Phenotypic correlates and prognostic outcomes of TET2 mutations in myelodysplastic syndrome/myeloproliferative neoplasm overlap syndromes: A comprehensive study of 504 adult patients
Authors: Giacomo Coltro, Guadalupe Antelo, Terra L. Lasho, Christy M. Finke, Animesh Pardanani, Naseema Gangat, Ryan M. Carr, Moritz Binder, Abhishek A. Mangaonkar, Rhett Ketterling, Martin E. Fernandez‐Zapico, Keith D. Robertson, Alberto Bosi, Alessandro M. Vannucchi, Ayalew Tefferi, Mrinal M. Patnaik
Journal: American Journal of Hematology (2020)

To the Editor: The 2016 iteration of the World Health Organization (WHO) classification of myeloid neoplasms identifies five distinct sub-types of myelodysplastic/myeloproliferative neoplasms (MDS/MPN), namely chronic myelomonocytic leukemia (CMML), atypical chronic myeloid leukemia, BCR/ABL1− (aCML), juvenile myelomonocytic leukemia (JMML), MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T), and MDS/MPN, unclassifiable (MDS/MPN-U).1 With the notable exception of JMML, a pediatric entity, currently considered a bona fide RASopathy, the remaining MDS/MPN are characterized by clinical heterogeneity that is at least in part supported by genome-wide molecular diversity. Mutations in the Ten Eleven Translocation 2 (TET2MT) are frequent in MDS/MPN overlap syndromes, especially CMML (∼60%).2 The TET2 gene (chromosome 4q24) encodes a Fe2+- and 2-oxoglutarate-dependent dioxygenase The enzyme is involved in the epigenetic regulation of the mammalian genome thorough the iterative oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC).3 Loss-of-function TET2MT are thought to represent early genetic driver events establishing pre-leukemic clonal hematopoiesis, but needing other gene mutations for the development of a fully penetrant leukemic phenotype.4 Recently, we investigated the phenotypic landscape and clinical outcomes of TET2MT in CMML,5 and in this manuscript extend the comparative assessments to adult patients with MDS/MPN overlap syndromes (excluding JMML) in a large molecularly annotated database. After approval by the Mayo Clinic (Rochester, MN, USA) institutional review board, adult patients with WHO-defined MDS/MPN (with the exception of JMML) were included in the study. The bone marrow (BM) morphology, cytogenetics and 2016, WHO diagnoses were retrospectively reviewed and all patients underwent targeted next generation sequencing for myeloid-relevant genes, obtained on BM mononuclear cells, at diagnosis, or at first referral, by previously described methods (Table S1).6 All variants were manually curated to assess their pathogenicity, as previously described.5 Statistical analyses considered clinical and laboratory parameters obtained at time of diagnosis or first referral (for details refer to Supplementary Material). Five hundred and four patients were included in the study: 387 (77%) with CMML, 48 (10%) with MDS/MPN-RS-T, 17 (3%) with aCML and 52 (10%) with MDS/MPN-U. The median age at diagnosis was 71 years (range 18-99 years), and 333 (66%) were male. TET2MT were seen in 212 (42%) patients, with the frequency of other commonly occurring mutations being ASXL1MT 45%, SRSF2MT 40%, NRASMT 15%, SF3B1MT 13%, CBLMT, RUNX1MT and SETBP1MT 12% each, and JAK2V617F 11% (Figure 1A,B). Among the MDS/MPN overlap syndromes, TET2 was more frequently mutated in CMML (49%) and aCML (47%) compared to MDS/MPN-RS-T (10%) and MDS/MPN-U (15%). The prevalence of patients with TET2MT increased with age, a finding consistent across all MDS/MPN subtypes (Figure S1A). Cumulatively, 343 TET2MT were identified in 212 patients (mean 1.6 variants per mutated patient; range 1-5). One hundred and twenty (24%) patients had >1 TET2MT, with 113 (22%), 5 (1%) and 2 (0.4%) harboring 2, 3 and 5 mutations, respectively. Both CMML and aCML patients were more likely to have an age-independent increase in multiple TET2MT (28% and 24%, respectively), in comparison to MDS/MPN-RS-T (4%) and MDS/MPN-U (8%). Thus, TET2MT spanned the entire coding sequence and were mostly truncating (78%): 61 (18%) were missense, 14 (4%) involved the splice-donor/acceptor sites, 2 (0.6%) were in-frame deletions, 129 (38%) were nonsense and 137 (40%) were frameshift mutations (Figure 1C). While truncating TET2MT were randomly spread across the entire coding sequence without identifiable hot spots, missense and splice site mutations were mainly located in the C-terminus catalytic domain. Overall, the distribution of TET2MT was superimposable across