Claudia Haferlach

Manja Meggendorfer

Sabine Jeromin

Torsten Haferlach

Wolfgang Kern

Haematologica

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The mutational landscape of 18 investigatedgenes clearly separates four subtypes of myelodysplastic/myeloproliferative neoplasmsAccording to the World Health Organization myelodys-plastic/myeloproliferative neoplasms (MDS/MPN) are aheterogeneous group of myeloid neoplasms showing clin-ical and morphological features that overlap betweenMDS and MPN.1 This category includes atypical chronicmyeloid leukemia (aCML), chronic myelomonocyticleukemia (CMML), MDS/MPN, unclassifiable(MDS/MPN, U) and MDS/MPN with ring sideroblastsand thrombocytosis (MDS/MPN-RS-T). In most cases ofMDS/MPN the karyotype is normal, while targetedsequencing revealed a high frequency of mutations inMDS/MPN.1-5 Although single entities have been investi-gated, no comprehensive comparative analysis ofMDS/MPN is available. Furthermore, MDS/MPN, U, inparticular, remains a heterogeneous mixture of pheno-types and genotypes, and in some cases difficult to distin-guish from aCML.5 We, therefore, aimed at discoveringentity-specific mutation patterns to enable a genetic dis-tinction of all four MDS/MPN entities for diagnostic pur-poses. We investigated 177 patients at diagnosis by cytomor-phology and genetic studies strictly following WorldHealth Organization criteria: 35 patients were diagnosedas having aCML, 58 as having CMML, 39 as havingMDS/MPN, U and 45 as having MDS/MPN-RS-T. Themedian age of the cohort was 75 years (range, 22-89years) and comprised 72 females and 105 males. Somecases were already included in former studies.2-4 Allpatients were analyzed by targeted next-generationsequencing for 18 genes.6 Chromosome banding analysiswas performed for 171/177 patients according to standardprocedures. Further information is available in the OnlineSupplementary Material.  Cytogenetically, 44/171 (26%) MDS/MPN patients hadan aberrant karyotype. An aberrant karyotype was mostfrequent in aCML (13/35, 37%), followed by MDS/MPN,U (11/38, 29%), CMML (14/55, 25%) and MDS/MPN-RS-T (6/43, 14%). The only significant difference in frequen-cy of aberrant karyotypes occurred between aCML andMDS/MPN-RS-T (P=0.032). The only recurrent aberra-tions detected were trisomy 8 (n=17), monosomy 7 (n=3)and loss of the Y-chromosome (n=8), reflecting the com-monly detected aberrations in MDS patients.7,8Studying the frequencies of mutations within the totalcohort revealed that ASXL1 was the most frequentlymutated gene (74/177, 42%), followed by TET2 (70/177,40%), the spliceosomal genes SF3B1 and SRSF2 (both53/177, 30%), JAK2 (37/177, 21%), NRAS (26/177, 15%),RUNX1 (22/177, 12%), CBL (21/177, 12%) and SETBP1(17/177, 10%). All other investigated genes showedmutation frequencies <10% (Figure 1). The number ofmutations per patient ranged from zero to six with amedian of two mutations. This was also true for the singleentities aCML, MDS/MPN, U and MDS/MPN-RS-T,while the median was three mutations per patient forcases of CMML (Figure 1).Looking at the mutational profile of the four separateentities revealed significant differences in mutation fre-quencies of single genes (Figure 2, Online SupplementaryTable S2). ASXL1 and TET2were frequently affected in allentities although there were significant differences:ASXL1 was less frequently mutated in MDS/MPN-RS-Thaematologica 2018; 103:e192LETTERS TO THE EDITORFigure 1. Molecular and cytogenetic characterization of patients with myelodysplastic/myeloproliferative neoplasms. Illustration of all 177 samples: each col-umn represents one patient. All 18 analyzed genes as well as karyotype information are given for each patient. Light gray: wild type or normal karyotype, red:mutated, black: aberrant karyotype, white: no data available. The number of mutations per patient is illustrated as a bar chart above the graph, the mutationfrequencies of single genes are given as a bar chart at the right.