Katharina Theophile

JAK2V617F 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.

Bone Morphogenetic Proteins Are Overexpressed in the Bone Marrow of Primary Myelofibrosis and Are Apparently Induced by Fibrogenic Cytokines

Primary myelofibrosis (PMF) is a myeloproliferative neoplasia characterized by progressive deposition of extracellular matrix components in the bone marrow. The involvement of members of the bone morphogenetic protein (BMP) family in aberrant bone marrow matrix homeostasis in PMF has not yet been investigated. Therefore, we analyzed expression of BMP1, an activator of latent transforming growth factor beta-1 (TGFbeta-1) and processor of collagen precursors, and other BMPs in bone marrow from PMF patients and controls (n = 95). Expression of BMP1, BMP6, BMP7, and BMP-receptor 2 was significantly increased in advanced stages of myelofibrosis compared with controls (P < or = 0.01), and enhanced levels of BMP6 expression were already evident in prefibrotic stages of PMF. Immunohistochemistry showed that bone marrow stromal cells and megakaryocytes were the major cellular sources of BMP1 protein. Because TGFbeta-1 and basic fibroblast growth factor have been shown to be important in the development of myelofibrosis, we studied the induction of BMPs by these cytokines in cultured fibroblasts. Fibroblasts treated with TGFbeta-1 showed a pronounced up-regulation of BMP6, suggesting that stromal cells may be susceptible to BMP activation by cytokines with a proven role in the pathogenesis of PMF. We conclude that BMP family members may play an important role in the pathogenesis of myelofibrosis in PMF and are apparently induced by cytokines such as TGFbeta-1.

Aberrant microRNA expression pattern in myelodysplastic bone marrow cells

The microRNA/miR system might contribute to deregulation of cell homeostasis/disease phenotype. This is the first approach to generate an expression profile of 365 microRNAs in myelodysplastic syndromes (MDS) with normal karyotype (n=12) and distinct cytogenetic aberrations (n=12). In MDS-del(5q), a series of microRNAs not in the 5q-region was increased. MicroRNAs encoded on chromosomes 5, 7 and 8 were not differentially expressed in MDS with del(5q), -7 or +8. Evaluation in a larger cohort could confirm the up-regulation of the miR-1 in MDS. These findings provide evidence that MDS-haematopoiesis is distinct in its microRNA-expression pattern from non-neoplastic cells.

Amplification of mRNA from laser-microdissected single or clustered cells in formalin-fixed and paraffin-embedded tissues for application in quantitative real-time PCR.

The determination of marker genes and gene clusters involved in disease pathogenesis is increasingly contingent on high-throughput methods of gene expression profiling. However, the concurrently increasing application of mRNA from formalin-fixed and paraffin-embedded (FFPE) tissue archives, as well as cell-type–specific approaches by laser-assisted microdissection, frequently results in very small and degraded quantities of RNA. Therefore, a successful amplification of cell-type–specific mRNA targets from FFPE tissues becomes more and more essential. To optimize the hitherto limited technical options, we applied 3 commercial amplification kits on FFPE single cells. We thereby determined the approach of target-specific cDNA amplification as being notably appropriate for subsequent real-time polymerase chain reaction, as a constant decrease of CT values by 14 polymerase chain reaction cycles could be demonstrated.

Significant inverse correlation of microRNA-150/MYB and microRNA-222/p27 in myelodysplastic syndrome

We investigated whether, in myelodysplastic syndromes (MDS), aberrant expression of miR-150/miR-221/miR-222 and their designated target mRNA molecules MYB, p27 and c-KIT may be involved in insufficient haematopoiesis. In a series of MDS (n=52), an aberrant increase of miR-150 was found only in MDS with associated del(5q) (n=9; p<0.01). The mRNA expression of transcription factor MYB, the designated target of miR-150, was shown to correlate inversely with the miR-150 level. Acute leukaemia evolving from MDS (n=11) showed significantly decreased levels of miR-221 but not miR-222. We conclude that inhibition of proliferation via over-expressed miR-150 might contribute to myelodysplastic haematopoiesis in MDS-del(5q).

