Mutational spectrum and prognosis in NRAS-mutated acute myeloid leukemia

The mutational spectrum and prognostic factors of NRAS-mutated (NRASmut) acute myeloid leukemia (AML) are largely unknown. We performed next-generation sequencing (NGS) in 1,149 cases of de novo AML and discovered 152 NRASmut AML (13%). Of the 152 NRASmut AML, 89% had at least one companion mutated gene. DNA methylation-related genes confer up to 62% incidence. TET2 had the highest mutation frequency (51%), followed by ASXL1 (17%), NPM1 (14%), CEBPA (13%), DNMT3A (13%), FLT3-ITD (11%), KIT (11%), IDH2 (9%), RUNX1 (8%), U2AF1 (7%) and SF3B1(5%). Multivariate analysis suggested that age ≥ 60 years and mutations in U2AF1 were independent factors related to failure to achieve complete remission after induction therapy. Age ≥ 60 years, non-M3 types and U2AF1 mutations were independent prognostic factors for poor overall survival. Age ≥ 60 years, non-M3 types and higher risk group were independent prognostic factors for poor event-free survival (EFS) while allogenic hematopoietic stem cell transplantation was an independent prognostic factor for good EFS. Our study provided new insights into the mutational spectrum and prognostic factors of NRASmut AML.

www.nature.com/scientificreports/ Treatment protocols. For M3 patients, all-trans retinoic acid and arsenic trioxide-based chemotherapy was given for the induction and consolidation therapy. Non-M3 patients received induction chemotherapy regimens include DA, IA, and MA regimens: a standard dose of cytarabine (Ara-C; 100 mg/m2/ day for 7 days) combined with daunorubicin (45 mg/m 2 /day for 3 days) or idarubicin (10 mg/m 2 /day for 3 days) or mitoxantrone (10 mg/m 2 /day for 3 days). After remission, young patients were consolidated with cytarabine (2-3 g/m 2 , once every 12 h for 3 days) based chemotherapy. For elderly patients, the chemotherapy consolidation was decided by the physicians in an individualized manner. A total of 24 patients underwent allogenic hematopoietic stem cell transplantation (allo-HSCT). Therapy recommendation was based on risk stratification and the results of minimal residual disease testing after 1-2 cycles of consolidation chemotherapy. The real treatment selection was based on both the physician's recommendation and the patient's preference. The last follow-up for surviving patients was conducted in December 2019.
Cytogenetics and fusion genes analysis. Cytogenetic

Results
Clinical and biological characteristics of NRAS mut AML. In the total cohort, NRAS mutations were found in 13% (152 of 1,149) cases. As shown in Table 1, median age was 44 (range 14-78) years, with 25 cases (16%) older than 60 years. Half of the cases were men. Twelve cases (8%) were M3 and 140 cases were non-M3 AML. The median white blood cell (WBC) count at diagnosis was 31 × 10 9 /L, and in 27 cases (18%) it was ≥ 100 × 10 9 /L. Forty-five cases (30%) had a bone marrow blast percentage of more than 80%. Forty-one cases (27%) was good-risk AML, 64 (42%) was intermediate-risk AML and 47 (31%) was poor-risk AML. Twenty-four cases (16%) received allogenic hematopoietic stem cell transplantation (allo-HSCT). Thirty-six cases failed to achieve CR after induction therapy and 61 cases died at the end of follow up. Thirty-five cases (16%) had more than two other recurrent genetic mutations. Forty-three fusion genes were detected in 135 cases and 16 cases were AML1-ETO positive; 15 cases were MYH11-CBFβ positive and 7 cases were MLL positive.

Comparison of OS and EFS between
Cases who accepted allo-HSCT had longer OS and EFS (P = 0.016, P = 0.001, respectively; Fig. 3c). Presence of U2AF1 was associated with shorter OS and EFS (P = 0.000, P = 0.000, respectively; Fig. 3d). Presence of RUNX1 and SF3B1 was associated with shorter OS (P = 0.014, P = 0.032, respectively). Number of co-mutations ≥ 3 and presence of IDH2 was associated with shorter EFS (P = 0.025, P = 0.043, respectively). However, both NRAS type and NRAS VAF had no effect on EFS and OS.  Table 4, independent prognostic factors for poor OS included age ≥ 60 years, non-M3 types and U2AF1 mutations. Multivariate analysis of factors related to EFS included age (≥ 60 vs. < 60 years), AML types (Non-M3 vs. M3), risk group (high vs. inter vs. low-risk), the time-dependent variable for allo-HSCT (yes vs.no), number of comutations (≥ 3 vs. < 3), IDH2 (mutated vs. wild type), RUNX1 (mutated vs. wild type) and U2AF1 (mutated vs. wild type). As shown in Table 4, age ≥ 60 years, non-M3 types and higher risk group were independent prognostic factors for poor EFS while allo-HSCT was an independent prognostic factor for good EFS.

