Background
Acute lymphoblastic leukemia (ALL), the most common childhood tumor, results from the clonal proliferation of lymphoid stem or progenitor cells with arrested maturation, with more than 80% originating from B cell progenitors [
1]. ALL is characterized by recurring structural chromosomal alterations, including aneuploidy (high-hyperdiploid, chromosomes ≥51; hypodiploid, chromosomes ≤44) and translocations (e.g., t (12;21)/
ETV6-RUNX1, t (1;19)/
TCF3-PBX1, t (9;22)/
BCR-ABL1, and
KMT2A (also known as
MLL) rearrangement). However, chromosomal changes alone are often insufficient to trigger leukemia, some additional genetic alterations must contribute to tumorigenesis [
2,
3].
Cytogenetic alterations or molecular abnormalities are frequent, and several molecular markers have been identified to stratify risk and predict prognosis, as they play key roles in ALL pathogenesis. Specific ALL subtypes exhibit different mutation distributions; for example,
TP53 mutations mostly occur in hypodiploidy [
4,
5].
PAX5/IKZF1 copy number abnormalities frequently exist in B-ALL, whereas mutations within
NOTCH1,
FBXW7, and
CDKN2A/CDKN2B are enriched in T-ALL [
1,
6‐
8]. Rare germline mutations in the genes
PAX5 [
9] and
ETV6 [
10] were found to be linked to familial leukemia, and some chemical agents or radiation exposure could increase the incidence of leukemia [
6]. In addition, some molecular alterations, such as
CREBBP [
11‐
13],
NT5C2 [
14,
15] and
PRPS1 mutations [
16], are associated with chemo-resistance. Thus, the identification of these abnormalities not only reveals molecular pathology, but also provides important therapeutic targets. Some targetable alterations or pathways have been used for therapeutic interventions in the clinic, especially kinase-activating alterations in
BCR-ABL1-positive or Philadelphia chromosome-like ALL patients who are amenable to tyrosine kinase inhibitors with improved survival rates [
17,
18]. However, a substantial percentage of patients classified as having a “good prognosis” (e.g., t (12;21)/
ETV6-RUNX1 or high-hyperdiploid) still experience relapse, which may be caused by the existence of additional or secondary molecular variants. Therefore, it remains important to further identify the repertoires of gene mutations and understand its clinical significance in pediatric ALL.
Recently, genetic profiling of several subtypes of pediatric ALL has been conducted with NGS [
4,
5,
11,
19,
20]. Numerous germline genetic variants and somatic alterations have been identified in newly diagnosed and relapsed childhood ALL or in specific subtypes, which may also have prognostic implications [
19,
20]. NGS has revealed changes in the microarchitecture and gene sequence, which advanced the understanding of the molecular basis of ALL and complemented genetic features of the ALL subtypes.
In this study, we used targeted exome sequencing technology to reveal the mutational spectrum in patients with ALL at initial diagnosis to better understand the cytogenetic and molecular classification of pediatric ALL in Chinese children, which may lead to the discovery of new therapeutic targets and enable the development of a tailored therapeutic regimen for each patient.
Discussion
In this study, we performed a genetic mutational analysis of Chinese children with ALL and identified an abundance of somatic mutations in essential genes, many of which were likely deleterious and may contribute to the pathogenesis of ALL. Although many of the most frequent mutations in pediatric ALL have been described previously, we identified distinct mutational characteristics and influenced different signaling pathways between B-ALL (
Ras pathway) and T-ALL (
Notch pathway) in this Chinese cohort.
Ras pathway mutations were recurrent in pediatric B-ALL [
24,
30,
31], and the vast majority of mutations occurred in
KRAS,
NRAS,
FLT3 and
NF1, revealing a central role of these genes in pediatric B-ALL.
Ras genes mutational sites included G12C/D/S/V, G13D/S/V, Q61K/H, A146T/P and K117N, which were also identified in the study by Ding LW, et al. [
32], suggesting that these mutational sites were common in Asian. one patient occurred
KRAS and
NRAS mutation simultaneously, these two mutations were close enough to be spanned by the same read-pair allowing the determination if the mutations are on either the same or different alleles [
32]. We also found that 75% high-hyperdiploid possessed
FLT3 mutations, which higher than 25% incidence as previous studies [
33,
34], indicating a higher incidence in the Chinese patients with ALL associated with hyperdiploidy. Consistent with previous reports [
20,
35], we also observed a high incidence of
Ras pathway mutations in high-hyperdiploid patients with low mutation rates in
TCF3-PBX1 and
MLL rearrangement cases. Similar research was showed that B-ALL patients carrying any of the recurrent translocations
ETV6-RUNX1,
BCR-ABL or
TCF3-PBX1 harbored few mutations compared to the other B-ALL patients [
36]. Overall, this further underscores the crucial role of
RAS mutations in ALL and highlight the genetic heterogeneity of pediatric ALL.
