Results
1. Clinical characteristics, treatment and prognosis data of ALL patients
Of 12 ALL patients, 11 had an initial onset of ALL, and one relapsed from testicular leukemia. The clinical manifestations of these patients at the first visit included fever, fatigue and subcutaneous hemorrhage, with a median age of 7 (3.3–14.8) years and a male-to-female ratio of 7:5. At the first visit, the median white blood cell (WBC) count was 5.96×109/L, the median hemoglobin (HGB) level was 85 g/L, and the median platelet count was 187×109/L. The average proportion of myeloid progenitor cells was 89.3%. The chromosome karyotype was predominantly a normal karyotype, and all patients had B-ALL. The immunophenotype was predominantly common B cells. One patient had central nervous system leukemia (CNS2). On average, 3.6 gene mutations occurred per patient, including 5 (41.7%)KRAS mutations, 4 (33.3%) NRAS mutations, and 2 (16.7%)CREBBP mutations. The fusion gene mutations included 3 (25.0%)ZNF384 mutations and one each of TEL-AML1 andDUX4::lgHJ6 (582). Risk assessment revealed that 75.0% of patients were classified as having intermediate risk. All patients received chemotherapy, and two patients received the targeted drug blinatumomab as part of induction therapy. None of the patients had undergone hematopoietic stem cell transplantation. The complete response (CR) rate of bone marrow after induction chemotherapy was 100%. Two patients had MRD<1×10-3 at Day (D) 15, and all patients had MRD<1×10-3 at D33. Moreover, the patients who entered the maintenance phase were assessed as MRD-negative before consolidation therapy. Currently, one patient has stopped the medicine for 1 year, and the other patients have remained in the chemotherapy stage. See Tables 1 and 2 for detailed information.
2. Clinical characteristics, treatment and prognosis of AML and APL patients
A total of 15 AML patients had an initial onset of AML. The clinical manifestations of these patients at the first visit included fever, upper respiratory tract infection, a pale face and fatigue. Patient AML-1 was complicated with Camurati-Engelmann disease and glucose-6-phosphate dehydrogenase deficiency. The median age was 11 (0.6–15.4) years, and the male-to-female ratio was 2:1. At the first visit, the median WBC count was 7.47×109/L. Most patients had anemia and thrombocytopenia. The proportion of myeloid progenitor cells was 47%. Two patients had myeloid sarcoma, one of whom had no bone marrow invasion. The chromosome karyotype was predominantly a normal karyotype. On average, each patient had 2.27 gene mutations. There were 3 (20.0%) patients with WT1 and PTPN11 mutations and 2 patients with TP53 mutations. There were 2 patients each with the fusion genes AML1-ETO andCBFβ-MYH11 each, and one patient each with KMT2A-MLLT3, MLL-AF9, NUP98-HoxA9 and FLT3-ITD. The risk assessment revealed that 66.7% of patients were at high risk. With respect to chemotherapy, seven patients received the targeted drugs venetoclax and gilteritinib, and nine underwent allogeneic hematopoietic stem cell transplantation. The bone marrow CR rate was 73.3% at D21 after the induction treatment and 86.6% at D28. Four (26.7%) patients had MRD<1×10-3 at D21, 5 (33.3%) had MRD<1×10-3 at D28, and 4 (26.7%) had 1×10-3≤MRD<1×10-2. Eleven (73.3%) patients survived at the end of follow-up; 2 patients died after induction therapy, one died due to severe infection and multiple organ failure after transplantation, and one died due to recurrence. There was one APL patient who was an 8-year-old girl, with 91% bone marrow blast cells, a normal karyotype, PML-RARA fusion genes and standard risk. She took arsenicals and retinoic acid throughout treatment, which has been stopped for 3 years. See Tables 1 and 3 for details.
3. Survival analysis of leukemia patients with NF1 gene mutations
The range of mutation frequencies of the NF1 gene was wide, ranging from 1.8% to 87.7%. The c.4676G>A site had the highest mutation frequency, and the c.6855C>A site had the lowest mutation frequency. Three children had multisite mutations. No duplicate or novel mutation sites were identified. The mutation types included nonsense mutations, missense mutations, frameshift mutations, and in-frame insertions. See Table 4 for details. Among the detected mutations, the median variant allele fraction (VAF) of the NF1 mutation was 0.27 in ALL patients and 0.39 in AML patients, suggesting that it plays a role in driving mutations in the leukemic clones but is not dominant. Children with germline NF1 mutations carried other adverse genetic factors such as TP53, KMT2A-MLLT3, MLL-AF9, and NUP98-HoxA9 . Children with somatic mutations carried mutations in FLT3, c-KIT, and TP53 . The overall treatment and outcomes of the 28 children with NF1 gene mutations are shown in Figure 1. The 5-year OS rate of the ALL group was better than that of the AML group; however, the difference was not statistically significant [100% vs. (67.7±14.8)%, =2.32,P =0.127; Figure 2]. In contrast, the difference in the 5-year OS rate between patients with germline and somatic NF1 mutations was significant [(33.3±25.5)% vs. (95.5±4.4)%, =6.34,P =0.012; Figure 3]. All the children with AML with germlineNF1 mutations died. The OS rate of children with AML was 73.3%. There was no difference in the 5-year OS rate between the NF1 mutation and non-mutation groups [(64±16.5)% vs. (66.6±7.9)%,P =0.994; Figure 4]. All ALL and APL patients survived at the end of follow-up, suggesting that the NF1 gene did not affect the OS prognosis of childhood leukemia patients.
4. Clinical characteristics of the NF1 gene and neurofibromatosis type 1
Six (21.4%) patients had germline NF1 mutations; all patients had ALL with a definite family history, and two patients were complicated with neurofibromatosis. Figure 5 displays the gene mutation sites of Patient ALL-2, showing germline mutations inherited from the mother. The remaining patients had somatic mutations, and one patient was complicated with neurofibromatosis. Patient ALL-1 had café-au-lait macules (CALM) (Figure 6), scoliosis (Figure 7) and intracranial involvement (Figure 8: right basal ganglia, bilateral thalamus). Patient ALL-2 had CALM, xanthogranuloma and intracranial involvement (left basal ganglia, right thalamus, midbrain, pons, brachium pontis, and bilateral cerebellar hemispheres). Patient ALL-3 had CALM, freckles and intracranial involvement (Figure 9: bilateral basal ganglia, thalamus, hypothalamus, bilateral temporal poles, hippocampus, cerebral peduncle, pons and cerebellum). The patient carried a nonsense mutation, and the sites were verified to be the wild type in the parents. The patient’s father had signs of CALM and suffered from leukemia in 2014; he carried an MLL fusion gene and WT1 mutations but no NF1 mutation. Therefore, the patient was considered to have somaticNF1 gene mutations, but the possibility of a chimeric mutation was not excluded. Three of the remaining patients had signs of CALM or freckles, but none met the diagnostic criteria for neurofibromatosis.