Jerome Schlué

Aberrant expression of transforming growth factor β-1 (TGFβ-1) per se does not discriminate fibrotic from non-fibrotic chronic myeloproliferative disorders

Transforming growth factor β‐1 (TGFβ‐1) is a potent inducer of fibrosis and has been shown to be essential for the development of bone marrow fibrosis in an animal model of idiopathic myelofibrosis (IMF). IMF belongs to the Philadelphia chromosome negative chronic myeloproliferative disorders (Ph− CMPD). Megakaryocytes and platelets have been suggested as the major cellular source of TGFβ‐1 in IMF. The osteoclastogenesis inhibitory factor osteoprotegerin (OPG) seems to be regulated by TGFβ‐1 and substantial involvement of OPG expression in the process of osteosclerosis in IMF has recently been suggested. In order to determine TGFβ‐1 expression in IMF and other Ph− CMPD, total bone marrow cells as well as laser‐microdissected megakaryocytes were quantitatively analysed by real‐time RT‐PCR. OPG mRNA expression in fibrotic IMF was correlated with TGFβ‐1 mRNA expression in a case‐specific manner. Both OPG and TGFβ‐1 were detected immunohistochemically in order to delineate cellular origin. When total bone marrow cells were investigated, TGFβ‐1 mRNA expression was increased in some but not all cases of IMF (n = 21), with highest values in fibrotic cases. Unexpectedly, increased values were also observed in essential thrombocythaemia (ET, n = 11) when compared to non‐neoplastic haematopoiesis (n = 38). Megakaryocytes isolated by laser microdissection displayed elevated TGFβ‐1 mRNA levels in most of the CMPD samples with no significant differences discernible between fibrotic IMF, polycythaemia vera (PV) and ET. TGFβ‐1 protein was predominantly expressed by the myeloid lineage in Ph− CMPD and non‐neoplastic haematopoiesis, which, however, displayed lower expression. IMF cases with advanced fibrosis concomitantly overexpressed TGFβ‐1 and OPG. Immunohistochemically, OPG expression was found in different stromal cells and a subfraction of megakaryocytes. In conclusion, enhanced TGFβ‐1 expression occurs in megakaryocytes as well as myeloid cells in Ph− CMPD. TGFβ‐1 may be necessary, but is not sufficient, to induce bone marrow fibrosis in IMF because non‐fibrotic Ph− CMPD entities share this feature with IMF and cannot be discriminated from each other on the basis of TGFβ‐1 expression. Copyright

Biclonal expansion and heterogeneous lineage involvement in a case of chronic myeloproliferative disease with concurrent MPLW515L/JAK2V617F mutation

To the editor: Chronic myeloproliferative diseases (CMPDs)/myeloproliferative neoplasms (MPNs) are caused by clonal mutations of tyrosine kinase or receptor genes such as BCR-ABL, JAK2V617F, and MPLW515L.[1][1],[2][2] Recently, we and others showed that these mutations may be combined in

The polycythaemia rubra vera-1 gene is constitutively expressed by bone marrow cells and does not discriminate polycythaemia vera from reactive and other chronic myeloproliferative disorders

Summary. Little is known about the expression of the polycythaemia rubra vera‐1 (PRV‐1) gene in bone marrow cells. To determine the expression level of PRV‐1 in the bone marrow, we analysed PRV‐1 quantitatively in Polycythaemia vera, other chronic myeloproliferative disorders, and reactive states. We demonstrated that PRV‐1 was constitutively expressed in both myeloproliferative and reactive states. We concluded that, rather than an upregulation of the PRV‐1 gene in the clonal haematopoiesis of polycythaemia vera, a failure to downregulate PRV‐1 in granulocytes emigrating from the bone marrow might be responsible for the increase of transcripts in peripheral cells.

