Recent research has significantly advanced our understanding of pediatric acute lymphoblastic leukemia (ALL), the most common childhood cancer. A detailed genomic study, focusing on the somatic mutation profiles at diagnosis and relapse, provides crucial insights into the complex nature of this disease. This study, utilizing whole-exome sequencing (WXS) at high coverage, examined samples from 20 pediatric patients with B-ALL, offering a comprehensive view of the genetic changes that occur as leukemia progresses and relapses after treatment. Understanding these changes is paramount for improving diagnosis and developing more effective, targeted therapies for pediatric leukemia.
Genomic Landscape of Pediatric Leukemia at Diagnosis and Relapse
The study meticulously analyzed samples collected at diagnosis, remission, and relapse from 20 pediatric patients enrolled in Children’s Oncology Group (COG) B-ALL trials. Eleven of these cases presented known oncogenic gene fusions and rearrangements at diagnosis, including TCF3-PBX1, TCF3-HLF, IGH-CRLF2, P2RY8-CRLF2, and ETV6-RUNX1. Importantly, these key fusions were found to persist at relapse, indicating their fundamental role in the leukemic clone’s origin and survival.
Beyond gene fusions, the research team identified a wide range of somatic mutations, including single-nucleotide variations (SNVs), insertions/deletions (indels), structural variations (SVs), and copy-number variations (CNVs). Through rigorous experimental verification, a total of 2,278 SNVs, 106 indels, and 68 SVs were confirmed across the patient cohort. The depth of sequencing, combining WXS and capture sequencing, was exceptionally high, ensuring the detection of even low-frequency mutations. This level of detail allowed for a robust analysis of the mutational landscape and its changes between diagnosis and relapse in pediatric leukemia.
A significant finding was that each case exhibited genetic alterations shared between diagnosis and relapse, reinforcing the concept of a common ancestral pre-leukemic clone. On average, a substantial majority (74%) of coding sequence mutations present at diagnosis persisted at relapse. Interestingly, a subset of relapsed tumors (four cases) displayed hypermutability, characterized by a dramatic increase in coding mutations. In these hypermutable cases, mutations in DNA mismatch repair genes like PMS2 and MSH6 were observed at relapse, suggesting a mechanism for the increased mutation burden. For the majority of non-hypermutable cases, the number of coding somatic sequence mutations was still significantly higher at relapse compared to diagnosis, indicating an accumulation of genetic changes over time.
Figure 1: Recurrently mutated pathways and genes identified in a cohort of 20 pediatric ALL patients, highlighting genetic alterations at diagnosis and relapse relevant to pediatric diagnosis.
Recurrently Mutated Pathways and Genes in Pediatric Leukemia Diagnosis
Integrated analysis of the genetic alterations revealed six key pathways recurrently mutated in pediatric ALL at diagnosis and/or relapse. These pathways are critical in cancer biology and include: Ras signaling, JAK-STAT signaling, transcriptional regulation of lymphoid development, nucleoside metabolism, epigenetic modification, and cell cycle regulation. The Ras pathway was frequently affected, with mutations present in 65% of cases overall. Notably, multiple subclonal Ras mutations were observed at diagnosis in several cases, sometimes within the same gene (NRAS, KRAS) or across different Ras pathway genes. In a subset of these cases, the study observed an intriguing phenomenon of convergence, where multiple subclonal Ras mutations at diagnosis evolved into a single clonal NRAS mutation at relapse.
JAK signaling pathway activation, driven by SNVs and SVs, was present in 25% of cases at both diagnosis and relapse. Genes regulating B-cell development were also frequently targeted, with loss-of-function mutations observed in a large proportion of cases at both time points. Cell cycle pathway alterations were also common, including CDKN2A/B deletions and relapse-specific TP53 mutations.
Of particular interest was the exclusive presence of sequence mutations in NT5C2, a gene involved in purine metabolism, at relapse in a significant proportion of cases. These mutations, including known activating mutations and a novel recurrent mutation, have been linked to chemotherapy resistance in relapsed ALL. Epigenetic regulators also showed a high mutation rate, with recurrent relapse-specific mutations in WHSC1 and CREBBP. In some instances, mutations in these epigenetic regulators were detectable at very low levels at diagnosis but became highly enriched at relapse, underscoring the dynamic nature of clonal evolution in pediatric leukemia.
