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)%, X² =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)%, X² =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.