Thrombopoietin receptor (Mpl) expression by megakaryocytes in myeloproliferative disorders

The thrombopoietin receptor (Mpl) is involved in the pathogenesis of chronic myeloproliferative disorders (CMPD). In this study, we determined Mpl expression by bone marrow cells and megakaryocytes in CMPD by applying laser microdissection, real‐time RT‐PCR, and immunohistochemistry. Mpl mRNA expression was significantly increased up to 9‐fold in total bone marrow cells (p < 0.001) and up to 4‐fold in megakaryocytes in chronic myeloproliferative disorders (n = 73) compared to normal controls (n = 26, p = 0.01). Immunohistochemistry revealed heterogeneous Mpl expression by megakaryocytes in CMPD with a stronger accentuation in idiopathic myelofibrosis (IMF) in comparison to polycythaemia vera (PV) and essential thrombocythemia (ET). In addition to megakaryocytes, the erythropoietic lineage was prominently labelled by Mpl antiserum, with considerably stronger staining in polycythaemia vera. We conclude that, in CMPD, megakaryocytes and erythroid cells exhibit increased Mpl expression levels which may contribute to the sustained proliferation of both cell lineages in CMPD. Copyright

Reliability of lymphoma classification in bone marrow trephines

Summary. 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.

SRSF2 mutation is present in the hypercellular and prefibrotic stage of primary myelofibrosis

To the editor: Myeloproliferative neoplasms (MPNs) are characterized by their propensity to progress from overproduction of 1 or more hematopoietic lineages to states of cytopenia associated with myelofibrosis and/or blast excess. Recently, it was demonstrated that in progressed primary

Reduced expression of H19 in bone marrow cells from chronic myeloproliferative disorders

Double minutes and c-MYC amplification in acute myelogenous leukemia: are they prognostic factors? Cancer Genet Cytogenet 2000; 120: 73–79. 4 Sen S, Sen P, Mulac-Jericevic B, Zhou H, Pirrotta V, Stass SA. Microdissected double-minute DNA detects variable patterns of chromosomal localizations and multiple abundantly expressed transcripts in normal and leukemic cells. Genomics 1994; 19: 542–551. 5 Tanke HJ, Wiegant J, van Gijlswijk RPM, Bezrookove V, Pattenier H, Heetebrij RJ et al. New strategy for multi-colour fluorescence in situ hybridisation: COBRA: COmbined Binary RAtio labelling. Eur J Hum Genet 1999; 7: 2–11. 6 Sambani C, Trafalis DTP, Vessalas G, Politis G, Peristeris P, Nakopoulou L et al. Trisomy 6 and double minute chromosomes in a case of chronic myelomonocytic leukemia. Cancer Genet Cytogenet 1998; 106: 180–181. 7 Crossen PE, Savage LM, Heaton DC, Morrison MJ. Characterization of the C-MYC amplicon in a case of acute myeloid leukemia with double minute chromosomes. Cancer Genet Cytogenet 1999; 112: 144–148. 8 Asker C, Mareni C, Coviello D, Ingvarsson S, Sessarego M, Origone P et al. Amplification of c-myc and pvt-1 homologous sequences in acute nonlymphatic leukemia. Leuk Res 1988; 12: 523–527.

[Myeloproliferative neoplasms: histopathological and molecular pathological diagnosis].

Myeloproliferative neoplasms (chronic myeloproliferative disorders according to former nomenclature) comprise chronic myeloid leukemia, polycythemia vera, essential thrombocythemia, primary myelofibrosis, chronic eosinophilic leukemia, chronic neutrophilic leukemia and systemic mastocytosis. All disorders have excessive proliferation of one or more hematopoietic lineages in common and progress with different probability to blast crisis or fibrosis. A further common feature is provided by the activating mutation of tyrosin kinases and associated pathways of signal transduction (BCR-ABL, JAK2(V617F), MPL(W515L/K), KIT(D816V) and FIP1L1-PDGFRA) causative for the abnormal proliferation. With regard to diagnosis and therapy these mutations are of utmost importance because they enable the exclusion of reactive processes, contribute with varying specificity to subtyping of MPN and are at least partly sensitive to targeted therapy. The molecular mechanisms of blastic and fibrotic progression are not yet understood.

