The role of adhesive interactions with the extracellular matrix components of the bone marrow (BM) stroma has been widely studied in the differentiation of erythroid and myelomonocytic cells, but not ...in the megakaryocytic lineage. The development of efficient culture techniques for the production of megakaryocytes (Mks) from CD34+ purified BM cells, enables the study of the expression and function of adhesion receptors for collagen (VLA‐2), fibronectin (VLA‐4 and VLA‐5) and laminin (VLA‐6) during the maturation of Mks. We have shown that a significant percentage of CFU‐MK (roughly 20%) adhere to fibronectin but not to collagen and laminin. The expression and adhesion of Mks developing in liquid culture from BM‐CD34+ cells were tested at days 4, 7 and 10 of incubation. The expression of VLA‐2, VLA‐5 and VLA‐6 on day 10 cultured Mks enabled purification of intermediate and large polyploid Mks by FACS sorting whereas VLA‐4 appeared to label only immature Mks and myeloid cells. We observed that only a small proportion of mature Mks was able to adhere to collagen without spreading at day 10 of culture, whereas 30% of Mks adhered to fibronectin as early as day 4 of incubation, 40% of which also attached to laminin. Our data suggest that VLA‐4 may be involved in the adhesion of CFU‐MK and immature Mks on fibronectin, then replaced by VLA‐5 in the final stages of maturation. The expression of VLA‐6 and the number of adherent Mks on laminin increased sharply between day 7 and 10 of incubation. A number of mature polyploid Mks found in day 10 of culture exhibited characteristic features of intense spreading on laminin and fibronectin which were not observed on collagen.
Nanoparticle (NP)-protein interactions in complex samples have not yet been clearly understood. Nevertheless, several studies demonstrated that NP's physicochemical features significantly impact on ...the protein corona composition. Taking advantage of the NP potential to harvest different subsets of proteins, we assessed for the first time the capacity of three kinds of superparamagnetic NPs to highlight the erythrocyte minor proteome. Using both qualitative and quantitative proteomics approaches, nano-liquid chromatography-tandem mass spectrometry allowed the identification of 893 different proteins, confirming the reproducible capacity of NPs to increase the number of identified proteins, through a reduction of the sample concentration range and the capture of specific proteins on the three different surfaces. These NP-specific protein signatures revealed significant differences in their isoelectric point and molecular weight. Moreover, this NP strategy offered a deeper access to the erythrocyte proteome highlighting several signaling pathways implicated in important erythrocyte functions. The automated potentiality, the reproducibility, and the low-consuming sample demonstrate the strong compatibility of our strategy for large-scale clinical studies and may become a standardized sample preparation in future erythrocyte-associated proteomics studies.
The diagnosis of polycythemia vera (PV) is based on clinical and biological criteria defined by either the Polycythemia Vera Study Group (PVSG) or the World Health Organization (WHO). Both the PVSG ...and WHO PV criteria have proved helpful and are extensively used, yet diagnostic strategies and scheduling of biological investigations vary. We assessed the value of measuring serum erythropoietin (Epo) as a first intention diagnostic test in patients with absolute erythrocytosis (AE).
Serum and bone marrow (BM) samples of 241 patients with a suspicion of erythrocytosis were collected in 8 hospital centers. One hundred and ninety had an absolute erythrocytosis (116 had PV, 66 had secondary erythrocytosis and 4 had idiopathic erythrocytosis). Serum Epo was assayed (ELISA) in 186. Statistical analysis (ROC curves) was used to define serum Epo thresholds that were specific for PV and secondary erythrocytosis and to analyze the diagnostic value of a low or high serum Epo level.
A large majority of PV patients (87% or 101/116) had a serum Epo level below the normal range in healthy patients (3.3 IU/L), giving this value a specificity of 97% with a 97.8% positive predictive value for the diagnosis of PV. Statistical analysis (ROC curves) defined two thresholds allowing a specific and direct diagnosis of 65.6% (65/99) of untreated PV (Epo < 1.4 IU/L) and 19.7% (13/66) of those with secondary erythrocytosis (Epo > 13.7 IU/L).
Based on these data, we propose that measurement of serum Epo level, a simple, reliable and inexpensive test, should be considered as a first intention diagnostic test for patients with absolute erythrocytosis.
The CD4+ CD56+ lin- immunophenotype characterizes rare malignancies, so far considered as arising from the transformation of NK progenitors, and therefore classified as blastic NK-cell ...leukemia/lymphoma by the WHO committee. Recently it was formally demonstrated that such malignancies do, in fact, develop from plasmacytoid dendritic cells (pDC), according to immunophenotypic and functional criteria. The clinico-biological features of this neoplasm were moreover recently summarized from a large series of 23 patients.
