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Besides histopathological findings, there are no indicators of increased risk for fibrotic progression in myeloproliferative neoplasms (MPNs). Age-related clonal hematopoiesis (ARCH) or clonal hematopoiesis of indetermined potential (CHIP) are frequent findings in the elderly and combinations with MPN driver mutations (JAK2, MPL, and CALR) have been described. To determine the impact of ARCH/CHIP-related mutations for the development of fibrosis in primary myelofibrosis (PMF), the mutational status of cases with fibrotic progression from grade 0 to grade 2/3 (n = 77) as evidenced by follow-up bone marrow biopsies (median 6.2 years) was compared to prefibrotic PMF samples without the development of fibrosis (n = 27; median follow-up 7.3 years). Frequent ARCH/CHIP-associated mutations (TET2, ASXL1, DNMT3A) demonstrable at presentation were not connected with fibrotic progression. However, mutations that are rarely found in ARCH/CHIP (SRSF2, U2AF1, SF3B1, IDH1/2, and EZH2) were present in 24.7% of cases with later development of fibrosis and not detectable in cases staying free from fibrosis (P = 0.0028). Determination of tumor mutational burden (TMB) in a subgroup of cases (n = 32) did not show significant differences (7.68 mutations/MB vs. 6.85 mutations/MB). We conclude that mutations rarely found in ARCH/CHIP provide an independent risk factor for rapid fibrotic progression (median 2.0 years) when already manifest at first presentation.

Myeloproliferative neoplasm (MPN)-associated myelofibrosis is a MPN characterized by bone marrow fibrosis, cytopenias, splenomegaly and constitutional symptoms. Pomalidomide, an immune-modifying drug, is reported to improve anaemia and thrombocytopenia in some patients with MPN-associated myelofibrosis. We designed a phase 2 study of pomalidomide in patients with MPN-associated myelofibrosis and anaemia and/or thrombocytopenia and/or neutropenia. Subjects received pomalidomide 2.0 mg/day in cohort 1 (n=38) or 0.5 mg/day in cohort 2 (n=58). Prednisolone was added if there was no response after 3 months in cohort 1 and based on up-front randomization in cohort 2 if there was no response at 3 or 6 months. Response rates were 39% (95% confidence interval (CI), 26-55%) in cohort 1 and 24% (95% CI, 15-37%) in cohort 2. In a multivariable logistic regression model pomalidomide at 2.0 mg/day (odds ratio (OR), 2.62; 95% CI, 1.00-6.87; P=0.05) and mutated TET2 (OR, 5.07; 95% CI, 1.16-22.17; P=0.03) were significantly associated with responses. Median duration of responses was 13.0 months (range 0.9-52.7). There was no significant difference in response rates or duration in subjects receiving or not receiving prednisolone. Clinical trial MPNSG 01-09 is registered at ClinicalTrials.gov (NCT00949364) and clinicaltrialsregister.eu (EudraCT Number: 2009-010738-23).

The thrombopoietin receptor gene (MPL) is expressed in megakaryocytes and exhibits the gain of function point mutation W515K/L in approximately 5% of patients with primary myelofibrosis/idiopathic myelofibrosis (PMF) representing one subtype of the chronic myeloproliferative disorders (myeloproliferative neoplasm). A series of primary and secondary acute myeloid leukaemias (AML) with megakaryoblastic phenotype and myelofibrosis unrelated to PMF (n=12) was analysed for the MPL(W515K/L) mutation by pyrosequencing. In three cases (25%), MPL(W515L) was found and in two of these a combination with trisomy 21 or the Philadelphia chromosome occurred. None of the secondary AML cases evolving from pre-existing PMF showed MPL(W515K/L) (n=4). We conclude that MPL(W515L) occurs in a considerable proportion of acute megakaryoblastic leukaemias with myelofibrosis unrelated to PMF.