Clonal Evolution and Mutation Dynamics in Pediatric Leukemia Relapse
To understand the evolutionary dynamics of pediatric leukemia, the researchers delved into clonal evolution analysis using deep sequencing data. They constructed clonal lineages based on mutation frequencies at diagnosis and relapse, employing a sophisticated binomial mixture model to define mutation clusters as diagnosis-specific, relapse-specific, or shared. This approach allowed for a detailed reconstruction of the clonal architecture in each case, revealing the complex shifts in subclonal populations during disease progression.
Case PAPSPN exemplifies the turnover of predominant driver mutations. This patient exhibited mutations in genes across multiple pathways, including JAK-STAT signaling (JAK2), Ras signaling (KRAS), and lymphoid development (PAX5). Clonal analysis identified six mutation clusters, with diagnosis-specific, shared, and relapse-specific clusters. At diagnosis, multiple JAK2 mutations were present, with one dominant clone and minor subclones each harboring distinct JAK2 mutations. Remarkably, at relapse, the dominant JAK2 mutation switched, highlighting clonal evolution and selection under therapy.
Figure 2: Illustration of three subclonal JAK2 mutations in patient PAPSPN at diagnosis, emphasizing the complexity of genetic alterations in pediatric leukemia.
Case PARJZZ presented a unique dual-lineage evolution, where two clones present at diagnosis persisted to relapse but reversed their clonal dominance. Both clones harbored distinct KRAS mutations and CDKN2A/B deletions. One clone was dominant at diagnosis, while the other, initially a minor subclone, became dominant at relapse after acquiring a relapse-specific NT5C2 mutation. This case highlights the diverse evolutionary paths leukemia can take in response to therapy.
Figure 3: Clonal architecture of diagnosis and relapse samples for PAPSPN, depicting mutation clusters and clonal lineages, crucial for understanding pediatric leukemia evolution.
Case PASLZM was one of the hypermutator cases, exhibiting a dramatic increase in mutations at relapse. Clonal analysis revealed relapse-specific mutation clusters, including a somatic splice site mutation in PMS2, a DNA mismatch repair gene. The PMS2 mutation was associated with a significant shift in mutation spectrum, with a strong enrichment for transition mutations, consistent with mismatch repair deficiency. This finding suggests that mutations in DNA repair genes can drive hypermutation and contribute to relapse in pediatric leukemia.
Figure 4: Clonal architecture for PARJZZ samples at diagnosis and relapse, showcasing dual lineage evolution and clonal dominance reversal in pediatric leukemia.
Figure 5: Clonal architecture for case PASLZM, illustrating mutation clusters and clonal lineages in a hypermutator case of pediatric leukemia.
Implications for Pediatric Leukemia Diagnosis and Therapy
Analyzing clonal lineages across the 20 cases revealed that in a majority of cases, the rising clone at relapse originated from a minor subclone present at diagnosis. This highlights the importance of understanding and targeting subclonal populations in initial therapy to prevent relapse. Several genes were found to harbor relapse-specific mutations in the founder clone at relapse, including NT5C2, USH2A, WHSC1, TP53, NRAS, IKZF1, and CREBBP. These genes represent potential targets for novel therapeutic strategies aimed at preventing or treating relapse in pediatric leukemia.
Figure 6: Pattern of subclone rise and fall from diagnosis to relapse in pediatric leukemia, summarizing clonal evolution across the study cohort.
In conclusion, this comprehensive genomic study provides valuable insights into the mutational landscape and clonal evolution of pediatric B-ALL at diagnosis and relapse. The identification of recurrently mutated pathways and relapse-specific mutations offers a foundation for improving diagnostic approaches and developing targeted therapies to overcome relapse, ultimately enhancing outcomes for children with leukemia. Further research building upon these findings is crucial to translate these genomic insights into clinical advancements in pediatric cancer care.