CMML, aCML, and MDS/MPN-U; the only exception being the absence of splice site mutations in the latter three entities (Figure S2). One hundred and eighty-nine (55%) TET2MT were secondary to pathogenic single nucleotide variants (SNV), while the remainders were secondary to deletions (28%) and insertions (17%). Transitions comprised the most frequent type of SNV (65%), with the C:G > T:A being the most common (56%; Figure S1B). Notably, this type of substitution is generally considered a mutational signature of aging, since it probably results from endogenous deamination of 5-mC that occurs spontaneously in normal and neoplastic cells.7 Median variant allelic fraction (VAF) was 45% (range, 6%-100%) for all evaluable TET2MT (n = 333). There were no significant differences in VAF among different types of TET2MT (median, 45% for missense, 42% for splice site, 46% for nonsense, and 43% for frameshift mutations), except for nonsense mutations which had a higher VAF compared to frameshift mutations (P = .0031; Figure S1C). The median TET2MT VAF was 45%, 45%, 47%, and 42% for CMML, MDS/MPN-RS-T, aCML, and MDS/MPN-U, respectively. There were no significant differences in the distribution of VAF among the different MDS/MPN entities (Figure S1D). Prominent clinical and laboratory characteristics of the study population, stratified by TET2 mutational status are summarized in Table S2. Among the 212 TET2MT patients, 146 (69%) were male and median age at diagnosis was 71 (range, 40-99) years. In comparison to their wild type counterparts, TET2MT patients were more likely to have higher hemoglobin levels (P < .0001), lower platelet counts (P < .0001), lower percentage of circulating blasts (P < .0001) and were less likely to be red blood cell transfusion dependent (P = .0007). By disease subtype, these findings were reproducible mainly in CMML (Table S3). Moreover, CMML patients with TET2MT were more likely to have higher white blood cell counts (P = .0157), higher absolute neutrophil counts (P = .0153), and lower BM blasts (P = .0060). Conversely, MDS/MPN-RS-T patients with TET2MT were more likely to be female (P = .0373) and had higher peripheral blood (P = .0127) and BM (P = .0278) blasts. With regards to the molecular landscape, MDS/MPN patients with TET2MT were more likely to have additional mutations in comparison to their wild type counterparts (mean mutation number 3.1 vs 2.1, P < .0001), with common co-mutations being SRSF2MT (51%), ASXL1MT (42%), CBLMT (17%), NRASMT (15%), and RUNX1MT (13%). CBLMT (P = .0050), SRSF2MT (P < .0001) and ZRSR2MT (P = .0366) significantly clustered with TET2MT, while IDH2MT (P = .0002), SETBP1MT (P = .0020), SF3B1MT (P = .0002) and U2AF1MT (P = .0023) were more common in TET2WT patients (Figure 1D). This is consistent with the reported exclusivity of TET2MT and IDHMT in myeloid neoplasms.8 By disease subtype, in CMML, TET2MT most often co-occurred with SRSF2MT (54%), ASXL1MT (40%), CBLMT (18%), NRASMT (14%), and RUNX1MT (12%). Compared to their wildtype counterparts, CMML patients with TET2MT more frequently had mutations in CBL (P = .0407), JAK2 (P = .0408), SRSF2 (P = .0014), and ZRSR2 (P = 0.0400), whereas ASXL1MT (P = .0104), IDH2MT (P < .0001), SETBP1MT (P = .0138), and U2AF1MT (P = .0007) significantly clustered with TET2WT (Figure 1D). In MDS/MPN-RS-T, TET2MT frequently co-occurred with SF3B1MT (100%), DNMT3AMT (40%), SETBP1MT (20%), JAK2MT (20%), and CSF3RMT (20%), and significantly clustered with CEBPAMT (P < .0001) and DNMT3AMT (P = .0495) (Figure 1D). In aCML, the most common concurrent mutations were ASXL1MT (88%), NRASMT (38%), EZH2MT (38%), and RUNX1MT (38%), with ASXL1MT being more frequent in TET2MT (P = .0235) (Figure 1D). In MDS/MPN-U, TET2MT frequently co-occurred with ASXL1MT (63%), SRSF2MT (50%), EZH2MT (38%), RUNX1MT (38%), and JAK2MT (25%), and significantly clustered with EZH2MT (P = .0036) and RUNX1MT (P = .0303) (Figure 1D). Finally, in CMML, but not in the other MDS/MPN entities, TET2MT were significantly associated with cytogenetic abnormalities (P < .0001), monosomal (P = .0032) and complex (P = .0058) karyotypes, in comparison to TET2WT patients. The median follow-up for the entire cohort was 73 months (95% confidence interval [CI], 56-87 months), and at last follow-up 330 (65%) deaths and 83 (16%) leukemic transformations were documented. The median overall survival (OS) was 29 months (95% CI, 24-32 months): 29 (95% CI, 23-32 months) for CMML, 63 (95% CI, 29-76 months) for