Bone marrow microvessel density in chronic myeloproliferative disorders: a study of 115 patients with clinicopathological and molecular correlations

Philadelphia-negative chronic myeloproliferative disorders (CMD) include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). Angiogenesis is critical in the pathogenesis of PMF. We studied angiogenesis in 115 patients with CMD (23 PV, 24 ET, 46 PMF, 12 post-PV and 10 post-ET myelofibrosis) by assessment of microvessel density (MVD) in bone marrow (BM). Kruskall–Wallis analysis of variance showed that patients with PMF had significantly higher values of MVD than those with PV ( P  <   0·001), ET ( P  <   0·001) and controls ( P  <   0·001). Mann–Whitney U -test demonstrated that patients with PMF at the prefibrotic stage had significantly higher MVD values than those with ET ( P  =   0·02). Patients with post-PV myelofibrosis showed significantly higher MVD values than those with PV ( P  <   0·001), as did patients with post-ET myelofibrosis compared with ET ( P  <   0·001). In patients with CMD, the multivariate generalized linear regression model showed that the JAK2 (V617F) mutational burden ( P  =   0·01), serum lactate dehydrogenase level ( P  =   0·003), and anaemia ( P  <   0·001) independently correlated with MVD. In summary, this study indicates that assessment of BM angiogenesis, as measured by MVD, may be a useful additional tool in the histopathological definition of CMD.     

Molecular mechanisms associated with leukemic transformation of MPL-mutant myeloproliferative neoplasms

Somatic activating mutations in MPL, the thrombopoietin receptor, occur in the myeloproliferative neoplasms, although virtually nothing is known about their role in evolution to acute myeloid leukemia. In this study, the MPL T487A mutation, identified in de novo acute myeloid leukemia, was not detected in 172 patients with a myeloproliferative neoplasm. In patients with a prior MPL W515L-mutant myeloproliferative neoplasm, leukemic transformation was accompanied by MPL-mutant leukemic blasts, was seen in the absence of prior cytoreductive therapy and often involved loss of wild-type MPL by mitotic recombination. Moreover, clonal analysis of progenitor colonies at the time of leukemic transformation revealed the presence of multiple genetically distinct but phylogenetically-related clones bearing different TP53 mutations, implying a mutator-phenotype and indicating that leukemic transformation may be preceded by the parallel expansion of diverse hematopoietic clones.     

TP53 overexpression is an independent adverse prognostic factor in de novo myelodysplastic syndromes with fibrosis

Bone marrow (BM) fibrosis is associated with poor prognosis in patients with de novo myelodysplastic syndromes (MDS). TP53 mutations and TP53 (p53) overexpression in MDS are also associated with poor patient outcomes. The prevalence and significance of TP53 mutations and TP53 overexpression in MDS with fibrosis are unknown. We studied 67 patients with de novo MDS demonstrating moderate to severe reticulin fibrosis (MDS‐F). Expression of TP53 was evaluated in BM core biopsy specimens using dual‐colour CD34/TP53 immunohistochemistry with computer‐assisted image analysis. Mutation analysis was performed using next‐generation sequencing, or Sanger sequencing methods. TP53 mutations were present in 47·1% of cases. TP53 mutation was significantly associated with TP53 expression (P = 0·0294). High levels of TP53 expression (3 +  in ≥10% cells) were associated with higher BM blast counts (P = 0·0149); alterations of chromosomes 5 (P = 0·0009) or 7 (P = 0·0141); complex karyotype (P = 0·0002); high‐ and very‐high risk IPSS‐R groups (P = 0·009); and TP53 mutations (P = 0·0003). High TP53 expression independently predicted shorter overall survival (OS) by multivariate analysis (P = <0·001). Expression of TP53 by CD34‐positive cells was associated with shorter OS and leukaemia‐free survival (P = 0·0428). TP53 overexpression is a predictor of poor outcome in patients with MDS‐F.     

87例骨髓增殖性肿瘤患者JAK2及MPL基因突变位点研究

目的:探讨JAK2V617F及MPLW515L/K点突变在骨髓增殖性肿瘤(MPN)患者中的发生情况及临床意义。方法:回顾性统计分析87例MPN患者的临床及实验室检查资料,应用等位基因特异性-聚合酶链反应(AS-PCR)及序列测定方法,检测MPN患者骨髓/外周血单个核细胞JAK2V617F及MPLW515L/K点突变的发生情况,结合JAK2V617F及MPLW515L/K点突变阳性与阴性2组患者的临床及实验室检查指标,探讨其在疾病诊断及分子发病机制中的意义。结果:87例MPN患者[真性红细胞增多症(PV)36例,原发性血小板增多症(ET)33例,原发性骨髓纤维化(PMF)18例]中共检出55例患者存在JAK2V617F突变,总突变率为63.2%(55/87),其中PV28例,突变率77.8%(28/36);ET17例,突变率51.5%(17/33);PMF10例,突变率55.6%(10/18)。JAK2V617F阳性PV和ET患者WBC及Hb水平高于阴性患者(P<0.05);JAK2V617F阳性PMF患者WBC及PLT高于阴性患者(P<0.05)。JAK2V617F阳性MPN患者血栓发生率高于阴性患者(P<0...     

