Myelodysplastic/ Myeloproliferative neoplasms 1(MDS/MPN) represent a category of myeloid neoplasms with overlapping pathologic and molecular features of both myelodysplastic syndrome (MDS) myeloproliferative neoplasms (MPN)1. They can manifest with both cytopenias and cytosis. The revised 4th edition of WHO 2 acknowledged the importance of identifying a clonal abnormality especially when dysplasia is minimal or absent. Recurrent cytogenetic abnormalities are noted in only about 30-40% of MDS/MPN. 3 Diagnosis can be very challenging with normal cytogenetics and borderline morphologic dysplasia especially in the presence of confounding factors such as infections, inflammation, medications, other underlying diseases such as liver/ kidney disease etc. The recent International Consensus Committee4 and the 2022 5th edition of WHO Classification 5 updated the criteria for the diagnosis of MDS/MPN. Although there are differences (see Table 1) between the two classifications, both recognize the increasing relevance of mutations in the diagnosis and prognostic stratification of MDS/MPNs and include the presence of mutations in addition to cytogenetic abnormalities as an evidence of clonality and as a supporting evidence for diagnosis. However; since mutations can be seen at low variant allelic frequency 6 in the setting of clonal hematopoiesis of indeterminate potential (CHIP) 4, 5, different cut-offs have been suggested for Variant allele frequency (VAF) 6 and the number of mutations required for diagnosis of a myeloid neoplasm (MDS or MDS/MPN). It has been shown that using ≥2 mutations at a VAF of ≥10% 7, 8 or ≥1 mutation with VAF of ≥20% gives a positive predictive value (PPV) of 100%, Negative predictive value (NPV)-38%, sensitivity- 60% and specificity -100% 8. On using ≥1 mutation or abnormal cytogenetics/ FISH, the sensitivity and NPV can be improved to 96% and 83% without significantly reducing the PPV (96%) 8.

In this review, we attempt to summarize the molecular genomic landscape of MDS/MPN neoplasms.

CMML is a clonal neoplasm associated with persistent monocytosis. It is more common in old age and in males, with an estimated incidence of approximately 1 in 100,000 9, 10, 11. As per both 2022 WHO and ICC classifications, the cutoff for absolute monocytosis required for this diagnosis has been lowered to 0.5×109/L 4, 5. However, to ensure diagnostic accuracy for cases with absolute monocytosis between 0.5×109/L and <1×109/L, detection of one or more clonal cytogenetic or molecular abnormality is a required criteria4, 5. ICC classification in addition emphasizes a VAF of at least 10% of the mutant allele. Two distinct disease subtypes have been recognized- myelodysplastic (CMML-MDS, leukocyte count <13×109/l) and myeloproliferative (CMML-MP, leukocyte count ≥13×109/l), with CMML-MP commonly associated with adverse outcomes 4, 5. Refer to Table 1 for criteria for diagnosis and Figure 1 for an example case.

