European Bone Marrow Working Group trial on reproducibility of World Health Organization criteria to discriminate essential thrombocythemia from prefibrotic primary myelofibrosis.

BACKGROUND: The World Health Organization classification of myeloproliferative neoplasms discriminates between essential thrombocythemia and the prefibrotic phase of primary myelofibrosis. This discrimination is clinically relevant because essential thrombocythemia is associated with a favorable prognosis whereas patients with primary myelofibrosis have a higher risk of progression to myelofibrosis or blast crisis. DESIGN AND METHODS: To assess the reproducibility of the classification, six hematopathologists from five European countries re-classified 102 non-fibrotic bone marrow trephines, obtained because of sustained thrombocytosis. RESULTS: Consensus on histological classification defined as at least four identical diagnoses occurred for 63% of the samples. Inter-observer agreement showed low to moderate kappa values (0.28 to 0.57, average 0.41). The percentage of unclassifiable myeloproliferative neoplasms rose from 2% to 23% when minor criteria for primary myelofibrosis were taken into account. In contrast, the frequency of primary myelofibrosis dropped from 23% to 7%, indicating that the majority of patients with a histological diagnosis of primary myelofibrosis did not fulfill the complete criteria for this disease. Thus, over 50% of cases in this series either could not be reproducibly classified or fell into the category of unclassifiable myeloproliferative neoplasms. CONCLUSIONS: World Health Organization criteria for discrimination of essential thrombocythemia from prefibrotic primary myelofibrosis are poorly to only moderately reproducible and lead to a higher proportion of non-classifiable myeloproliferative neoplasms than histology alone.

Opposite expression pattern of Src kinase Lyn in acute and chronic haematological malignancies.

Lck/yes-related novel (Lyn) tyrosine kinase overexpression has been suggested to be important for leukaemic cell growth making it an attractive target for therapy. By contrast, Lyn deficiency was shown to be responsible for a phenotype resembling myeloproliferative neoplasm (MPN) in mice. We aimed to shed more light on Lyn's role in haematological neoplasm and systematically investigated Lyn expression in MPN, acute and chronic leukaemia subtypes (n = 236). On top, B-cell chronic lymphocytic leukaemia (B-CLL) and chronic myeloid leukaemia significantly overexpressed Lyn when compared to de novo acute lymphoblastic leukaemia, de novo acute myeloid leukaemia (AML) and Philadelphia-chromosome-negative myeloproliferative neoplasms (p < 0.001). Most of acute leukaemia subtypes showed a notable down-regulation of Lyn mRNA but anyhow individual cases were labelled for the active form of Lyn protein. Intriguingly, secondary AML evolved in myelodysplastic syndromes revealed almost undetectable Lyn. Overexpression of Lyn in B-CLL was associated with a significant down-regulation of microRNA-337-5p suggesting that aberrant expression of this particular microRNA could be involved in post-transcriptional control of Lyn mRNA fate. We conclude that tyrosine kinase Lyn contributes to the malignant phenotype in certain leukaemia subtypes and therefore attracts targeted therapy.

The suppressor of cytokine signalling-1 (SOCS-1) gene is overexpressed in Philadelphia chromosome negative chronic myeloproliferative disorders.

The suppressor of cytokine signalling-1 (SOCS-1) is a negative regulator of signal transduction mediated by cytoplasmic tyrosine kinases such as the Janus kinases (JAKs). We investigated SOCS-1 expression in bone marrow cells from Philadelphia chromosome negative chronic myeloproliferative disorders (Ph(-) CMPD) and normal haematopoiesis (n=121), and additionally in peripheral blood samples (n=18). Except for chronic idiopathic myelofibrosis harbouring wild-type JAK2, other Ph(-) CMPD expressed significantly higher SOCS-1 levels of up to 14-fold compared to the control group (p<0.001) independent of the JAK2 status. The mononuclear cell fraction but not granulocytes in patients with Polycythaemia vera also significantly overexpressed SOCS-1. We conclude that up-regulation of the SOCS-1 gene might reflect a compensatory feedback mechanism with different emphasis among Ph(-) CMPD subtypes independent of an underlying JAK2 (V617F) mutation.

Different involvement of the megakaryocytic lineage by the JAK2 V617F mutation in Polycythemia vera, essential thrombocythemia and chronic idiopathic myelofibrosis.

Atypical megakaryocytes provide the histomorphological hallmark of all Philadelphia-chromosome negative chronic myeloproliferative disorder (Ph(-) CMPD) subtypes and have not been studied so far for the JAK2(V617F) mutation. The mutant gene dosage was determined in isolated megakaryocytes from 68 cases of JAK2(+)/Ph(-) CMPD by a pyrosequencing assay. Megakaryocytes from essential thrombocythemia (ET) showed significantly lower levels of mutated JAK2 alleles compared to patients with chronic idiopathic myelofibrosis (cIMF) with manifest fibrosis and polycythemia vera (PV) but not to prefibrotic cIMF. Solely, ET JAK2V617F in megakaryocytes is associated with a PV-like phenotype, and at least in one patient, the JAK2 mutation was exclusively acquired within the megakaryocytic lineage. The overt differences between prefibrotic and fibrotic cIMF suggested a causative role of the gene dosage of mutant JAK2 in fibrotic progression. Megakaryocyte analysis of a follow-up of eight individual cases with sequential biopsies, however, showed that progression to homozygosity of V617F mutated JAK2 and onset of manifest fibrosis appeared to be independent events. We conclude that megakaryocytes might be the predominant or even the exclusive lineage that acquires the JAK2(V617F) mutation in ET and that the JAK2(V617F) evolution to higher gene dosages represents a dynamic and complex process substantially involving megakaryocytes.