De novo CSF3R mutation associated with transformation of myeloproliferative neoplasm to atypical CML

/L), circulating myeloid precursors (>10 %), BCR-ABLnegativity [2, 3]) [4, 5]. We found a newly occurring CSF3R(exons 14, 17) mutation during progression in a case of MPNunclassifiable (MPNu). The 65-year-old male presented withthrombocytosis, splenomegaly, a leukoerythroblastic bloodpicture, anemia, and signs of hemolysis (thrombocytes 602×10

Evolution of chronic myelomonocytic leukemia to myeloproliferative neoplasm

Dear Editor, Chronic myelomonocytic leukemia (CMML) is characterized by sustained non-reactive monocytosis (≥1 × 10μl, ≥10 % monocytes of peripheral blood leucocytes), frequently accompanied by thrombocytopenia and/or anemia. Progression to acute leukemia (AML) takes place in 30– 40 % of cases [1]. Here, we present for the first time a series of four CMML cases which did not progress to AML but evolved to myeloproliferative neoplasm (MPN). The four patients were followed by sequential bone marrow biopsies and mutational studies for 2–11 years (median 5 years, 4–6 sequential biopsies per patient). CMML had been known for 2–6 years (median 3.5 years). During follow-up, all four patients showed an increase of platelet counts (from median 99.5 × 10μ l -1 , range 49–279 × 10μ l -1 to median 497.5×10μl, range 210–560×10μl Table 1). In two patients, previous thrombocytopenia was converted into thrombocytosis (cases 1 and 4, Table 1). By contrast, an improvement of anemia could not be noticed (Table 1). Leucocyte counts increased in three patients while absolute and relative monocytosis persisted in all four patients. The monocyte counts before increase of platelet counts (median 3.2 × 10μl, 20 % of peripheral leucocytes; range 1.0– 7.8×10μl, 18.5–28.9 % of peripheral leucocytes) did not differ from the values afterwards (median 3.3 × 10μl, 16.6 % of peripheral leucocytes; range 1.1–7.4 × 10μl, 10.9–18.7 % of peripheral leucocytes, Table 1). Evolution to MPN became evident by a profound change of bone marrow morphology as shown in Fig. 1. Before, as typical for CMML, there had been a predominant proliferation of granuloand monopoiesis accompanied by a small number of dysplastic small megakaryocytes (Fig. 1a, b). The most impressive change in bone marrow histology, indicating evolution to MPN, was provided by an increase in megakaryocyte number and size with cluster formation and fibrosis (Fig. 1c, d). The transformation of bonemarrowmorphology was paralleled by the occurrence of MPN driver mutations such as JAK2 or MPL (Table 1). Mutation profiling of 23 genes was performed as previously described [2]. All four patients exhibited TET2 mutations with a high allelic burden, probably affecting both alleles (Table 1). Three cases showed SRSF2 mutations, which are typically found in CMML [3]. Furthermore, a mutation of CBL was noticed (Table 1). Case 1, which unexpectedly revealed a homozygous SRSF2 mutation (96.2 % p.P95_P96del) was studied with Sanger sequencing and PyrosequencingTM with a different primer set and yielded identical results. The four cases demonstrate that CMML can undergo transformation to a clinical presentation and histological picture that fulfill the criteria of MPN. The phenotypic transformation appears not to be caused by a replacement of the CMML clone by a MPN clone because the characteristic SRSF2 and TET2 mutations persisted. The MPNtypical driver mutations, such as JAK2 and MPL appeared to be acquired by the pre-existing neoplastic clone which had given rise to CMML. In one case, the JAK2 mutation was detectable already at presentation but revealed an increase of allelic burden from 4.7 to 64.8 % which probably caused the transformation of the histological picture. In all the other cases, evolution to MPN could be explained by de novo mutation of either JAK2 or MPL. The detection threshold of NGS is approximately 1 %; consequently, it cannot be excluded that very small sub-clones had * H. Kreipe Kreipe.Hans@MH-Hannover.de