(20%) than in aCML (60%; P<0.001) and CMML (52%;P=0.001). TET2was more often mutated in CMML (53%)than in MDS/MPN, U (26%; P=0.007) or MDS/MPN-RS-T (31%; P=0.028). Spliceosomal genes are one of the mostfrequently mutated genes in MDS but different frequen-cies can be noted between the MDS subgroups.9 Withinthe MDS/MPN entities SRSF2 mutations appeared moreoften in CMML (53%) than in MDS/MPN-RS-T (9%;P<0.001) or MDS/MPN, U (15%; P<0.001). SF3B1 muta-tions prevailed in MDS/MPN-RS-T (91%) compared to allother entities (aCML: 11%, CMML: 5%, MDS/MPN, U:13%; for all P<0.001). Interestingly, in MDS/MPN, U allthree investigated splicing genes (SF3B1, SRSF2 andU2AF1) appeared mutated with a slight predominance inU2AF1 mutations, while in aCML and CMML SRSF2mutations and in MDS/MPN-RS-T SF3B1mutations weremost often observed. Although the distribution was notstatistically significantly different, SETBP1 mutationsoccurred most often in aCML (23%), followed byMDS/MPN, U (10%) and CMML (9%); this gene was notmutated in MDS/MPN-RS-T. SETBP1 mutations havebeen described as a marker pointing to a diagnosis ofaCML, which is in line with the data presented here.2,10,11The incidence of JAK2, the MPN entity-defining muta-tion, was significantly higher in MDS/MPN-RS-T (51%)than in MDS/MPN, U (23%, P=0.013), CMML (7%,P<0.001) and aCML (3%, P<0.001). Furthermore, JAK2mutations were also significantly more frequent inMDS/MPN, U than in aCML and CMML (P=0.015 andP=0.032, respectively). Investigating the RAS signalinggenes showed that NRAS is not mutated in MDS/MPN-RS-T and MDS/MPN, U but was quite frequently mutatedin aCML (31%, P<0.001) and CMML (26%, P<0.001).Furthermore, CBL was more often mutated in CMML(24%) than in MDS/MPN-RS-T (7%, P=0.030), aCML(6%, P=0.025) and MDS/MPN, U (5%, P=0.013). TheCBL mutation frequency therefore makes the differencebetween the quite similar aCML and CMML cases.Overall, these mutation frequencies are in line with pub-lished data, except for a slightly higher frequency of CBLmutations in CMML and the absence of NRAS mutationsin MDS/MPN, U.2-5,10,12-14 In previous studies the frequencyof the CBL mutation was found to be ~10% in CMMLinstead of the 24% in our study and RAS mutations weredetected in 14% of MDS/MPN, U, contrasting with the0% in the present study.14,15 These differences might behaematologica 2018; 103:e193LETTERS TO THE EDITORFigure 2. The mutation frequencies of all 18 analyzed genes as well as karyotype information for the separate subcohorts of myelodysplastic/myeloprolifer-ative neoplasms. Each of the four subcohorts, aCML, CMML, MDS/MPN, U and MDS/MPN-RS-T, is shown separately. In the MDS/MPN-RS-T graph the mutationfrequency of SF3B1 (91%) is cropped for more clarity of other gene mutation frequencies.caused by overall small cohort sizes slightly over- orunderestimating mutation frequencies. Taking the overallmutational landscape into account, Figure 2 shows thataCML and CMML share a similar mutation pattern, differ-entiated by the frequencies of SRSF2, TET2, NRAS andCBL mutations (upper panel Figure 2), reflecting the typi-cally described mutation profiles.2,3,10,12,15MDS/MPN-RS-T,however, showed a different pattern dominated by over-all low mutation frequencies except for SF3B1 and JAK2(lower right panel Figure 2), as described previously.4,13MDS/MPN, U cases seem to have an intermediate muta-tion profile, showing features of both groups (lower leftpanel Figure 2): overall, there were lower mutation fre-quencies, but a higher frequency of JAK2 mutations, incontrast to aCML and CMML, and no NRAS mutationssimilar to MDS/MPN-RS-T.14One can postulate that casesso far classified as MDS/MPN, U may be categorizedaccording to their respective molecular patterns in thenear future and that this would be a refinement comparedto the criteria currently in use. It is, therefore, worth men-tioning that the related entity chronic neutrophilicleukemia (CNL) shows