Detection of the single hotspot mutation in the JH2 pseudokinase domain of Janus kinase 2 in bone marrow trephine biopsies derived from chronic myeloproliferative disorders.

The recent discovery of a single point mutation in the JH2 pseudokinase domain of Janus kinase 2 (JAK2) in a considerable fraction of patients has shed light on the molecular pathomechanism in Philadelphia chromosome-negative chronic myeloproliferative disorders (Ph- CMPDs). We established a robust and reliable method for detection of the JAK2 mutation in bone marrow cells derived from archival bone marrow trephines based on polymerase chain reaction and subsequent restriction site analysis. In a series of proven Ph- CMPDs classified according to World Health Organization criteria (n = 79), we detected the JAK2 mutation in 90% of polycythemia vera, 22% of cellular prefibrotic chronic idiopathic myelofibrosis, 60% of advanced chronic idiopathic myelofibrosis, and 27% of essential thrombocythemia. JAK2 mutation was not detected in Ph+ chronic myeloid leukemia (n = 5), acute myeloid leukemia (n = 10), acute lymphoblastic leukemia (n = 10), secondary erythrocytosis (n = 10), or normal bone marrow (n = 10). Restriction site analysis was also suitable for unfixed cell populations derived from peripheral blood and bone marrow aspirates. Besides providing support in the differential diagnosis of reactive versus neoplastic myeloproliferations, this newly developed assay reveals considerable overlaps between histologically different disease entities, indicating that additional genetic alterations might be responsible for the established differences of CMPD subentities.

Evolution of myelofibrosis in chronic idiopathic myelofibrosis as evidenced in sequential bone marrow biopsy specimens.

Although the new World Health Organization (WHO) classification acknowledges "prefibrotic" phases, progression of myelofibrosis in chronic idiopathic myelofibrosis (cIMF) is controversial because there are only a few studies about sequential biopsy specimens, and they yield conflicting results. The conflicting results might be due to a mixture of different degrees of myelofibrosis and therapy regimens within the respective groups studied. To prove this hypothesis, we studied sequential bone marrow biopsy specimens from patients with cIMF and compared 3 groups with different degrees of myelofibrosis at initial diagnosis with a group of patients with primarily unfibrosed disease who met the WHO criteria for prefibrotic cIMF. Patients receiving chemotherapy were considered separately from patients without treatment. Our results favor a steady progression of myelofibrosis unrelated to therapy modalities, whereas confusing literature data can be explained: fibrosis may remain static or lessen, especially in more advanced stages of cIMF.

Analysis of risk factors of the evolution of myelofibrosis in pre-fibrotic chronic idiopathic myelofibrosis: a retrospective study based on follow up biopsies of 70 patients by using the RECPAM method.

The advanced, fibrotic phase of chronic idiopathic myelofibrosis (CIMF) is preceded by a pre-fibrotic stage. However, the factors which may influence or predict the development of myelofibrosis are not well established. Thus we investigated follow up biopsies of 70 patients with CIMF, diagnosed in stages of no or only scant fiber increase, for the development of myelofibrosis. The influence of histopathological (megakaryocytes, initial fiber content), clinical (age, gender, splenomegaly, chemotherapy) and hematological (Hb, leukocyte- and platelet count) parameters on the development of myelofibrosis was evaluated by using the univariate Log Rank method and the multivariate recursive partition and amalgamation (RECPAM) analysis. Surveying a mean observation period of 47 months we found a development of significant myelofibrosis in 30 of the 70 patients. In the univariate analysis, the development of myelofibrosis was associated with increased megakaryocytes, initial fiber content and age of the patient. In the RECPAM analysis, the patients with a high megakaryocytic count and older age showed the highest risk of developing myelofibrosis (mean 58.3 and 60.1 months). The group with the best prognosis comprised the patients under 60 years which have a low content of megakaryocytes (mean 137 months). We found that the development of myelofibrosis in CIMF was best predicted by an increase of megakaryocytes within the bone marrow, possibly reflecting the release of growth factors by these cells. The next important risk factor was the age of the patients.

JAK2(V617F) allele burden discriminates essential thrombocythemia from a subset of prefibrotic-stage primary myelofibrosis.

