Myelofibrosis (MF) is a clonal Philadelphia chromosome negative myeloid neoplasm characterized by cytopenias with or without proliferative hematologic changes, extramedullary hematopoiesis and an increased propensity for transformation to acute myeloid leukemia. Identification of somatic mutations in Janus kinase 2 (JAK2) has revolutionized our understanding of the pathogenesis of some MF patients. However, there is a wide range of clinical heterogeneity in MF patients and somatic mutations of JAK2 (exon 14 or exon12) and MPL are only found in 50–60%/1–2% and 5–10% of MF patients respectively. Therefore, a large proportion of MF patients remain molecularly unaccounted after sequencing for these commonly analyzed mutations. We hypothesized that MF patients who are lack of JAK2 and MPL mutations, collectively called “triple-negative MF,” have a distinct clinicopathologic profile compared to their mutant counterparts.
Our study included 76 MF patients (primary MF=48, post-essential thrombocythemic-MF=21, post-polycythemic-MF=7), with amedian age of 65 years (25–80) and predominantly intermediate 1 or 2 risk by the D-IPSS grouping (low=8, Int-1=20, Int-2=42, high=6). We compared hematologic and clinical variables between JAK2 mutant (N=40) and triple-negative MF (N=36) and found that triple-negative MF patients had a higher WBC count (14.4±23.6. vs 11±14.9) and higher platelet counts (365±434.1 vs 252±220.5) but similar hemoglobin levels, LDH levels and D-IPSS plus risk profiles. No difference in overall survival was noted (25.5 months in triple-negative vs 24 months in JAK2 positive).
Newly discovered molecular mutations in myeloid malignancies have provided insight into the biology of MDS and some MPN cases. We performed direct sequencing on 9 genes including TET2 (Exon 3), DNMT3A (Exon 18/19, 20, 21, 22 and 23), IDH1/2 (Exon4), LNK (exon2–8), EZH2 (Exon 18/19, N/KRAS (Exon 1–2), CBL (Exon 8–9) and did not find any mutation in the triple-negative cohort. Recently, L3MBTL1 has been reported to be important in the pathogenesis of polycythemia vera, but sequencing of this gene in MF patients did not yield any mutations.
Next generation sequencing technologies have been instrumental in the discovery of new genes important in disease pathogenesis in myeloid neoplasias. We performed whole exome sequencing in the triple-negative cases and found somatic mutations in the following genes, ODZ1 (cellular signal transducer), ZNFE391 (transcriptional regulator), DENND3 (protein-coding), ARHGEF11 (modulator of cellular process through G protein) and PIK3CD (Phosphorylate Inositol Lipids regulator involving in Immune response) in one patient and subsequently confirmed as somatic by direct sequencing. This is the first time, which these genetic mutations were reported in hematologic malignancies specifically in MF. However, these mutations did not recur when screening, a limited cohort of patients (N=20).
For better understanding of this disease subtype, we compared the cytokine profile in these two groups based on the fact that, cytokines have been crucial in the pathogenesis of MF, and we found marked elevation in the levels of IL-1B (1.63 vs 0.42), IL-8 (2.89 vs 0.56), IL-17A (2.56 vs 1.12) and IFN-δ (2.07 vs 0.37) in the JAK2 positive patients compared to the triple-negative cases. Comparison of both groups showed similar responses to JAK inhibitors by spleen size shrinkage and constitutional symptoms.
In conclusion, despite the discovery of JAK2 and MPL mutations, a subset of MF patients remains molecularly uncharacterized. We found that triple negative MF patients had higher leukocyte and platelet counts, but a similar prognosis. Moreover, we identified novel somatic mutations in 1 triple-negative MF patient, which has not been previously described interestingly, despite lower cytokine levels; responses to JAK-inhibitors were still noted. Whole exome sequencing and other immunogenetic studies may reveal novel genes which may elucidate the pathogenesis of these MF cases.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.