CMML is commonly associated with TET2, SRSF2 and ASXL1 mutations 12, 13, 14. Association of certain mutation patterns have been shown to be predictive of prognosis. Truncating ASXL1 mutations are known to be deleterious and have an adverse impact on both overall survival and leukemia free survival (LFS) 15, 16. Mutations in RUNX1, NRAS, SETBP1 and ASXL1 are independently associated with overall survival15, 16. On the contrary, TET2 mutations have been demonstrated to have favorable outcomes with survival advantages in cases of CMML, especially in the absence of ASXL1 mutation17, 18. TET2 mutations are frequently seen in older age groups, more commonly in dysplastic CMMLs and have a higher number of co-occuring mutations.17 In one study, the survival data demonstrated a significant difference in favor of ASXL1 wild type (wt)/ TET2 mutated group compared to ASXL1wt/TET2wt, ASXL1mut/TET2wt, ASXL1mut/TET2mut.18 Some of the mutations associated with CMML are also seen in elderly patients not fulfilling the criteria for CMML (as defined in 2016 WHO classification). Presence of these mutations correlated with similar immunophenotype and overall survival, irrespective of whether the patient was diagnosed to have CMML or not.12 Apart from TET2, SRSF2 and ASXL1, other genes mutated at low frequency include SETBP1, RUNX1, JAK2, DNMT3A, IDH1, IDH2, STAG2, PHF6, CEBPA, ETNK1 and E2H2.12,18,19DNMT3A mutations are seen in about 6% of the cases of CMML but with concurrent other gene mutations and abnormal karyotype (in ∼30% of cases) and are predictive of shortened leukemia free survival.19 No effect on survival or leukemia transformation has been shown with SETBP1 mutations 15. EZH2 mutations are found to be infrequent, co-occur with ASXL1 mutation and are not associated with DNMT3A or SF3B1 mutations.20 They themselves have not shown to impact either overall survival or LFS but ASXL1/EZH2 co-mutated patients have been found to have a shorter survival than ASXL1 mutated patients alone.20 The two subtypes of CMML myelodysplastic and myeloproliferative also differ in their mutation profile 4, 5. Mutations in the RAS pathway have been associated with a proliferative phenotype (CMML-MP) with marked leukocytosis/ monocytosis, constitutional symptoms, splenomegaly and lower survival21. TET2, ASXL1 and RUNX1 are commonly identified in both CMML-MDS and CMML-MP. SF3B1, DNMT3A, STAG2 and CBL mutations were exclusively seen in CMML-MDS while SETBP1, JAK2, SRSF2 and NRAS mutations were seen only in CMML-MP. Secondary mutations occurring downstream contributed to the clinical heterogeneity of these cases. 22SF3B1 mutations occur in <10% of CMML and are associated with better overall survival 23. RUNX1 mutation was detected in 11% CMML cases. No phenotypic or survival differences were noted between patients with one or two RUNX1 mutations. Overall, RUNX1 mutation was associated with a greater degree of thrombocytopenia and negatively impacted overall and LFS 24. TP53 mutations are rare in CMML, mostly seen in therapy related setting25.

NPM1 mutations have been rarely described in cases of CMML. While ICC still recognizes these cases as CMML (it is unclear if the blast count should be <10% or could be between 10-19% for the case to be still considered as a CMML in the presence of NPM1 mutation) 26, in 2022 WHO presence of NPM1 trumps and these cases would be classified as acute myeloid leukemia (AML) with mutated NPM1 4, 5. It is well known that NPM1 mutations present in cases of AML with normal cytogenetics are associated with a better prognosis especially in absence of FLT3.27 However, cases of CMML with NPM1 mutations are associated with severe anemia, high bone marrow monocyte percentage, increased tendency for AML progression and an inferior overall survival.28 They tend to be associated with normal cytogenetics, a dysplastic CMML phenotype and increased incidence of DNMT3A and demonstrate blast transformation in greater than 50% of the cases. These cases of CMML with NPM1 mutation that progress to AMLs do not carry the good prognosis associated with denovo AML with mutated NPM1.29

While mutation in at least one gene is seen in the vast majority of the CMML cases12 cytogenetic alterations are seen in only about a third of the CMML cases. These include trisomy 8, -Y, abnormalities of chromosome 7, trisomy 21 and complex karyotypes 30. Various cytogenetic scoring systems including the Spanish CMML specific cytogenetic risk stratification30 and Mayo-French cytogenetic risk stratification systems 31 have been shown to define distinct prognostic categories in CMML based on the cytogenetic abnormalities. Some clinical trials are underway to test novel therapies in CMML based on the molecular genomic findings. Some of the novel therapies tested include the use of ruxolitinib (JAK inhibitor)32, Tipifamib, a farnesyl transferase inhibitor14 and Tagraxofusp, a recombinant fusion protein in CMML 14.

Clonal monocytosis of undetermined significance has been recognized under the ICC classification4. It is identified on the basis of monocytosis with the same cut off used for CMML in the presence of myeloid neoplasm associated mutations but without bone marrow morphologic findings needed for a diagnosis of overt CMML. Essentially like Clonal Cytopenia of Undetermined Significance (CCUS) with monocytosis. These patients are at an increased risk of developing overt CMML. 9,12,33 The spectrum of mutations in cases of clinically significant monocytosis without a confirmed myeloid malignancy was found to be similar to the cases with malignancy. 12