MDS/MPN-RS-T, 14 (95% CI, 5-16 months) for aCML and 24 (95% CI, 16-36 months) for MDS/MPN-U. On univariate analysis, there were no survival differences between TET2MT and TET2WT patients in the entire MDS/MPN cohort (Figure 1E). By disease subtype, while OS was not significantly different in aCML, MDS/MPN-RS-T, and MDS/MPN-U (Figures S3A-S3C), as previously published, in CMML, TET2MT were associated with a survival advantage in comparison to wild type patients (35 vs 21 months, P < .0001) (Figure 1F). There were no differences in leukemia-free survival (LFS) between TET2MT and TET2WT patients across all the MDS/MPN overall syndromes. The current study confirms that TET2MT are among the most frequent mutations in patients with MDS/MPN overlap syndromes, especially CMML and aCML, with an age-dependent increase in the frequency and number of TET2MT. Nonsense and frameshift mutations are the most common and span the entire coding sequence, whereas non-truncating TET2MT are less frequent and localize mainly in the C-terminus catalytic domain. Among TET2 SNV, the C:G > T:A transition is the most frequent; a finding congruous with the fact that most patients are elderly and that this change is considered a signature of aging.7 The survival benefit and favorable phenotypic characteristics associated with TET2MT in CMML patients are not seen in other MDS/MPN overlap syndromes, further strengthening the clause that CMML indeed has unique disease biology. Future studies assessing the mechanisms behind these findings are much needed. Current publication is supported in part by grants from the "The Henry J. Predolin Foundation for Research in Leukemia, Mayo Clinic, Rochester, MN, USA". Mrinal Patnaik has served on the advisory board of StemLine Pharmaceuticals. Figure S1. A, Graphic representation of age-related increases in the prevalence of TET2MT within the entire MDS/MPN cohort and each disease subtype. B, Distribution of base changes among the 189 TET2MT consequent to single nucleotide variants (SNV). C and D, Box plot showing the distribution of variant allele frequency (VAF) of TET2MT by mutation type (C) and disease subtype (D). Abbreviations: aCML, atypical chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia, MDS/MPN, myelodysplastic/myeloproliferative neoplasms; MDS/MPN-U, MDS/MPN, unclassifiable; MDS/MPN-RS-T, MDS/MPN with ring sideroblasts and thrombocytosis. Figure S2. Lollipop plot showing type and location along the protein sequence of all the 343 TET2MT in the combined MDS/MPN cohort and separately in the CMML, MDS/MPN-RS-T, aCML and MDS/MPN-U cohorts. The number of recurrently detected alterations is indicated by the text within each disc, as well as by disc size. Colors indicate the type of mutation: blue, missense; purple, splice site; orange, nonsense; red, frameshift; gray, in-frame insertion/deletion. The N-terminal region (yellow), cysteine-rich (CysR) domain (red), double-stranded β-helix (DSBH) domain (sea green), and low-complexity insert (LCI, gray) are shaded on the band. All TET2MT were merged to construct the mutation profile using the web-based tool ProteinPaint (https://pecan.stjude.cloud/proteinpaint/). Abbreviations: aCML, atypical chronic myeloid leukemia; CMML, chronic myelomonocytic leukemia; MDS/MPN, myelodysplastic/myeloproliferative neoplasms; MDS/MPN-U, MDS/MPN, unclassifiable; MDS/MPN-RS-T, MDS/MPN with ring sideroblasts and thrombocytosis. Figure S3. Kaplan-Meier estimates of overall survival in the MDS/MPN-RS-T cohort (A), aCML cohort (B) and MDS/MPN-U cohort (C) stratified by the presence or absence of TET2MT. Abbreviations: aCML, atypical chronic myeloid leukemia; CI, confidence interval; CMML, chronic myelomonocytic leukemia; MDS/MPN, myelodysplastic/myeloproliferative neoplasms; MDS/MPN-U: MDS/MPN, unclassifiable; MDS/MPN-RS-T: MDS/MPN with ring sideroblasts and thrombocytosis; NR: not reached; OS: overall survival. Table S1. Details on genes tested, coverage and read depth of every NGS panel from the three center included in the study. Table S2. Clinical and laboratory features and subsequent events in 504 patients with WHO-defined MDS/MPN stratified by TET2 mutational status. Table S3. Clinical and laboratory features and subsequent events in 504 patients with WHO-defined MDS/MPN stratified by disease subtype and TET2 mutational status. Appendix S1. Supplementary Material. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. 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