo-SCT; UPN, unique patient identifier.

Figure 2.

Oncogenomes are exclusive to individual patients and relapse states. (A) Mutational spectra on SNV level in OG1, OG2, and OG3. (B) Recurrently mutated genes (≥3 patients) in OG2 (also see supplemental Table 6). UPNs as earlier. (C) Recurrently mutated genes (≥3 patients) in OG3 (also see supplemental Table 7). n.d., not done.

Figure 3.

Mutational clusters show profound clonal dynamics. Shown are the 7 patients for whom information on all 3 oncogenomes was available (A: 316, 318, 337, 514; B: 590, 660, 685). Different mutational patterns can be observed, both within as well as across the patients. Individual clusters are indicated and marked in different colors.

Figure 3.

Mutational clusters show profound clonal dynamics. Shown are the 7 patients for whom information on all 3 oncogenomes was available (A: 316, 318, 337, 514; B: 590, 660, 685). Different mutational patterns can be observed, both within as well as across the patients. Individual clusters are indicated and marked in different colors.

Figure 4.

Clonal dynamics and selective pressure of NT5C2 and TP53 mutations. (A) Graphs indicate the variant allele frequencies in the SNVs in NT5C2 as detected by whole-exome sequencing (supplemental Table 9). Patient identifiers (UPN) as well as amino acid exchanges are indicated. Below each graph, the results of the amplicon sequencing (AS) are indicated. (B) SNVs detected in TP53 (also supplemental Table 10). The Li-Fraumeni patient (590) is not shown.

Figure 5.

Mutations in cancer genes and targetable genetic lesions in the post–allo-SCT relapses. (A) Mutated cancer genes across all patients and oncogenomes. (B) Details on those cancer genes, which were mutated in at least 2 patients. UPNs as earlier. (C) Targetable genes carrying mutations in the OG3 of the respective patients, with possible therapeutic options indicated. SNVs are indicated in red, copy number losses in orange, hypermutated oncogenomes (defined by ≥200 SNVs) in blue. (D) Sensitivity testing of patient cells/patient-derived xenografts against APR-246 in different concentrations (1-5-10-100 micro molar). neg. ctrl., negative control (primograft X116; TP53 wild-type); pos. ctrl., positive control (TP53 mutant primograft X172 carrying 2 TP53 mutations on different alleles).