In this study, we dissected the genetic landscape, analyzed the mutational spectrum of various immunological ALL lineages, and explored the correlations between mutational and clinical features, including patient characteristics, risk stratification, and treatment outcomes in a Chinese pediatric ALL cohort. A number of gene mutations were identified, which provided a comprehensive genomic profile of Chinese pediatric ALL. Consistent with previous reports, B-ALL and T-ALL presented a distinct mutation spectrum; Ras pathway mutations were enriched in B-ALL, while Notch pathway mutations were enriched in T-ALL (Al-Kzayer et al. 2015; Girardi et al. 2017; Irving et al. 2014; Roberts et al. 2019; Tasian and Hunger 2017).
As described previously, mutations involved in the Ras signaling pathway (NRAS, KRAS, FLT3, PTPN11, and NF1) occurred in more than half of B-ALL patients (Al-Kzayer et al. 2015; Irving et al. 2014). Also, a higher incidence of mutations was detected in NRAS rather than KRAS. This finding was contradictory to the previous studies in the Chinese cohort but was in agreement to that from the USA, Sweden, and Japan (Liang et al. 2006; Paulsson et al. 2008; Perentesis et al. 2004; Shu et al. 2004; Yamamoto et al. 2006). These discrepancies might be related to the population distribution and environmental factors, which highlighted the genetic heterogeneity of pediatric ALL. Compared to CBL, TET2, CDKM2A, and BCORL1 genes with a higher median VAF, Ras signaling pathway-related genes, such as FLT3, NRAS, KRAS, and PTPN11, displayed a lower median VAF of 5–20%. The lower VAF indicated that Ras mutations were more likely subclones rather than a major clone (Oshima et al. 2016), suggesting that B-ALL is driven by other fusion genes. Reportedly, Ras pathway functioned as a molecular switch for signaling pathways that regulated cell proliferation, survival, growth, migration, and differentiation (Zhang et al. 2020a). Moreover, Ras pathway mutations were prevalent in other tumor types, including colorectal cancer, pancreatic cancer, and thyroid cancer (Laghi et al. 2002; Nikiforov and Nikiforova 2011; Saif and Shah 2009). Thus, we speculated that Ras pathway mutations occurred during B-ALL progression rather than tumorigenesis. Based on genetic testing of a large number of ALL patients, Shu et al. and Perentesis et al. demonstrated that RAS mutations did not present any unique clinical manifestation nor predicted clinical outcomes (Perentesis et al. 2004; Shu et al. 2004). Moreover, some recent studies showed that ALL patients with Ras pathway mutations, especially KRAS/NRAS mutations, present high-risk features, including early relapse and CNS involvement (Irving et al. 2014; Ney et al. 2020; Zhang et al. 2011). In our cohort, no correlation was established between the presence of Ras mutation and clinical characteristics, risk stratification, and MRD level. This phenomenon could be attributed to the neutralization effect of other genomic variations, such as low-risk hyperdiploidy and high-risk hypodiploidy on prognosis (Case et al. 2008; Davidsson et al. 2010; Paulsson et al. 2008; Wiemels et al. 2010, 2005; Zhang et al. 2011). For instance, the association between poor outcome and Ras pathway mutations was not detected in hyperdiploid ALL (Paulsson et al. 2008) but in MLL rearranged infants (Driessen et al. 2013). In order to exclude the interference of cytogenetics, the coexistence between Ras pathway mutations and cytogenetics were analyzed. Except for KRAS and FLT3 mutations that were enriched in patients with hyperdiploidy, NRAS, PTPN11, FLT3 and NRAS mutations were detected in patients with normal cytogenetics. To determine whether Ras pathway status influences the clinical characteristics and risk stratification, additional studies are warranted on various cytogenetic subgroups of B-ALL. Notch pathway mutations, especially NOTCH1 and FBXW7, were enriched in T-ALL patients, as previously reported (Eguchi-Ishimae et al. 2008; Erbilgin et al. 2010; Sulis et al. 2007; Valliyammai et al. 2018). NOTCH1 was the most common mutated gene in about 60.9% of all T-ALL cases, followed by PTEN (21.7%) and FBXW7 (21.7%). Notch signaling pathway, especially NOTCH1, plays a crucial role in all stages of T lymphocyte development and can promote the differentiation of lymphoid precursor