OBJECTIVE: Among Philadelphia chromosome-negative myeloproliferative neoplasms (Ph(-) MPN), essential thrombocythemia (ET) and the prefibrotic phase of primary myelofibrosis (PMF) represent two subtypes with considerable overlap. MATERIALS AND METHODS: In this study, histopathological classification of 490 MPN cases was correlated with the allelic burden of JAK2(V617F) and MPL(W515L). RESULTS: Ph(-) MPN entities largely overlap with regard to JAK2(V617F) and MPL(W515L) allele burden, but ET displayed mutant allele burden <50%. PMF with different stages of myelofibrosis all yielded similar JAK2(V617F) allele burden. At initial presentation one-quarter of prefibrotic PMF cases exhibited an allele burden exceeding 50% (38% median JAK2(V617F) alleles, n=102). In ET, its main differential diagnosis, not a single case was found with >40% JAK2(V617F) alleles (median, 24% JAK2(V617F) alleles; n=90; p<0.001). Increase in JAK2(V617F) alleles during follow-up could not be linked to fibrosis or blastic progression but was related to polycythemic transformation in ET. MPL(W515L) was found in 3% of ET and 8% of PMF, with a significantly higher percentage of mutated alleles in fibrotic than prefibrotic PMF (median, 78% MPL(W515L) alleles; p<0.05). CONCLUSION: Histopathological categories ET and prefibrotic PMF correlate with significant differences in mutant allelic burden of JAK2(V617F), but not of MPL(W515L) which, by contrast to JAK2(V617F), shows a higher percentage of mutated alleles in fibrotic than in prefibrotic cases. Thus, for Ph(-) MPN in which ET and prefibrotic PMF represent the most probable diagnoses, a JAK2(V617F) allele burden >50% favors a diagnosis of prefibrotic PMF.

Aberrant collagenase expression in chronic idiopathic myelofibrosis is related to the stage of disease but not to the JAK2 mutation status.

Bone marrow fibrosis in chronic idiopathic myelofibrosis (cIMF) most likely represents an imbalance between synthesis and turnover of collagen fibers. Because the JAK-STAT signaling pathway is involved in the regulation of genes encoding matrix metalloproteinases (MMPs), we examined the expression of MMPs, their tissue inhibitors (TIMPs), and collagen types in relation to the JAK2 status (V617F mutation versus wild-type) in cIMF (n = 64). Whereas no correlation was found between the JAK2 status and MMP gene products, there was an evident association with the stage of disease. Membrane type 1-MMP (MMP-14) was overexpressed by up to 80-fold in advanced stages that progressed to fibrosis (P < 0.001), and megakaryocytes and endothelial cells were unmasked as the major cellular source. By contrast, a significantly higher expression of neutrophil collagenase (MMP-8) was encountered in the prefibrotic stages of cIMF (P < 0.001). Altogether, the stepwise progress of myelofibrosis in cIMF was associated with expression of a defined subset of target genes as shown in sequential trephine biopsies of cIMF patients. We conclude that the expression of matrix-modeling genes in cIMF is not influenced by the JAK2 mutation status but is predominantly related to the stage of disease.

The incidence of myelofibrosis in essential thrombocythaemia, polycythaemia vera and chronic idiopathic myelofibrosis: a retrospective evaluation of sequential bone marrow biopsies.

The incidence of myelofibrosis (MF) among the three major Philadelphia chromosome-negative chronic myeloproliferative disorders, i.e. essential thrombocythaemia (ET), polycythaemia vera (PV) and chronic idiopathic myelofibrosis (CIMF), is not well documented since the diagnostic criteria have recently been redefined by the WHO. Therefore we performed a retrospective analysis of follow-up biopsies of 275 patients with ET, PV and CIMF according to the WHO classification of chronic myeloproliferative disorders. In the diagnostic bone marrow biopsies, MF was observed in 57 of the 136 CIMF patients (42%), 4 of the 73 PV patients (5%) and none of the 66 patients with ET. Within a median observation time of 2.9 years, 34 of the 79 patients with CIMF (43%), 13 of the 69 patients with PV (19%) and 1 of the 66 patients with ET (1.5%)--each initially without MF--developed MF regardless of myelosuppressive therapy.

Reliability of lymphoma classification in bone marrow trephines.

The aim of this study was to test and establish the accuracy and reliability of lymphoma classification in bone marrow trephines according to the new World Health Organization (WHO) classification by considering predominantly the morphology and immunophenotype. Therefore, we retrospectively compared lymphoma diagnoses, rendered exclusively on bone marrow trephines without knowledge of lymph node diagnosis in 124 patients, with the results of the reference centres that had reviewed lymph node (n = 90) or extranodal biopsies (n = 34). The overall concordance rate was higher than 85% and 91%, respectively, when patients with discordant malignancy grades were excluded. The concordance rate for low-grade B-cell lymphomas was 93% and for high-grade B-cell lymphomas 84%. The main reasons for discordant diagnoses were divergent immunophenotypes among low-grade B-cell lymphomas (6 out of 81, i.e. 7.4%) and discrepant malignancy grades within high-grade B-cell lymphomas (6 out of 31, i.e. 19.4%). No relationship between discordant diagnoses and chemotherapy given during the course of the disease with the site of biopsy (i.e. lymph nodes, extranodal sites) was noted. We conclude from our results that bone marrow trephines are a reliable tool, not only for establishing bone marrow infiltration, but also for the subtyping of lymphomas.