MicroRNA expression profiling of megakaryocytes in primary myelofibrosis and essential thrombocythemia

In primary myelofibrosis (PMF) and essential thrombocythemia (ET) the megakaryocytic lineage characteristically shows aberrant proliferation and maturation in which the regulatory microRNA (miR) system might be involved. Laser-microdissected PMF and ET megakaryocytes were analysed with real-time polymerase chain reaction (PCR) low density arrays comprising 365 microRNAs. The highest megakaryocytic expression levels were observed for miR-223, which is known to be expressed also in granulopoiesis. Cluster analysis revealed a tendency of disease-specific megakaryocytic microRNA expression profiles indicating that a complex shift of microRNA expression appears to be the underlying defect. Increased accumulation of miR-146b was observed in cellular stage PMF (p = 0.0125) but not ET megakaryopoiesis. In conclusion, this is the first microRNA profiling of in situ-derived PMF, ET and normal megakaryocytes.

MPLW515L mutation in acute megakaryoblastic leukaemia.

The thrombopoietin receptor gene (MPL) is expressed in megakaryocytes and exhibits the gain of function point mutation W515K/L in ∼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 MPLW515K/L mutation by pyrosequencing. In three cases (25%), MPLW515L 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 MPLW515K/L (n=4). We conclude that MPLW515L occurs in a considerable proportion of acute megakaryoblastic leukaemias with myelofibrosis unrelated to PMF.

Obliterative Airway Remodeling: Molecular Evidence for Shared Pathways in Transplanted and Native Lungs

Obliteration of the small airways is a largely unresolved challenge in pulmonary medicine. It represents either the irreversible cause of functional impairment or a morphologic disorder of limited importance in a multitude of diseases. Bronchiolitis obliterans is a key complication of lung transplantation. No predictive markers for the onset of obliterative remodeling are currently available. To further elucidate the molecular mechanisms of airway remodeling, compartment-specific expression patterns were analyzed in patients. For this purpose, remodeled and nonremodeled bronchioli were isolated from transplanted and nontransplanted lung explants using laser-assisted microdissection (n = 24). mRNA expression of 45 fibrosis-associated genes was measured using quantitative real-time RT-PCR. For 20 genes, protein expression was also analyzed by immunohistochemistry. Infiltrating cells were characterized at conventional histology and immunohistochemistry. Obliterative remodeling of the small airways in transplanted and nontransplanted lungs shared similar grades of chronic inflammation and pivotal fibrotic pathways such as transforming growth factor β signaling and increased collagen expression. Bone morphogenetic protein and thrombospondin signaling, and also matrix metalloproteinases and tissue inhibitor of metalloproteinases, were primarily up-regulated in obliterative airway remodeling in nontransplanted lungs. In transplanted lungs, clinical remodeled bone morphogenetic protein but nonremodeled bronchioli were characterized by a concordant up-regulation of matrix metalloproteinase-9, RANTES, and tissue inhibitor of metalloproteinase-1. These distinct expression patterns warrant further investigation as potential markers of impending airway remodeling, especially for prospective longitudinal molecular profiling.

Megakaryocytic expression of miRNA 10a, 17-5p, 20a and 126 in Philadelphia chromosome-negative myeloproliferative neoplasm

Micro RNA (miRNA) are small non-coding RNA molecules which have a post-transcriptional inhibitory regulation function, e.g. in megakaryopoiesis. A characteristic of Philadelphia chromosome-negative myeloproliferative neoplasm (Ph− MPN) is the abundance of morphologically aberrant megakaryocytes. Based on previously published in vitro megakaryocytic differentiation assay data, we selected miRNA 10a, 17-5p, 20a and 126 and potential target proteins (HOXA1, RUNX1) for analysis of laser-microdissected bone marrow megakaryocytes from Ph− MPN and controls (n = 66). Furthermore, we tested a potential influence of cytoreductive treatment on miRNA expression in bone marrow cells during the course of Ph− MPN (n = 18). In summary, miRNA 17-5p, 20a and 126 are constitutively expressed in Ph− MPN megakaryopoiesis while low or absent miRNA 10a appeared to correlate with strong megakaryocytic HOXA1 protein expression. No association to thrombocytosis, JAK2V617F mutations or cytoreductive treatment (bone marrow cells) were observed.

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