Atypical CML was originally described as a disease closely resembling chronic myeloid leukemia (CML) but lacking the BCR::ABL1 translocation.34 Subsequent molecular and cytogenetic studies helped to recognize this as a separate entity that is different from other myeloid neoplasms 35. It's relative incidence is estimated at one to two cases for every 100 patients with BCR::ABL1 positive CMLs. It has been described in adults and commonly in males 35. The nomenclature for atypical CML was changed to MDS/MPN with neutrophilia, as per the 5th edition WHO classification. This change was made to better clarify the MDS/MPN nature of this entity while avoiding potential confusion with CML.5 On the other hand, the ICC recommended dropping the use of BCR::ABL1 negative terminology as all subtypes of MDS/MPN require the absence of BCR::ABL1 fusion gene. The ICC classification also acknowledges that aCML is not characterized by eosinophilia.4 Refer to Table 1 for criteria for diagnosis and Figure 2 for an example case. aCML has been demonstrated to show genomic mutations in all cases and cytogenetic abnormalities in about 1/3rd of the cases36. None of the aCML cases showed JAK2V617F mutation compared to other BCR::ABL1 negative MPNs 37 Thus, absence of MPN related mutations is helpful in differentiating aCML from advanced stages of MPN.4 Mutations in ASXL1, TET2, NRAS, SETBP1, RUNX1, ETNK1 and PTPN11 have been identified in varying proportions and TET2, NRAS and PTPN11 mutations have been found to impact survival.36ASXL1, SETBP1 and ETNK1 mutations have been found to not impact overall survival.36 Cytogenetic abnormalities seen include trisomy 8, 9 and 21.36 Recurrent somatic ETNK1 mutations have been found in a small proportion of aCML cases.38CSF3R mutations can also be seen in cases of aCML in about 6% of the cases bringing in the differential diagnosis with chronic neutrophilic leukemia (CNL)39. The difference between aCML and CNL may be related to the other contributing mutations, the allele burden or diverse downstream effects of the gene activation. It is believed that CSF3R is the main driver in cases of CNL while aCML tends to be genetically heterogeneous.40 Comparing aCML and CMML, CBL mutation was found to be more common in CMML while SETBP1 mutation was found to be more common in aCML.39CSF3R was not present in any case of CMML39.

Patients of MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T) were included as a distinct entity under the 2016 WHO classification2. MDS/MPN-RS-T was distinguished from myelodysplastic syndrome with ringed sideroblasts by the presence of thrombocytosis and megakaryocytic morphology similar to myeloproliferative neoplasms 41.

According to the 2022 WHO classification and ICC, the MDS/MPN-RS-T category has been further refined by including the presence of SF3B1 mutation (ICC mentions the VAF of SF3B1 to be ≥10%). 4, 5 and are now classified as MDS/MPN with SF3B1 mutation and thrombocytosis (MDS/MPN-T-SF3B1). The cases without SF3B1 mutation (or where the molecular information is not present) but with >15% ring sideroblasts are designated as MDS/MPN with ring sideroblasts and thrombocytosis (MDS/MPN-RS-T). 4, 5 It is further stated in the ICC that anemia and thrombocytosis should be present at the time of initial diagnosis in both these categories.4 However, WHO classification5 mentions that rare cases of myeloid neoplasm with SF3B1 mutation and ring sideroblasts that later evolve to MDS/MPN-RS-T upon acquisition of another mutation association with MPN (such as JAK2, MPL or CALR) may be classified as MDS/MPN-with SF3B1 mutation and thrombocytosis. Presence of ring sideroblasts is no longer required for the diagnosis of MDS/MPN-RS-T-SF3B1 as long as SF3B1 mutation is present with a VAF>10%.4

In most cases of MDS/MPN-RS-T (∼99% of cases), at least one mutation can be found.42 The commonest mutations identified are SF3B1 (90.2%) (hence the rationale of including SF3B1 in the diagnostic criteria), JAK2V617F (58.7%), TET2 (23.3%), DNMT3A (16.7%) and ASXL1 (14.3%)42 Further, nearly all cases which had a wild type SF3B1 demonstrated JAK2V617F and ASXL1 mutation. Most of these cases also demonstrated SRSF2 or U2AF1 mutations.42, 43. CALR and MPL mutations are rare. 4CALR mutations are usually associated with non-mutated JAK2 and MPL.44 Cytogenetic abnormalities are seen in a minority of cases and the common abnormalities include trisomy 8, complex and monosomal karyotypes.43 In multivariate survival analysis, abnormal karyotype along with anemia was predictive of inferior survival43 whereas univariate analysis demonstrated a poor survival associated with the presence of SETBP1 or ASXL1 mutation.43 Studies have also suggested the association of adverse overall survival (OS) specifically with frameshift and non-sense ASXL1 mutations.41 A prognostic model incorporating abnormal karyotype, ASXL1 or SETBP1 mutation status and hemoglobin <10 gm/dl has been suggested.43

