Juvenile myelomonocytic leukemia (JMML) is a fatal, mixed myeloproliferative and myelodysplastic disorder occurring in infancy and early childhood. Children with JMML have mutually exclusive genetic abnormalities in granulocyte-macrophage colony-stimulating factor (GM-CSF) signaling pathways, inactivation of the NF1 or mutations in PTPN11, NRAS, KRAS and CBL.1, 2 A whole-exome sequencing study, performed by Sakaguchi et al.,3 has recently demonstrated that in addition to the high frequency of RAS pathway mutations, mutations in SETBP1 and JAK3 are common recurrent secondary events, and that these events may be involved in tumor progression, and are associated with poor clinical outcomes. The SETBP1 and JAK3 mutations have also been reported in the other hematological malignancies.4, 5, 6, 7, 8, 9
We have previously reported seven cases of patients (five with PTPN11 mutation; one with NRAS mutation) with significant chromosomal changes after chemotherapy or allogeneic hematopoietic stem cell transplantation (HSCT).10 In addition, we observed a loss of wild-type NRAS locus and monosomy 7 after blastic crisis in a patient with JMML and a heterozygous NRAS mutation.11
The present study aimed to evaluate whether JMML clones with the RAS pathway-associated gene mutation coexist at the onset with those harboring both the RAS pathway-associated and nonRAS pathway gene mutations, and examine 6-mercaptopurine (6-MP)-susceptibility of these two clone types.
First, we examined the presence of JAK3 and SETBP1 mutations in 29 patients with JMML (20 patients with PTPN11 mutations, and nine with NRAS or KRAS mutations), including seven patients who acquired chromosomal abnormalities during the clinical course.10 The study was approved by the Institutional Review Board of Shinshu University. Informed consent was obtained from the guardians of the patients in accordance with the institutional guidelines. DNA was extracted from peripheral blood mononuclear cells (PBMNCs) obtained at diagnosis and/or after chemotherapy. Exons 2–6 of SETBP1 and exons 2–24 of JAK3 were amplified by PCR, using primer pairs described previously.3, 6 The amplicons were subjected to direct sequencing from both directions using an automatic DNA sequencer. Among 29 patients, four patients with PTPN11 mutations had heterogeneous JAK3 mutation and/or SETBP1 mutation (Table 1). These genetic data were obtained from the PBMNCs collected at diagnosis in cases nos. 1 and 2, and from those after chemotherapy in case nos. 3 and 4. The PTPN11 mutations in the four cases were considered to be acquired according to the data reported previously.12, 13 Case nos. 1 and 3 harbored JAK3 R657Q, and case no. 2 harbored SETBP1 D868N mutations. Case no. 4 harbored both JAK3 R657Q and SETBP1 G870R. Two patients were older than 24 months at the onset of the disease. Only one patient had platelet counts of <33 × 109/l, whereas fetal hemoglobin levels of 3 patients were >15%. Chromosomal changes were observed in two patients (case nos. 3 and 4) after chemotherapy. Three patients who received allogeneic HSCT are alive and disease free. The lack of residual disease was confirmed by allele specific quantitative PCR14 for PTPN11 mutation in case nos. 1 and 2, and by fluorescence in situ hybridization for sex chromosomes using more than 500 cells in case no. 3.
We then investigated whether JMML clones harboring both PTPN11 mutation and the nonRAS pathway gene mutations coexisted with those harboring only PTPN11 mutation at onset. PBMNCs (1 × 104) maintained in liquid nitrogen were plated in dishes containing methylcellulose medium supplemented with 10 ng/ml of GM-CSF. GM colonies were individually lifted after 12 days, and single cell suspensions were prepared. Sequence analyses were then performed on individual GM colony-constituent cells, as described previously.10 As presented in Figure 1, 16 of 34 GM colonies derived from PBMNCs obtained at diagnosis of case no. 1 had both JAK3 mutation and PTPN11 mutations. The identical number of GM colonies was positive for PTPN11 mutation but negative for JAK3 mutation. In case no. 2, both PTPN11 and SETBP1 mutations were found in 16 of 27 GM colonies derived from PBMNCs obtained at onset, whereas the remaining 11 GM colonies had only PTPN11 mutation. There were no GM colonies harboring the mutated nonRAS pathway gene and wild-type PTPN11 gene in these patients. Interestingly, the frequency of GM colonies with both PTPN11 mutation and the nonRAS pathway mutation significantly increased (>80%) between 1.5 and 4 months after treatment with only 6-MP in both the cases (P=0.0032 in case no. 1 and P=0.0093 in case no. 2). The χ2-test was used to determine the significance of differences. PBMNCs from case no. 3 were obtained 22 months after treatment with 6-MP, which also yielded two types of GM colonies (Figure 1c). In case no. 4, we found heterogeneous mutations in all three gene types (PTPN11, JAK and SETBP1) in 38 of 40 GM colonies grown from PBMNCs obtained 16 months after repeated chemotherapy including 6-MP (Figure 1d). The remaining two colonies had mutated PTPN11 and JAK3, where the SETBP1 was wild type.