Unlabelled: Marrow fibrosis (MF) has rarely been studied in myelodysplastic syndromes (MDS). There are no data on occurrence and significance of MF in the context of the World Health Organization (WHO) classification of disease. In total, 349 bone marrow biopsies from 200 patients with primary MDS were examined for MF and its prognostic relevance. MF correlated with multilineage dysplasia, more severe thrombopenia, higher probability of a clonal karyotype abnormality, and higher percentages of blasts in the peripheral blood (P<0.002). Its frequency varied markedly between different MDS types ranging from 0 (RARS) to 16% (RCMD, RAEB, P<0.007). Two patients with MF showed a Janus kinase-2 mutation (V617F). Patients with MF suffered from marrow failure significantly earlier with shortening of the survival time down to 0.5 (RAEB-1/-2), and 1-2 (RCMD, RA) years in median (P<0.00005). The prognostic relevance of MF was independent of the International Prognostic Scoring System and the classification of disease., Conclusion: The risk of MF Differs markedly between various subtypes of MDS. MF indicates an aggressive course with a significantly faster progression to fatal marrow failure and should therefore be considered in diagnosis, prognosis and treatment of disease.

In chronic myeloid leukemia (CML), imatinib may reverse bone marrow fibrosis (MF). Whether the unfavorable prognosis of MF is also reversed and whether imatinib guarantees against evolution of MF are unclear as yet. Fifty-nine patients with Ph+ CML treated with > or = 400 mg imatinib/day were examined for MF in 6- to 12-month intervals. Imatinib effectively reversed initial MF (P<0.0005). However, during a follow-up period of up to 4.8 years, small foci with abnormal fiber increase (FFI) emerged in 8 of 30 pretreated and 6 of 29 non-pretreated patients. Patients with FFI showed a significantly lower probability of achieving a complete cytogenetic or major molecular response (36 versus 81%; P<0.007). During the further follow up, 57% of patients with FFI but none of the other patients suffered from full-blown MF (P=0.00005). None of the patients with FFI or MF showed a Janus kinase-2 mutation (V617F). Evolutions of FFI and MF were independent significant predictors of imatinib failure (P=0.0031), accelerated phase and death of patients (P=0.0001; multivariate analyses). Imatinib effectively reverses initial MF in CML, but neither eliminates its unfavorable prognosis nor guarantees completely against new evolution of MF.

Overt myelofibrosis in bone marrow biopsies as a diagnostic postulate in cIMF has been discarded only recently by the WHO. Therefore, only a few studies have been performed on the evolution of myelofibrosis in "prefibrotic" cIMF by means of sequential bone marrow biopsies. We carried out this study on 38 patients, split up into two groups, A and B according to treatment modalities, to evaluate the dynamics and frequency of myelofibrosis in both groups. Our results indicate a step-wise development of myelofibrosis from a "prefibrotic" to a "classical" cIMF, as 75-80% of the respective patients in both groups progressed to myelofibrosis. However, this evolution seems to be heterogeneous and unpredictable in individual patients, since myelofibrosis could be seen as early as less than 2 years after diagnosis in 12/38 (31.6%) patients, whereas 3 patients remained "prefibrotic" even after up to 6 years of follow-up.

Agnogenic myeloid metaplasia (AMM) is a chronic myeloproliferative disorder that leads to a sustained proliferation of megakaryocytes and an increase of reticulin fibers within the bone marrow. Blood and bone marrow samples from patients with advanced AMM with fully developed myelofibrosis as well as cases in the cellular phase of the disease were investigated for clonality. Clonality was studied by X-linked restriction length polymorphism in conjunction with DNA methylation patterns. Granulocytes and total bone marrow cells proved to be monoclonal in origin whereas at least a minor portion of the peripheral lymphocytes were not clonally derived. Our findings indicate that the cellular phase of AMM as well as the fully developed disease progressed to myelofibrosis represent a monoclonal proliferation of pluripotent hematopoietic stem cells.

Myelofibrosis with myeloid metaplasia (MMM) belongs to the group of myeloproliferative syndromes. It is characterized by a sustained proliferation of megakaryocytes and increased medullary reticulin fibers. Until now the cellular phase at onset of the disease has not been analyzed for clonality of the hematopoietic cells. In this study we used X-linked restriction length polymorphism (RFLP) analysis to investigate the clonality of granulocytes and bone marrow cells from the cellular phase and advanced stages of the disease. In each of 12 heterozygous females, monoclonality of granulocytes or total bone marrow cells could be demonstrated. These results show that the cellular phase represents a monoclonal, and hence a probably neoplastic, proliferation of a pluripotent stem cell. The monoclonality of granulocytes present at the onset of disease should allow analysis of DNA of these easily accessible peripheral cells for the detection of specific clonal aberrations.