Circulating YKL‐40 in myelofibrosis a potential novel biomarker of disease activity and the inflammatory state

Chronic myeloproliferative neoplasms (MPN), encompassing essential thrombocythaemia (ET), polycythaemia vera (PV) and myelofibrosis (PMF), are featured by a chronic inflammatory state which is pronounced in myelofibrosis The value of YKL‐40 as a biomarker of disease burden has been demonstrated in several different diseases, including cancer, diabetes mellitus and cardiovascular diseases. A state of chronic inflammation is shared by them all, YKL‐40 also being involved in the severity of chronic endothelial inflammation, which today is considered of crucial importance for the development of atherosclerosis. The MPNs being cancers with a heavy burden of cardiovascular diseases we hypothesised that circulating YKL‐40 might reflect the inflammatory process and potentially serve as a novel disease marker. Using ELISA, we measured YKL‐40 in 15 patients with ET, 16 patients with PV, 17 patients with PMF and 30 healthy controls. YKL‐40 was significantly elevated in PMF vs. control subjects, PMF levels median 43 ng/mL vs. controls median 28 ng/mL, P = 0.033. An increase from ET over PV may reflect the integrated impact of disease processes in MPNs.     

A patient with a 20-year lag phase between JAK2-V617F+ myeloproliferation and NPM1-mutated AML arguing against a common origin of disease

We have sought to unravel the molecular biology of a female patient who in 1985 at the age of 55 was diagnosed with a chronic myeloproliferative neoplasm (MPN) and in whom overt acute myeloid leukemia (AML) developed in 2005. To this end, DNA and RNA (extracted from either paraffin-embedded bone marrow (BM) or from BM and/or peripheral blood stored in an RNA/DNA-preserving buffer) were analyzed by qPCR and by capillary gel electrophoresis of PCR products. We found the patient to be JAK2-V617F mutation positive throughout the course of disease, while a mutation of the nucleophosmin (NPM1) gene emerged at AML diagnosis and relapse. The 20-yr lag phase between the polycythemia vera and the AML adds indirect evidence to the growing realization that the leukemic transformation in patients with MPN occurs from in a JAK2 wild-type stem cell.     

Elevated TRIB2 with NOTCH1 activation in paediatric/adult T-ALL

TRIB2 is a potent oncogene, elevated in a subset of human acute myeloid leukaemias (AML) with a mixed myeloid/lymphoid phenotype and NOTCH1 mutations. Although rare in AML, activating NOTCH1 mutations occur in 50% of all T cell acute lymphoblastic leukaemias (T-ALL). TRIB2 is a NOTCH1 target gene that functions in the degradation of key proteins and modulation of MAPK signalling pathways, implicated in haematopoietic cell survival and proliferation. This study showed that TRIB2 expression level is highest in the lymphoid compartment of normal haematopoietic cells, specifically in T cells. Analysis of TRIB2 expression across 16 different subtypes of human leukaemia demonstrated that TRIB2 expression was higher in ALL phenotypes versus all other phenotypes including AML, chronic lymphocytic leukaemia (CLL), myelodysplastic syndrome (MDS) and chronic myeloid leukaemia (CML). A T cell profile was distinguished by high TRIB2 expression in normal and malignant haematopoiesis. High TRIB2 expression was seen in T-ALL with normal karyotype and correlated with NOTCH signalling pathways. High TRIB2 expression correlated with NOTCH1/FBXW7 mutations in a paediatric T-ALL cohort, strongly linking NOTCH1 activation and high TRIB2 expression in paediatric T-ALL. The relationship between TRIB2 and T cell signalling pathways uniquely identifies leukaemia subtypes and will be useful in the advancement of our understanding of T cell and ALL biology.     

