Transcription factor mutations in myelodysplastic/myeloproliferative neoplasms

Abstract

Background: Aberrant activation of tyrosine kinases, caused by either mutation or gene fusion, is of major importance for the development of many hematologic malignancies, particularly myeloproliferative neoplasms. We hypothesized that hitherto unrecognized, cytogenetically cryptic tyrosine kinase fusions may be common in non-classical or atypical myeloproliferative neoplasms and related myelodysplastic/myeloproliferative neoplasms.

Design and methods: To detect genomic copy number changes associated with such fusions, we performed a systematic search in 68 patients using custom designed, targeted, high-resolution array comparative genomic hybridization. Arrays contained 44,000 oligonucleotide probes that targeted 500 genes including all 90 tyrosine kinases plus downstream tyrosine kinase signaling components, other translocation targets, transcription factors, and other factors known to be important for myelopoiesis.

Results: No abnormalities involving tyrosine kinases were detected; however, nine cytogenetically cryptic copy number imbalances were detected in seven patients, including hemizygous deletions of RUNX1 or CEBPA in two cases with atypical chronic myeloid leukemia. Mutation analysis of the remaining alleles revealed non-mutated RUNX1 and a frameshift insertion within CEBPA. A further mutation screen of 187 patients with myelodysplastic/myeloproliferative neoplasms identified RUNX1 mutations in 27 (14%) and CEBPA mutations in seven (4%) patients. Analysis of other transcription factors known to be frequently mutated in acute myeloid leukemia revealed NPM1 mutations in six (3%) and WT1 mutations in two (1%) patients with myelodysplastic/myeloproliferative neoplasms. Univariate analysis indicated that patients with mutations had a shorter overall survival (28 versus 44 months, P=0.019) compared with patients without mutations, with the prognosis for cases with CEBPA, NPM1 or WT1 mutations being particularly poor.

Conclusions: We conclude that mutations of transcription and other nuclear factors are frequent in myelodysplastic/myeloproliferative neoplasms and are generally mutually exclusive. CEBPA, NPM1 or WT1 mutations may be associated with a poor prognosis, an observation that will need to be confirmed by detailed prospective studies.

Figure 1.

Targeted array CGH and sequencing profiles. (A) Control experiments demonstrating that targeted arrays were readily able to identify the 800kb FIP1L1-PDGFRA deletion in EOL1 cells in a background of normal cells. EOL1 cells harboring two copies of the del(4) and one normal chromosome 4. (B) 1:2.5 mixtures of EOL1 DNA with normal DNA, simulating a heterozygous deletion with 50% background normal cells. (C) RUNX1 deletion in a patient with atypical CML. Targeted array CGH profile of chromosome 21 (left) with zoom of the respective region (right) showing a hemizygous deletion of RUNX1. (D) Whole-genome 244K array CGH results confirm this observation and further characterize the deletion as a 841kb deletion including RUNX1. (E) CEBPA deletion in a patient with atypical CML. Targeted array CGH profile of chromosome 19 (left) with zoom of the respective region (right) showing a hemizygous deletion of CEBPA. (F) Whole-genome 244K array CGH results further characterize the deletion as a 6.3Mb deletion including CEBPA and several other genes. (G) Sequencing result of the atypical CML patient with a CEBPA deletion. A homozygous 2 bp insertion was observed resulting in a frameshift at amino acid threonine 60 (bottom panel). The CEBPA reference sequence is shown in the top panel.

Figure 2.

Schematic presentation of (A) RUNX1 protein and (B) CEBPA protein with locations of mutations. The majority (60%) of RUNX1 mutations were found within the RUNT domain. CEBPA mutations were spread throughout the coding sequence. ▵ Missense mutation; ▴ frameshift insertion/deletion mutation; ● nonsense mutation; TAD, transactivation domain; DNA, DNA binding domain.

Figure 3.

Clinical significance of transcription factor (TF) mutations in MDS/MPN. Kaplan-Meier estimates of (A) overall survival and (B) progression-free survival for 127 MDS/MPN patients with or without mutations of transcription factors. Overall survival was significantly lower in mutation-positive compared to mutation-negative cases (P=0.0190, log-rank test). Kaplan-Meier estimates of (C) overall survival and (D) progression-free survival for each specific mutation. Patients with CEBPA, NPM1 or WT1 mutations collectively showed a highly significant shorter overall survival (P<0.001) and progression-free survival (P<0.001) compared to cases without transcription factor mutations. Considering each gene individually, the P values for CEBPA, NPM1 and WT1 were 0.0047, 0.0024 and 0.0583, respectively, for overall survival and 0.0096, 0.0018 and 0.174, respectively for progression-free survival.