Time Course of Acquisition of a CSF3R Mutation and Subsequent Development of AML in a Patient with Cyclic Neutropenia

Acquired CSF3R (colony stimulating factor 3 receptor, granulocyte) mutations have been detected with a high frequency (17-34%) in severe congenital neutropenia (CN) patients. Moreover, in CN patients who developed overt acute myeloid leukemia (AML) the frequency of CSF3R mutations in the intracellular critical region reaches more than 80%. Up to now there was no report on the detection of acquired mutations in CSF3R or development of AML/MDS in cyclic neutropenia patients (CyN). This group of patients reveals the same genotype with mutations in the ELANE gene as CN patients. In contrast, both groups of patients have a clearly distinct disease penetration.

Using ultra-deep sequencing approach we screened 22 CyN patients for the presence of genetic abnormalities in the critical region of the CSF3R gene. Interestingly, we found only in one CyN patient a clone with acquired mutation in G-CSF receptor (NP_000751.3, p.Gln741X) at 3% of allele frequency. The cycling neutrophils and platelets were well documented and verified the diagnosis of CyN. Within two years after the first detection, the mutant clone harboring the CSF3R mutation expanded and was detected in 16% of patient’s bone marrow (BM) MNCs.

The sequencing of the ELANE gene in the CyN patient with CSF3R mutation revealed the presence of two mutations p.Ala233Pro and p.Val235TrpfsX (NP_001963.1). Intriguingly, both ELANE mutations were absent in DNA isolated from each of parents indicating their de novo origin. Additionally, the patient was found to be heterozygous for synonymous SNP rs17216649 (dbSNP build 138) on the same ELANE exon permitting determination of the parental origin of mutations. The sub-clone analysis in E.coli showed that both mutations were always found on the paternally-derived allele. The missense mutation p.Ala233Pro (NP_001963.1) is well known to be associated with CN phenotype, whereas the single-base deletion p.Val235TrpfsX (NP_001963.1) was reported as cause of CN only once. There are no reports about detection of these mutations in CyN patients.

Three years after the first detection of the CSF3R mutation the patient presented with 14% blast cells in bone marrow acquiring monosomy 7 and trisomy 21. Based on results of cDNA sequencing all CSF3R expressing cells in the sample were positive for p.Gln741X (NP_000751.3) mutation.

Recently, we demonstrated acquisition of cooperative of RUNX1 and CSF3R mutations in more than 60 % of CN-AML/MDS patients. We also found RUNX1 mutation p.Asp171Asn (NP_001001890.1) at 10% allele frequency in BM MNCs of CyN-AML patient.

To evaluate at which stage of hematopoietic differentiation acquisition of CSF3R mutations appeared leading to the leukemogenic transformation, we performed colony forming unit (CFU) assays using patients BM MNCs. We observed the appearance of abnormal «CFU-blast» colonies after cultivation of BM MNCs in MethoCult H4434 Enriched medium supplemented with rh SCF, rh G-CSF, rh IL-3, rh IL-6 and rh EPO (72,2% of CFU-blasts (n=232), 21,8% of CFU-G (n=70), 5% of CFU-GM (n=16) and 1% of BFU-E (n=3)). Interestingly, we found that all sequenced CFU-G (n=4), CFU-GM (n=6) and «CFU-blast» colonies (n=20) were positive for p.Q741X CSF3R mutation. At the same time, no CSF3R mutations were identified in BFU-E colonies.

In summary, we report a first case of acquisition of CSF3R mutation and subsequent development of AML in a CyN patient underlying the complex nature of genotype:phenotype relations in CN and CyN patients. This case also represents an example of an extremely rare situation of double de novo ELANE mutations and provides a unique opportunity to investigate pathogenic molecular mechanisms of leukemia development in CyN patients. The ultra-deep next generation sequencing (NGS) for identification of CSF3R mutations is a useful tool to study the frequency of CSF3R mutations not only in CN but also in CyN patients and is clinically important for the identification of patients with the risk of progression to leukemia.