A Novel Somatic Mutation in IKZF1 Is Common in Patients Undergoing Allogeneic Hematopoietic Stem Cell Transplant for Myelofibrosis

Background:

Myeloproliferative neoplasms (MPN) including polycythemia vera, essential thrombocytosis, and primary and secondary myelofibrosis (MF) are characterized by driver mutations in JAK2, CALR, and MPL. Additional somatic variants can be seen. Currently allogeneic hematopoietic stem cell transplant (HCT) is the only treatment with curative potential, but it is associated with a high risk of mortality, exceeding 50% in some patient subsets. The risk of disease progression usually outweighs the risk of transplant in patients with advanced MF. Here, we have evaluated the mutational profile using a targeted next-generation sequencing panel of 54 genes involved in myeloid neoplasms in patients with MF treated with HCT.

Patients and Methods:

48 patients with a diagnosis of primary (n=18) or secondary (n=30) MF who received a HCT at our center and from whom genomic DNA was available from pre-HCT marrow aspirates were evaluated. DNA was isolated from whole marrow aspirates obtained prior to HCT. Paired-end indexed libraries were sequenced at an average read depth of 1000x on Illumina MiSeq platform. Sequences were aligned to hg19 using the Burrows-Wheeler Aligner, and nucleotide variants were identified using the Genome Analysis Toolkit and Picard tools. Variants were annotated with ANNOVAR. Variants present at >5% variant allele frequency (VAF) with at a minimum of 25 variant reads were selected for further analysis. Variants were called "mutations" if they resulted in (1) non-synonymous changes in amino acid sequence, including nonsense and missense mutations, (2) the variant was known to be associated with hematologic malignancies in COSMIC database, or (3) the variant was present at < 0.5% in 1000 genomes and ExAC databases. Comparisons between clinical variables and mutations were analyzed with chi-square tests, adjusted for multiple testing.

Results:

The average age of the 48 patients was 55 years (range 23-72) and 52% of patients were female. Disease risk was estimated with DIPSS-Plus (High 33%, INT-2 53%, INT-1 7%, Low 7%). Unfavorable cytogenetics were present in 27% of patients. Donors included a matched sibling donor in 30% (n=14), a matched unrelated donor in 60% (n=29), and an alternative donor in 10% (n=5). At 3 years, overall survival was 52%, 17% of patients had relapsed disease, and 31% had non-relapse mortality.

In our cohort, 38% had a mutation in CALR (n=15 type-I and n=3 type-II), 29% in JAK2 (n=14), 0% in MPL, and 33% were triple-negative (n=16; no evidence of CALR, JAK2, or MPL mutation). The average VAF of CALR mutations was 50% (range 20-82%) and JAK2 mutations was 58% (range 25-81%). In addition to driver mutations, 94% of patients had at least one other mutation in 26 of the 54 genes sequenced. On average, patients had 2.1 other mutations (range 0-5 mutations in addition to driver mutation). The most common other mutations were in ASXL1 (31%), IKZF1 (23%), ETV6 (17%), and U2AF1 (17%). The VAF of these other mutations spanned 9-76%.

Patients with triple-negative MF were more likely to have primary MF (p=0.019) and platelets <100 (p<0.001), than patients with JAK2 and CALR mutations. Patients with ≥3 other mutations were more likely to have hemoglobin <10 (p=0.02) and a higher rate of relapse (p<0.01) than patients with <3 other mutations. Patients with ASXL1 mutations were more likely to have peripheral blood blasts (p<0.001).

Mutations in ASXL1, ETV6, and U2AF1 have been previously reported in myeloid malignancies. Like other groups, we found ASXL1 mutations associated with worse prognosis. Here we report a novel, recurrent mutation in the gene IKZF1. All patients with IKZF1 mutation carried the same insertion of 11 nucleotides leading to an early frameshift and ultimate nonsense mutation (L92fs), which would lead to deletion of the zinc-finger domain. Such a mutation likely leads to loss of IKZF1 tumor suppressor function.

Conclusions:

Somatic mutations, in addition to driver mutations, are common in patients with MF treated with HCT. Mutation profiling in this patient subset may improve relapse risk prediction. Loss of heterozygosity on chromosome 7 mapping to the IKZF1 locus has been found by others to be associated with leukemic transformation in patients with MPN (Jaeger et al. 2010. Leukemia. 24: 1290). Given their frequency in this high risk MF population, further study of the role of IKZF1 mutations in MPN is warranted.