Discussion
High frequencies of NRAS mutations had been seen in AML patients 11 , indicating its important function in the pathogenesis and progression of AML. Although the prognostic value of NRAS mutations in AML patients remains inconclusive 6,12 , several large cohort studies indicated that NRAS mutations in AML did not influence the prognosis of patients 11,13,14 . A recently published meta-analysis also suggested that NRAS oncogene mutations were not correlated with the prognosis of patients with AML 6 . However, given the prevalence of NRAS mutations www.nature.com/scientificreports/ in AML, there is urgently need to explore the clinical characteristics, companion gene mutations and possible prognostic factors of NRAS mut AML patients to provide evidence for clinical stratified diagnosis and treatment. Our data showed that NRAS mutations were found in 13% of cases, which is consistent with findings in other literature that showed a range of 9.7% to 13.9% NRAS mutations 11,14-16 . The median age of NRAS mut AML cases was 44 years and the median WBC counts was 31 × 10 9 /L, which was consistent with a large cohort study in China in 2013 14 . In our study, most NRAS mutations were found at codon 12 and the most frequent change were from glycine to asparagine, which was supported by previous reports 11,17 . Interestingly, we found that some samples have two NRAS mutations such as Q61K + Q61R, which have been confirmed by Sanger sequencing. This situation is very rare, it is not ruled out that two mutations have occurred in the same gene, but it may also be caused by mutations in the same allele. Nearly 90% of the cases had at least one companion gene mutation, which suggests that the molecular mechanism of patients with NRAS mut AML is complicated, and multiple molecular interactions may exist. However, previous studies often focused on comparing the difference between NRAS mut and NRAS wild-type patients 11,14 , with little attention on the molecular mutation spectrum. We observed that mutations of DNA methylation-related genes occurred in 62% NRAS mut AML, the most common of which is TET2. This indicated that DNA methylation may play an important role in the pathogenesis in NRAS mut AML and provided a basis for demethylation treatment of NRAS mut AML patients.
AML in older patients generally had poorer prognosis due to poorer baseline performance status, and comorbidities 18 . In our cohort of NRAS mut AML, age ≥ 60 years also had a negative impact on both response to induction therapy and survival. Allogeneic HSCT which was usually considered the cure for AML, also showed survival benefit in our study. Traditional risk stratification schemes based on genetics and molecular biology were still applicable in patients with NRAS mut AML and could well predict patients' prognosis. Mutation Gene VAF of FLT3-ITD or NPM1 was reported to be significantly correlated with prognosis of AML 19,20 . However, we found that NRAS VAF had no correlation with either response to induction therapy or survival. FLT3-ITD was www.nature.com/scientificreports/ associated with increased risk of relapse while NPM1, AML1-ETO, MYH11-CBFβ were good prognostic factors 18 . In our study, however, recurrent genetic mutations including FLT3-ITD, NPM1, DNMT3A, TET2 and KIT and fusion genes including AML1-ETO and MYH11-CBFβ were not associated with survival. The discrepancy may be related to possible interplay of mutated genes. U2AF1 belongs to mutations in splicing factor (SF) genes. Mutations in U2AF1 is a poor prognostic indicator in myelodysplastic syndrome 21 . Recently many studies proved that mutations in U2AF1 predict poor prognosis in patients with de novo AML [22][23][24][25] . Our study showed that U2AF1 was also an independent poor prognostic factor for survival in NRAS mut AML patients. In a large study of 664 AML patients conducted by the German AML Cooperative Group, mutations in U2AF1 were one of the independent risk factors for achievement of CR1 26 . Similar to this result, in our study, 90% U2AF1-mutated AML patients failed to achieve complete remission.
The limitations to our study need to be acknowledged. First, our study was retrospective and susceptible to selection biases. Second, some gene mutations may not be detected due to the limitation of technique. Prognostic effects of some important gene mutations may be ignored. Third, whether these findings are restricted to NRAS mut AML need to be further explored by parallel comparison with non NRAS mut AML. Fourth, the small sample sizes of some subgroups resulted in relatively low statistical power and the univariate analyses were not adjusted for multiple comparisons which may result in false positive results, especially in small subgroups. Because of these limitations, our conclusion needs validation in a larger and prospective cohort.
In conclusion, our study provided new insights into the mutational spectrum and prognostic factors of NRASmut AML. These individuals companied with U2AF1 mutations experienced poor responses to chemotherapy and the mechanisms need to further evaluate. More detailed mutational spectrum information and large prospective studies are needed in the future for better prognostication of NRAS mut AML. www.nature.com/scientificreports/