an overlapping but different muta-tion pattern to the entitites addressed here. CNL can beclearly discriminated by the high occurrence of CSF3Rmutations (43%).11Since the most prominent differences between the fourentities occurred in spliceosomal genes, JAK2 and NRAS,we grouped the genes by their respective pathways inattempt to gain further insights into the different mecha-nisms involved in disease manifestation. We, therefore,grouped together the genes ASXL1, TET2 and DNMT3Ainvolved in epigenetic regulation, the spliceosomal genesSF3B1, SRSF2 and U2AF1, the genes JAK2, CALR, MPLaffecting the JAK-STAT pathway and finally the genesNRAS, KRAS and CBL from the RAS signaling pathway.All four MDS/MPN entities were affected by mutations indifferent pathways (Figure 3). Although the most fre-quently mutated genes belong to the epigenetic regulatorsin MDS/MPN, these genes were less frequently mutatedin MDS/MPN-RS-T (58%) than in CMML (84%, P<0.001)and also less frequently in MDS/MPN, U (51%) than inaCML (77%, P=0.030) or CMML (84%, P<0.001). Thegroup of spliceosomal genes were more often mutated inMDS/MPN-RS-T (100%) than in MDS/MPN, U (44%,P<0.001), CMML (62%, P<0.001) or aCML (51%,P<0.001). Mutations in JAK2/CALR/MPL (the JAK-STATpathway) occurred at a higher prevalence in MDS/MPN,U (33%) and MDS/MPN-RS-T (53%) than in aCML (9%)or CMML (7%; P<0.001). In contrast, NRAS/KRAS/CBL(the RAS pathway) were more often mutated in aCML(37%) and CMML (52%), as compared to MDS/MPN, U(5%) and  MDS/MPN-RS-T (9%; P<0.001). Here differentgenetic profiles became obvious (Figure 3). While thesame pathways were affected in aCML and CMML,namely the RAS pathway, epigenetic regulation and splic-ing with hardly any involvement of the JAK-STAT path-way, in MDS/MPN-RS-T epigenetic regulation, the JAK-STAT pathway and splicing were affected but rarely theRAS signaling pathway. Once again, MDS/MPN, U seemsto be a mixture of both with affected epigenetic regula-tion, JAK-STAT pathway and splicing but a marginallyaffected RAS pathway. Considering the mean variantallele frequencies of the gene mutations in the differentaffected pathways revealed similar variant allele frequen-cies for all the pathways in all four entities pointing todriver mutations in the main cell clone (OnlineSupplementary Figure S1). Signaling mutations (the JAK-STAT and RAS signalingpathways) result in aberrant activation of proliferation aswell as anti-apoptotic pathways and therefore correlatewith a myeloproliferative phenotype. Overall our datareflect the described incidences of signaling mutations(80% JAK-STAT mutations in MDS/MPN-RS-T and 50%signaling mutations in CMML): the here presented slight-ly lower mutation frequencies of 53% in the JAK-STATpathway in MDS/MPN-RS-T and 53% signaling muta-tions in CMML might be caused by different grouping ofanalyzed genes to more subdivided pathways.5In conclusion, genes involved in epigenetic regulationare overall the most frequently mutated genes inMDS/MPN. For discrimination, SRSF2 is most frequentlymutated in CMML, but also in aCML, while SF3B1 is pre-dominantly mutated in MDS/MPN-RS-T. The JAK-STATpathway is more often affected in MDS/MPN, U andMDS/MPN-RS-T in contrast to the RAS pathway, whichis more often affected in aCML and CMML. The two enti-haematologica 2018; 103:e194LETTERS TO THE EDITORFigure 3. The frequencies of patients showing at least onemutation within genes grouped according to their functionalproperties. The gene groups were: epigenetic regulation(ASXL1, TET2, DNMT3A), splicing (SF3B1, SRSF2, U2AF1), theJAK-STAT pathway (JAK2, CALR, MPL) and the RAS pathway(NRAS, KRAS, CBL). ties aCML and CMML show very similar mutation pro-files, which are clearly different from the profile ofMDS/MPN-RS-T. MDS/MPN, U seems to be a mixture ofboth groups with a specific mutation profile and would bebetter classified according to gene patterns than by phe-notype.Manja Meggendorfer, Sabine Jeromin, Claudia