WHO‐histological criteria for myeloproliferative neoplasms: reproducibility,diagnostic accuracy and correlation with gene mutations and clinical outcomes

Bone marrow histology is included in the diagnostic criteria of myeloproliferative neoplasms (MPNs). However, some concerns have emerged about its reproducibility. To evaluate the diagnostic accuracy of histology and to assess its correlation with presence of mutations and clinical outcomes, two pathologists reviewed the bone marrow biopsies corresponding to 211 patients with MPN. Despite the low agreement in the evaluation of individual histopathological characteristics, the concordance among pathologists when establishing the diagnosis was good (Kappa index 0·67). The specificity of histology was 100%, 98·5% and 98% in polycythaemia vera (PV), essential thrombocythaemia (ET) and primary myelofibrosis (PMF), respectively, whereas the sensitivity of histological diagnosis was low in PV and ET (32·5% and 54% respectively) and acceptable in PMF (75%). Thirteen out of 146 (9%) patients with clinical ET were diagnosed as prefibrotic PMF. No histological agreement or MPN otherwise unspecified was more frequently observed in JAK2 V617F‐positive ET than in CALR‐mutated cases, whereas megakaryocytic abnormalities and prefibrotic PMF were more frequently observed in CALR‐mutated ET. In conclusion, histological criteria of MPN have a limited diagnostic accuracy due to low sensitivity. Patients with JAK2 V617F‐positive MPN have a heterogeneous histology while CALR‐positive ET is associated with megakaryocyte abnormalities and prefibrotic PMF.     

Clinical significance of Th1/Th2 ratio in patients with myelodysplastic syndrome

In this study, we attempted to evaluate the clinical significance of T helper 1 (Th1)/T helper 2 (Th2) ratio in patients with myelodysplastic syndrome (MDS), five refractory anaemia (RA), four refractory anaemia with ringed sideroblasts (RARS), 31 refractory cytopenia with multilineage dysplasia (RCMD), nine refractory anaemia with excess blast-1 (RAEB-1) and seven refractory anaemia with excess blast-2 (RAEB-2). Intracellular interleukin-4 (Th2 cytokine) and interferon-gamma (Th1 cytokine) production was assessed in CD4+ T lymphocytes activated by phorbol 12-myristate 13-acetate and ionomycin using flow cytometry. Mean Th1/Th2 ratios in each MDS group were as follows: RA/RARS, 8.8 (95% CI, 5.8-11.8), RCMD, 14.7 (95% CI, 9.5-19.9), RAEB-1, 10.6 (95% CI, 4.6-16.6), RAEB-2, 12.8 (95% CI, 3.0-22.7) and control 12.8 (95% CI, 9.6-16.1). There were no significant differences in Th1/Th2 ratio in the RA/RARS, RCMD, RAEB-1 and RAEB-2 subgroups when compared to controls. Because Th1/Th2 ratio in the RCMD group was widely distributed, we divided RCMD patients according to Th1/Th2 ratio into three groups (low, normal and high Th1/Th2 groups). There were no differences in severity of cytopenia among the three above groups. However, the percentage of CD8 cells in the low Th1/Th2 group was significantly lower than those in the high group (P < 0.01). These data suggest that Th1/Th2 imbalance induces CD4/CD8 imbalance, and serves as a marker of the biological interplay in immune regulation.     

Postallogeneic monitoring with molecular markers detected by pretransplant next‐generation or Sanger sequencing predicts clinical relapse in patients with myelodysplastic/myeloproliferative neoplasms

Relapse is the major cause of treatment failure after allogeneic stem‐cell transplantation (AHSCT) for patients with myelodysplastic syndrome/myeloproliferative syndrome neoplasms (MDS/MPN). We evaluated the impact of molecular mutations on outcome and the value of molecular monitoring post‐transplantation. We screened 45 patients with chronic myelomonocytic leukemia (n = 39 patients, including seven with transformed‐acute myeloid leukemia), MDS/MPN unclassifiable (n = 5), and atypical BCR‐ABL1‐negative CML (n = 1) for mutations in ASXL1, CBL, NRAS, and TET2 genes by molecular genetics including a sensitive next‐generation sequencing (NGS) technique. In 36 patients, sufficient DNA was available for molecular analyses. In particular, TET2 and CBL mutations were screened applying amplicon deep sequencing. In 89% of cases, at least one mutation could be detected: ASXL1: n = 18 (50%); CBL: n = 7 (19%); TET2: n = 15 (42%); and NRAS: n = 11 (32%). Survival after AHSCT at 5 yr was 46% (95% CI 28–64%) and was not influenced by any mutation. After a median of 6 months after AHSCT in 33% of the patients, one of the molecular markers was still detectable, resulting in a higher incidence of relapse than in patients with undetectable mutations (50% vs. 15%, P = 0.04). In conclusion, pretransplant molecular mutation analysis can help to detect biomarkers in patients with MPN/MDS, which may be subsequently used as minimal residual disease markers after AHSCT.