Using liquid cultures, we finally examined whether GM progenitor cells with both nonRAS pathway mutation and PTPN11 mutation exhibited a susceptibility to 6-MP different from those with only PTPN11 mutation. Appropriate aliquots of 6-MP (Sigma Chemical, St Louis, MO, USA) were dissolved in 1 N sodium hydroxide, and then diluted with alpha-medium.10 To examine susceptibility to 6-MP, PBMNCs (1 × 104) were cultured in a dish containing 10 ng/ml of GM-CSF with or without 6-MP (30 μM). Number of GM colonies from PBMNCs obtained at onset in case no. 1 was decreased to one-third by the addition of 6-MP (30 μM). Nevertheless, exposure to 6-MP significantly increased the proportion of GM colonies with both PTPN11 and JAK3 mutations (P=0.0130, Figure 1e).
From the data that SETBP1 and JAK3 mutations have lower allele frequencies (difference not statistically significant for SETBP1) than the RAS pathway mutations (PTPN11, NF1 and NRAS/KRAS), Sakaguchi et al. inferred that the SETBP1 and JAK3 mutations represent secondary genetic hits that contribute to clonal evolution after the main tumor population is established.3 In this study, genetic analyses of individual GM colonies clearly revealed that GM progenitor cells harboring both PTPN11 and the nonRAS pathway gene mutations (JAK3 or SETBP1), and cells harboring only PTPN11 mutation coexisted at onset (cases nos. 1 and 2). Nevertheless, there were no GM colonies harboring the mutated nonRAS pathway gene and wild-type PTPN11. Thus, SETBP1 and JAK3 mutations appear to be the second genetic aberration in some JMML children with PTPN11 mutation. Nevertheless, it is necessary to exclude a possibility of prenatal origin of JMML clone with both PTPN11 and nonRAS pathway gene mutations, which can be confirmed using Guthrie cards (dried blood spots), as we previously described.14
In case nos. 1 and 2, the percentage of GM colonies with both PTPN11 and nonRAS pathway mutations increased substantially several months after treatment with only 6-MP in comparison with the percentage at diagnosis. Furthermore, the addition of 6-MP to a liquid culture containing PBMNCs obtained at onset of case no. 1, and supplemented with GM-CSF significantly increased the proportion of GM colonies with PTPN11 and JAK3 mutations. As treatment with 6-MP was continued up to the beginning of preparative conditioning for allogeneic HSCT in case nos. 1, 2 and 3, we could not examine whether the growth advantage of the subclone harboring both the mutated nonRAS pathway gene and PTPN11 mutation decreased in the absence of therapeutic pressure. Accordingly, JMML clones with SETBP1 mutation and/or JAK3 mutation in addition to PTPN11 mutation appear to be refractory to 6-MP. Allogeneic HSCT may be capable to eliminate such 6-MP-resitant JMML clones because three of the children are alive and disease free after HSCT. Further large-scale studies are needed to accurately establish the relationship between acquisition of the nonRAS pathway mutations and post-transplant outcomes in patients with PTPN11 mutations.
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We thank Ms Yumiko Oguchi, Department of Pediatrics, Shinshu University School of Medicine, for her technical support.
The authors declare no conflict of interest.
KK and KM designed and performed the research, collected the samples, analyzed the data and wrote the paper. YN designed the research. CI performed the research. TK, KH, SS, MT, KY, RY, KS and SS collected the samples and analyzed the data. All the authors read and approved the manuscript.
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Matsuda, K., Nakazawa, Y., Iwashita, C. et al. Myeloid progenitors with PTPN11 and nonRAS pathway gene mutations are refractory to treatment with 6-mercaptopurine in juvenile myelomonocytic leukemia. Leukemia 28, 1545–1548 (2014). https://doi.org/10.1038/leu.2014.58
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