In our cohort,
NOTCH1 mutations occurred in 23.1% of T-ALL cases, which was significantly lower than previously reported values [
26,
29]. However, it is interesting that 2 cases with
PHF6 mutations co-occurred with
NOTCH1 mutations and were significantly correlated with the
NOTCH1 mutation in Chinese adult T-ALL (
PHF6 mut
NOTCH1mut vs
PHF6 wt
NOTCH1mut, 75.0% vs 44.2%;
P = 0.035) [
37]. This discrepancy could be caused by the limited number of T-ALL cases enrolled in this study (
n = 26), or possible coverage bias impairing ability to call gene sequence [
38], and the detection of sequence mutations in ALL was insufficient. Frequently, some genes are affected by more than one type of alterations such as point mutation, copy number alterations (CNAs), focal aberrations/small insertions/deletions (INDEL), or structural variations (SVs). So, only one type of analysis lead to the underestimation of the mutation frequency of
NOTCH1. Similarly, we underestimated the mutation frequencies of
CDKN2A/2B,
ETV6 and
PAX5, due to lack of analysis of somatic copy number gains or losses. Copy losses of
CDKN2A/2B (9p21),
PAX5 (9p13) and
ETV6 (12p13) were prevalent in children, while copy gains of
RUNX1 (21q22.3) were more enriched in children [
39]. So, large deletion, amplification and structural variant should be warranted; no single type of sequencing is capable of detecting the same alterations; WES is useful for point mutation investigation, whereas WGS can reveal SVs. Besides, NGS is increasingly being used to monitor drug response and treatment toxicity [
40], contributing to the refinement of diagnosis and prognosis for 34% of patients with hematologic malignancies and blood disorders [
41]. Incorporating pharmacogenomics and pharmacotranscriptomics can provide an enormous of molecular markers responsible for the efficacy, side effects, and toxicity of the chemotherapeutic drugs to improve the treatment protocols [
42]. Then, utilizing genomic technology can better management and potential improve the survival rate in pediatric ALL patients.
In our findings, the most frequently mutated gene of epigenetic regulators was
KMT2D, which encodes histone methyltransferase for methylates the Lys-4 position of histone H3, and its mutation can cause Kabuki syndrome, an autosomal dominant disease [
43].
KMT2D is a key regulator of transcriptional enhancer function and plays an important role in maintaining genomic stability [
44], and it is mutated in a large number of different cancers (e.g., diffuse large B cell lymphoma, small cell undifferentiated lung cancer, and medulloblastoma) [
45‐
47]. As
KMT2D is a predicted tumor driver gene in ALL [
19] and it overexpressed in ALL, when
KMT2D is knocked down, it significantly decreased leukemia cell growth, promoted cell apoptosis, and inhibited cell proliferation. A related study also showed that
KMT2D was overexpressed in primary gastrointestinal diffuse large B cell lymphoma (PGI-DLBCL) and appeared as a prognostic factor for patients older than 60 years old [
48].
KMT2D overexpression was observed in esophageal squamous cell carcinoma (ESCC), predicting poor clinical outcomes and facilitating ESCC tumor progression [
49]. In addition,
KMT2D can interact with
KMT2A in acute myeloid leukemia, its deletion reduced
MLL-AF9 leukemia cell survival, and the codeletion of both
KMT2A and
KMT2D resulted in more severe reductions in survival, proliferation, and gene expression than either individual gene deletion [
50]. Hence, the
KMT2D gene plays an important role in hematological tumors and may act as a drug target in MLL-rearranged leukemia. However, there existed limitation in our research, the off-target effect remains one of the major obstacles in
KMT2D-shRNA experiment and it is insufficient to research the function of
KMT2D in ALL. So, we should generate a
KMT2D knock-out cells by CRISPR-Cas9-mediated genome editing to demonstrate its potential molecular pathogenesis in ALL in the future study.
As the main part of this study, we intend to show the genomic landscape of pediatric ALL from a single center in China, and our results provided a substantial number of genetic variants contributing to accumulate genetic data of Chinese children and explore molecular determinants in the future. However, there are some limitations in the present study. The number of patients enrolled in the present study was limited, and sample selection may be biased, which may contribute to the discrepancies in the findings between our study and others, and collaborative efforts with larger sample sizes are needed. Structural alterations may play important roles in leukemogenesis; thus, the absence of this information leads to incomplete understanding of the genetic basis of ALL. More comprehensive approaches, such as WGS, RNA-seq, pharmacogenomics and pharmacotranscriptomics, and larger integrative studies, can be warranted to dissect the underlying complexity of ALL in the future. The frequencies and distributions of abnormalities of ALL patients between children and adult, Chinese and western should further be compared in a larger cohort.
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