Nucleophosmin 1 (<i>NPM1</i>) mutations in chronic myelomonocytic leukemia and their prognostic relevance
Authors: Rangit Vallapureddy, Terra L. Lasho, Katherine Hoversten, Christy M. Finke, Rhett Ketterling, Curtis Hanson, Naseema Gangat, Ayalew Tefferi, Mrinal M. Patnaik
Journal: American Journal of Hematology (2017)

Chronic myelomonocytic leukemia (CMML) is an aggressive hematologic malignancy with overlapping features of myelodysplastic syndromes (MDS) and myeloproliferative neoplasms.1, 2 Patients with CMML, display between 10 and 15 mutations per kilobase of coding DNA regions; with majority of these (>90%) involving epigenetic regulator genes (TET2 60%, ASXL1 40%), splicing machinery (SRSF2 40%), and cell signaling (oncogenic RAS pathway 30%).3-5 Nucleophosmin 1 (NPM1,chromosome 5q35) is a gene encoding for the NPM1 protein that primarily resides in the nucleus, but has the ability to shuttle between the nucleus and the cytoplasm.6 NPM1 is required for critical cellular functions including; ribosome biogenesis, regulation of genomic stability, p53/MDM2 (mouse double minute 2 homolog) dependent stress response and the modulation of growth suppressive pathways.6 NPM1 mutations are seen in ≈ 30% of patients with acute myeloid leukemia (AML) with normal cytogenetics, with common phenotypic associations being; high bone marrow (BM) blasts, high white blood cell counts, reduced CD34 expression, and the presence of extramedullary manifestations.6, 7 The presence of NPM1 mutations in the absence of FLT3-ITD (fms related tyrosine kinase 3- internal tandem duplications) in cytogenetically normal AML is associated with favorable outcomes.8 NPM1 mutations are infrequent in CMML (<5%) and MDS (<5%) and have an unclear prognostic impact.9-11 We carried out this study to assess the (i) frequency and clinical correlates, (ii) prognostic impact and (iii) survival outcomes related to NPM1 mutations in CMML. Patients were selected according to the 2016 World Health Organization (WHO) diagnostic criteria for CMML and identified from the institutional database.1 All patients had BM biopsies and cytogenetic studies performed at diagnosis. A 29 gene panel next generation sequencing assay was carried out on BM DNA specimens obtained at diagnosis for the following genes; TET2, DNMT3A, IDH1, IDH2, ASXL1, EZH2, SUZ12, SRSF2, SF3B1, ZRSR2, U2AF1, PTPN11, Tp53, SH2B3, RUNX1, CBL, NRAS, KRAS, JAK2, CSF3R, FLT3, KIT, CALR, MPL, NPM1, CEBPA, IKZF, ETNK1, and SETBP1, by previously described methods.5, 11 Statistical analyses considered parameters obtained at time of CMML diagnosis. Differences in the distribution of continuous variables between categories were analyzed by either Mann–Whitney or Kruskal–Wallis tests. Patient groups with nominal variables were compared by chi-square test. Overall survival (OS) was calculated from the date of first referral to date of death or last contact. Leukemia-free survival (LFS) was calculated from the date of first referral to date of leukemic transformation or death/last contact. OS and LFS curves were prepared by the Kaplan–Meier method and compared by the log-rank test. Cox proportional hazard regression model was used for multivariable analysis. A total of 373 WHO-defined CMML patients were included in the study; median age 71 years (range, 20–95), 66% males. NPM1 mutations were identified in 8 (2%) patients (Table 1). All (100%) NPM1 mutations occurred in patients with normal cytogenetics, involved exon 12, with 4 of 8 (50%) being the classical type A NPM1 mutations resulting in a duplication of TCTG (nucleotides 956–959) creating a 4 base pair insertion at position 960. Morphological subtypes included; CMML-0 2 (25%), CMML-1 3 (38%), and CMML-2 3 (38%). Risk stratification by the Mayo Molecular Model included; intermediate-1 4 (50%) and high risk 4 (50%).4 In comparison to their wild type counterparts, NPM1 mutant CMML patients were more likely to be anemic (P = .02), have a "dysplastic CMML subtype" (87%, P = .04), have an increased incidence of DNMT3A (50%, P < .0001), FLT3 ITD (13%, P = .04) and a lower frequency of TET2 (13%, P = .03) and ASXL1 (13%, P = .03) mutations. Cytogenetics (n = 344) Abnormal; n (%) At last follow up (median, 14 months), 249 (67%) deaths and 69 (18%) leukemic transformations were documented. Median survival in NPM1 mutated CMML patients was 12.5 months versus 20.5 months in their wild type counterparts (P = .12, Figure 1A). Five of 8 (62%) NPM1 mutated CMML patients underwent blast transformation (BT), at a median of 5 months (range, 1–16). In 2 (of 5) evaluable patients, NPM1 mutations were detected at both, CMML diagnosis and at BT, without additional cytogenetic or molecular clonal evolution. Four (of 5) patients with BT were treated with AML-like induction chemotherapy, with 2 patients subsequently undergoing allogeneic stem cell transplantation (HCT). Median survival after BT was 3 months (range, 1–12), with only one patient that underwent HCT being alive at last follow up. In univariate analysis, LFS was adversely impacted by age (P < .0001), low hemoglobin (P = .0009), high absolute lymphocyte count (P = .02), higher serum lactate dehydrogenase levels (P = .023), PB blast % (P < .0001), BM blast % (P < .0001), 2016 WHO morphological subtype CMML-2 (P < .0001), abnormal cytogenetics (P = .002), Mayo-French cytogenetic risk stratification (P = .01),12 NPM1 (P = .0004), and Tp53 (P = .002) mutations. ASXL1, DNMT3A, TET2, and FLT3 ITD mutations did not impact LFS; in multivariable analysis that included the aforementioned significant variables, only PB blasts % (P < .0001, HR 1.2, 95% CI 1.13–1.3), NPM1 mutations (P = .03, HR 4.7, 95% CI 1.7–12.8), and Tp53 mutations (P = .001, HR 5.4, 95% CI 1.6–18.4) remained significant. A, Overall survival of 373 patients with chronic myelomonocytic leukemia stratified by their NPM1 mutational status. B, Leukemia-free survival of 373 patients with chronic myelomonocytic leukemia stratified by their NPM1 mutational status The current study confirms the infrequent occurrence of NPM1 mutations in CMML; also, the number of informative cases was large enough to allow demonstration of clustering of NPM1 mutations in CMML with normal cytogenetics, "dysplastic CMML phenotype" and DNTM3A mutations. More importantly, we show a significantly higher risk of BT in patients with CMML harboring NPM1 mutations. 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