MDS/MPN, unclassifiable has been renamed as MDS/MPN-not otherwise specified with the intention to remove the qualifier “unclassifiable” from the classification in the 5th edition of the WHO classification.5 The ICC classification also recommends the use of the terminology MDS/MPN, NOS. Lack of specific gene rearrangements or fusions associated with myeloid/ lymphoid neoplasms with eosinophilia with tyrosine kinase gene fusions is required for the diagnosis. Proven clonality is expected, although the diagnosis can be made if there is morphological evidence of MDS/MPN and all other entities are excluded.4 A significant proportion of these cases have been shown to be associated with abnormal karyotypes. In a study by Mangaonkar et al, trisomy 8, monosomy 7/ deletion 7q, deletion 20q were the commonest cytogenetic abnormalities detected.6ASXL1, SRSF2, SETBP1, JAK2V617F, NRAS and TET2 are the commonest mutations reported to be associated with MDS/MPN-NOS.6TP53 mutations are seen in about 6% of the cases and are associated with a higher frequency of monosomal karyotype6TP53 and CBL mutations are found to be associated with an inferior overall survival. 6 In contrast to some other types of MDS/MPN neoplasms, ASXL1 mutations were not found to be predictive of overall survival. 6 In another study, apart from ASXL1, TET2, JAK2 and SRSF2 mutations, EZH2, U2AF1 and RUNX1 mutations were also identified 6,45CALR mutations were seen in 3% of the cases and correlated significantly with improved survival. 45ZRSR2 mutations were associated with a better overall survival while STAG2, CEBPA and EZH2 mutations were associated with an inferior overall survival. 45

MDS/MPN with i (17q) has been recognized as a new provisional subentity under the umbrella of MDS/MPN, NOS in the ICC classification.4 While acknowledged in WHO 5th edition this is not considered a separate entity.5 It shares genomic signature with aCML and hence it is important to understand if this truly represents a separate entity One additional cytogenetic abnormality (non complex karyotype) other than deletion (7q)/-7 is accepted in this diagnosis 46. Morphologically, MDS/MPN with i(17q) have marked hypolobation of the neutrophils 47. These neoplasms have been found to have an aggressive clinical course with a median survival of 11 months and most patients undergoing rapid progression to acute myeloid leukemia. 48 There is a high frequency of SRSF2, SETBP1, ASXL1 and NRAS mutations.48 They present in younger age group (median age of 67 years), with lower platelet and absolute neutrophil counts, higher circulating blasts and higher frequency of SETBP1 and SRSF2 mutations which can coexist. 49 It is interesting to note that in spite of loss of one allele of 17p, TP53 mutations were rare in the uninvolved TP53 allele. 49 Further, cases with i(17q) were found to have a shorter medial overall survival and hence, recognition of this distinct entity has been suggested. 49

MDS with isolated del(5q) is a recognized entity. A small proportion of these cases show concomitant JAK2V617F mutation. While this is not a recognized entity in either the 2022 WHO or the ICC but has been shown to demonstrate mostly MDS/MPN morphology. Sangiorgio et al described 6 such cases (12.7% of the MDS with isolated del(5q) studied). These are enriched in women, have heterogeneous blood counts, pleomorphic morphology of megakaryocytes and prominent reticulin fibrosis. High VAF of JAK2 in most of the cases and presence of del(5q) in majority of the cells analyzed suggest presence of both abnormalitues in the same clone. No difference in leukemic transformation or overall survival was seen between JAK2 wt and JAK2 mutated cases. JAK2 mutation probably confers a proliferative phenotype to this disease presentation and it might be of interest in the future to explore whether this group of cases qualify for a consideration under the MDS/MPN umbrella. 50

JMML has been reclassified into pediatric disorders and/or germline mutation associated disorders according to the ICC classification 4 and among the myeloproliferative disorders in the 2022 WHO classification. 5 Germline NF1, RAS, CBL mutations and somatic PTPN11, NRAS, KRAS, RRAS mutations have been described in association with JMML. 51