Haferlach,Wolfgang Kern and Torsten HaferlachMLL Munich Leukemia Laboratory, GermanyAcknowledgments: We thank all clinicians for sending samples toour laboratory for diagnostic purposes and for providing clinical infor-mation and follow-up data. In addition, we would like to thank allthe co-workers at the MLL Munich Leukemia Laboratory forapproaching together many aspects in the field of leukemia diagnos-tics and research.Correspondence: manja.meggendorfer@mll.com   doi:10.3324/haematol.2017.183160Information on authorship, contributions, and financial & other dis-closures was provided by the authors and is available with the onlineversion of this article at www.haematologica.org.References1. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to theWorld Health Organization (WHO) classification of myeloid neo-plasms and acute leukemia. Blood. 2016;127(20):2391-2405.2. Meggendorfer M, Bacher U, Alpermann T, et al. SETBP1 mutationsoccur in 9% of MDS/MPN and in 4% of MPN cases and are strong-ly associated with atypical CML, monosomy 7, isochromosomei(17)(q10), ASXL1 and CBL mutations. Leukemia. 2013;27(9):1852-1860.3. Meggendorfer M, Roller A, Haferlach T, et al. SRSF2 mutations in275 cases with chronic myelomonocytic leukemia (CMML). Blood.2012;120(15):3080-3088.4. Jeromin S, Haferlach T, Weissmann S, et al. Refractory anemia withring sideroblasts and marked thrombocytosis (RARS-T) cases har-bor mutations in SF3B1 or other spliceosome genes accompaniedby JAK2V617F and ASXL1 mutations. Haematologica. 2014;100(4):e125-e127.5. Mughal TI, Cross NC, Padron E, et al. An International MDS/MPNWorking Group's perspective and recommendations on molecularpathogenesis, diagnosis and clinical characterization of myelodys-plastic/myeloproliferative neoplasms. Haematologica. 2015;100(9):1117-1130.6. Delic S, Rose D, Kern W, et al. Application of an NGS-based 28-gene panel in myeloproliferative neoplasms reveals distinct muta-tion patterns in essential thrombocythaemia, primary myelofibro-sis and polycythaemia vera. Br J Haematol. 2016;175(3):419-426.7. Haferlach T, Nagata Y, Grossmann V, et al. Landscape of geneticlesions in 944 patients with myelodysplastic syndromes. Leukemia.2014;28(2):241-247.8. Papaemmanuil E, Gerstung M, Malcovati L, et al. Clinical and bio-logical implications of driver mutations in myelodysplastic syn-dromes. Blood. 2013;122(22):3616-3627.9. Yoshida K, Sanada M, Shiraishi Y, et al. Frequent pathway muta-tions of splicing machinery in myelodysplasia. Nature.2011;478(7367):64-69.10. Piazza R, Valletta S, Winkelmann N, et al. Recurrent SETBP1 muta-tions in atypical chronic myeloid leukemia. Nat Genet. 2013;45(1):18-24.11. Meggendorfer M, Haferlach T, Alpermann T, et al. Specific molec-ular mutation patterns delineate chronic neutrophilic leukemia,atypical chronic myeloid leukemia, and chronic myelomonocyticleukemia. Haematologica. 2014;99(12):e244-e246.12. Cazzola M, la Porta MG, Malcovati L. The genetic basis ofmyelodysplasia and its clinical relevance. Blood.2013;122(25):4021-2034.13. Reiter A, Invernizzi R, Cross NC, Cazzola M. Molecular basis ofmyelodysplastic/myeloproliferative neoplasms. Haematologica.2009;94(12):1634-1638.14. Wang SA, Hasserjian RP, Fox PS, et al. Atypical chronic myeloidleukemia is clinically distinct from unclassifiable myelodysplas-tic/myeloproliferative neoplasms. Blood. 2014;123(17):2645-2651.15. Itzykson R, Kosmider O, Renneville A, et al. Prognostic scoreincluding gene mutations in chronic myelomonocytic leukemia. JClin Oncol. 2013;31(19):2428-2436.haematologica 2018; 103:e195LETTERS TO THE EDITOR

The mutational landscape of 18 investigated genes clearly separates four subtypes of myelodysplastic/myeloproliferative neoplasms

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The mutational landscape of 18 investigated genes clearly separates four subtypes of myelodysplastic/myeloproliferative neoplasms

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