cells into T lymphocytes and inhibit their differentiation into B lymphocytes (Radtke et al. 2013; Vanderbeck and Maillard 2020). Except for the excessive activation of the Notch pathway, impaired CDKN2A/2B cell cycle regulators also played a prominent role in T-ALL pathogenesis. Strikingly, CDKN2A/2B deletions were detected in > 50% of T-ALL cases (Girardi et al. 2017; Yeh et al. 2019). However, the copy number variations were not detected and analyzed in the present study, and only a few CDKN2A gene point mutations were identified in T-ALL. Recent sequencing studies demonstrated that T-ALL was an aggressive malignancy caused by the accumulation of genomic lesions. On average, 10–20 mutations were detected in T-ALL cells (De Keersmaecker et al. 2013; Girardi et al. 2017; Holmfeldt et al. 2013; Zhang et al. 2012). Although our study showed that T-ALL had a significantly higher mutation level than B-ALL, the average number of mutations was still lower than the expected value. This deviation could be attributed to the scope of sequencing, the evaluated variation types, the sensitivity of the test, and the filter criteria of mutation calling. Consistent with the findings of previous studies, several genes, such as DNM2, PHF6, WT1, and RPL10 were found to be involved in increased kinase signaling, transcription factors, epigenetic factors, translation, and RNA stability at a low frequency in our cohort (Belver and Ferrando 2016; Girardi et al. 2017). We also found that the accumulation of mutations in T-ALL did not occur randomly (Vogelstein et al. 2013). Interestingly, the coexistence of NOTCH1-PTEN-FBXW7 and DNM2-USP7-PHF6 mutations was observed in our T-ALL cohort. The coexistence phenomenon suggested that those Notch pathway and non-Notch pathway genes interconnect physiologically and cooperate during the development and progression of the T-ALL, respectively.
MLL translocations and PIK3R1 mutations were common in infant ALL, a group characterized as immature cytologically, resistant to conventional therapies, and showing poor prognosis. In addition, the MLL gene arrangement is the hallmark of infant leukemia, associated with a high incidence (approximately 80%) (Eguchi et al. 2003). Moreover, a significant coexistence between MLL gene arrangement and PIK3R1 mutations was detected in our cohort. This observation indicated that PI3K/AKT is a secondary hit for partial MLL-positive ALL. Furthermore, ALL patients carrying mutations in NOTCH1 and PTEN presented significantly high initial WBC counts. Combined with the data of previous studies, a significant correlation was established between higher initial WBC counts and T-ALL compared to B-ALL (Zhang et al. 2019). In addition, patients with FLT3 mutations showed lower platelet counts and hemoglobin levels, while patients with NOTCH1 mutations had higher hemoglobin levels. In terms of therapeutic effect, patients with ETV6 or PHF6 mutations were sensitive to steroid treatment. Additionally, PIK3R1 and PTEN mutations were characteristics of high risk in ALL patients. MRD analysis showed a high level of MRD in patients with PIK3R1, TET2, and KMT2D mutations, indicating a high risk of relapse. Both TET2 and KMT2D belong to epigenetic regulator genes, which play key roles in DNA demethylation and histone H3 methylation, respectively (Ichiyama et al. 2015; Zhang et al. 2020b). This finding suggested that mutations in epigenetic regulator genes elevate the MRD level.
However, our study also has some limitations. First, the enrolled patients were from our single center, which could not well reflect the gene mutation level of the whole Chinese pediatric ALL, and further multi-center studies are needed. Second, long-term follow-up of patients is needed to explore the relationship between mutations and prognosis.
In summary, our study depicted the specific genomic landscape and revealed the relevance between mutational spectrum and clinical features of Chinese pediatric ALL in a single cohort, including patient characteristics, cytogenetics, genetic subtypes, risk stratification and treatment outcomes. The discovery of this mutational spectrum highlights the need for molecular classification, risk stratification, and prognosis evaluation and also provide the basis for the development and application of new targeted therapy for pediatric ALL.