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T. Kaburagi et al.
Recently, aberrations of NF1 and PTPN11 were reported to be associated with a poor prognosis in adult patients with AML.3,4 NF1 and PTPN11 are the family of RAS path- way genes and constitute the granulocyte-macrophage colony stimulating factor signaling pathway. Among the broad family of RAS pathway genes, mutations of CBL, NRAS and KRAS were also commonly detected in AML.2 These RAS pathway alterations have also been implicated in the causation of juvenile myelomonocytic leukemia (JMML).5
Mutations of PTPN11, NRAS, and KRAS have been reported in 3–4%,6,7 7–13%, 6–11%8,9 of pediatric patients with AML, respectively. However, there is no clear consen- sus on the clinical significance of RAS pathway gene muta- tions especially NF1 and CBL mutations.10,11 The reported frequency of detection of CBL mutations and NF1 muta- tions or deletions in adult patients with AML is 0.6–0.7%12,13 and 3.5–10.5%,14-16 respectively. However, the prognostic relevance of these mutations is not well characterized, par- ticularly in pediatric AML patients.
In this study, we analyzed NF1, PTPN11, CBL, NRAS, and KRAS alterations in 328 pediatric patients with AML to determine the clinical significance of these alterations. We also examined the correlation of RAS pathway alterations with other genetic aberrations, cytogenetic alterations, and clinical characteristics.
Methods
Patients
Between November 2006 and December 2010, 443 pediatric patients with de novo AML (age <18 years) participated in the Japanese AML-05 trial conducted by the Japanese Pediatric Leukemia/Lymphoma Study Group (JPLSG). Treatment, data col- lection, and other details of the AML-05 study are presented in the Online Supplementary Appendix and the Online Supplementary Figure S1. This study was conducted in accordance with the Declaration of Helsinki and approved by the Institutional Review Board of the Gunma Children’s Medical Center and the Ethical Review Board of the JPLSG.
Mutation analysis of RAS pathway alterations
We analyzed PTPN11 (exons 2–4, and 13), CBL (exons 8–9), NRAS (exons 1–2), and KRAS (exons 1–2) mutations using Sanger sequencing as previously described.9,17,18 All coding exons of the NF1 were captured using the SureSelect custom kit (Agilent Technologies, Santa Clara, CA, USA), and sequenced using Hiseq 2500. Somatic mutations in NF1 were identified as described else- where.19
Molecular characterization
We analyzed KIT (exons 8, 10, and 17),20 NPM1 (exon 12),21 CEBPA (exons 1–4),22 CSF3R (exons 14 and 17),23 WT1 (exons 7– 10),24 ASXL1 (exon 12), ASXL2 (exons 11 and 12),25 all exons of BCOR, BCORL126, RAD21, SMC3, STAG2,27 RUNX1,28 FLT3-ITD,29 and gene rearrangement of NUP98-NSD130 and FUS-ERG31 using Sanger sequencing. KMT2A-partial tandem duplication (PTD) was analyzed using the multiplex ligation-dependent probe amplifica- tion (MLPA) method.32 Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of the PRDM16 and MECOM genes was performed using the 7900HT Fast Real Time PCR System, TaqMan Gene Expression Master Mix, and TaqMan Gene Expression Assay (Applied Biosystems, Foster City, CA, USA), as described elsewhere.33
Copy number analysis
Copy number (CN) analysis was performed as previously reported34 using an in-house pipeline CNACS (https://github.com/papaemmelab/toil_cnacs); the total number of reads covering each bait region and the allele frequency of het- erozygous single-nucleotide polymorphisms (SNP) (n=1,216) detected by targeted sequencing were used as input data. Based on the previous reports,15 we set the total CN <1.5 as the definition of NF1 deletion.
Statistical methods
All statistical analyses were performed using the EZR software (version 1.35; Saitama Medical Center, Jichi Medical University, Saitama, Japan).35 Between-group differences with respect to clin- ical characteristics were assessed using the Fisher’s exact and Mann-Whitney U tests. Survival rates were estimated using the Kaplan–Meier method and compared using the log-rank test. Overall survival (OS) was defined as the time from diagnosis to death or last follow-up. Event-free survival (EFS) was defined as the time from diagnosis to the date of failure (induction failure, relapse, second malignancy, or death) for patients who experi- enced treatment failure or to the date of last contact for all other patients. Cox proportional hazards model was used to estimate hazard ratios and 95% Confidence Intervals (CI). For all analyses, two tailed P-values <0.05 were considered indicative of statistical significance.
Results
Frequencies of RAS pathway alterations in 328 pediatric acute myeloid leukemia patients
Out of the 443 patients, 115 patients were excluded from this study because of unavailability of genomic DNA samples. Therefore, 328 samples were analyzed in this study. We did not analyze germline alterations because of the lack of non-hematological or remission samples. The clinical characteristics of patients with available samples (n=328) and those with no available samples (n=115) are summarized in the Online Supplementary Table S1. White blood cell (WBC) count at diagnosis was significantly higher in the “sample available group” than in the “sample unavailable group” (P<0.001). There were more patients who were at a low risk and there were less patients who were at an intermediate risk in the “sample available group” as compared with the “sample unavailable group” (low risk, P=0.046; intermediate risk, P=0.003). Cytogenetic features and prognosis were not significantly different between the available and unavailable samples (Online Supplementary Table S1).
RAS pathway alterations were detected in 80 (24.4%) of the 328 patients; most of these alterations were mutually exclusive (Figure 1). The mutation sites and clinical char- acteristics of patients with RAS pathway alterations are summarized in Figure 2, Tables 1 and 2 and the Online Supplementary Tables S2 and S3, respectively.
We detected six NF1 mutations in four patients; all of these were frameshift or nonsense mutations (Figures 1 and 2). Two patients concomitantly had two types of mutations, respectively (Table 1). In addition, we also detected four patients with a microdeletion within chro- mosome 17q containing NF1 (Table 1; Online Supplementary Figure S2). One patient had both an NF1 mutation and CN alteration and NF1 alterations were detected in seven (2.1%) patients (Figure 1; Table 1). Two
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