The main symptoms at presentation were cutaneous lesions and bone marrow failure, due to invasion by blastic cells, all of which were EBV negative and agranular. Most patients were initially sensitive to chemotherapy regimens, but they rapidly relapsed and died within 3 years. Only 2 allotransplanted patients were long survivors. Recurrent chromosomal aberrations involving chromosomes 5q, 6q, 12p, 13q, 15q and 9 were described and it was characteristic that these were associated in the same cell. In the present review we compared these findings to those in the literature.
The concordant characteristics led us to confirm that this neoplasm actually represents a new entity, that we propose to rename early pDC leukemia/lymphoma. The diagnosis is primarily based on a characteristic immunophenotypic profile i.e. CD4+ CD56+ CD3- CD13- CD33- CD19-. Complementary analyses assessing the expression of more specific pDC-related markers showed the cells to be HLA-DR+, CD123high, CD116low, CD45RA+, BDCA-2+ or BDCA-4+. Such complementary investigations are necessary only in the case of an atypical phenotype, in order to confirm a pDC origin and exclude another hematologic disease. This presently regards the expression of CD33 or cytoplasmic CD3e (cyCD3e) and the negativity of CD56.
Recent advances in myeloproliferative neoplasms (MPN) have highlighted the prevalence of mutations in the calreticulin gene (CALR), bringing a major new actor in these disorders. CALR mutations were ...reported in 25% of ET and in 35% of MF patients, which were non-mutated for JAK2 and MPL. CALR mutations lead to a frame-shift generating a common 36 amino acids C-terminal end and loss of the KDEL motif. Two variants account for 85% of the CALR mutations in ET and PMF: type 1, a 52-bp deletion and type2, a 5-bp insertion.
572 MPN patients negative for JAK2 and MPL mutations were collected from several French and Belgian hospitals. In our series, 396 patients were diagnosed as ET, 108 as MF and 68 as mixed MDS/MPN. We identified mutations of CALR in 368 patients (63.3%). The remaining 204 patients were designated as triple negative. In MF there was an over representation of type 1 mutation (70%) and an under representation of type 2 mutation (13%) as compared to patients with ET. This bias was associated with a higher allelic burden of CALR mutation in MF.
MF patients represent a quite homogeneous group, mostly composed of men diagnosed at a median age of 62.5 with a low hemoglobin concentration (10.1 g/dl) and a low platelet count (median at 237 x 109/l). In ET patients the clinical presentation was more heterogeneous. They were mostly women (more than 61%) at a median age of diagnosis of 57 with a median platelet count of 724 x 109/l. In CALR mutated patients there were no sex prevalence and a more important thrombocytosis (785 x 109/l). The type of CALR mutation impacted also age and platelet count.
We report the caracterisation of triple negative patients. In ETs they were mostly women (76.9%), particularly for ET patients under 50 years old that were almost exclusively women (27/28). In MF, triple negative patients presented a low hemoglobin concentration (8.85 g/dl) and a low leukocyte count (1.995 x 109/l). A striking characteristic is their platelet count, which was significantly lower than their group mates either in ET or in MF. This lower platelet count may suggest that in the general population, putative asymptomatic triple negative ET male patients could be retrieved, which would only be diagnosed at more advanced age with a symptomatic MF.
Taken together, our results underline the differences between the two most frequent types of CALR mutation and show that CALR mutated patients should not be considered as a single entity.
No relevant conflicts of interest to declare.
Abstract 3856
The gene and protein expression profiles in myeloproliferative neoplasms (MPN) may reveal gene markers of a potential clinical function in diagnosis and prediction of response to ...therapy. Using cDNA microarray analysis, involving 25,100 unique genes, we studied the gene expression profile of hematopoietic CD34+ progenitor cells and granulocytes obtained from peripheral blood of patients with essential thrombocythemia (ET), polycythemia vera (PV) and primary myelofibrosis (PMF) compared with healthy individuals. The microarray analyses of the hematopoietic progenitor cells and granulocytes have been performed on 9 patients with ET, 8 patients with PV, 4 patients with PMF and 8 healthy donors. The granulocytes for proteomic studies have been pooled in 4 groups: PV with JAK2 mutant allele burden above 80%, ET with JAK2 mutation, PMF with JAK2 mutation and ET/PMF with no JAK2 mutation. We focused our analysis to hematopoiesis related genes. In the collected patient samples, the increased number of granulocytes allowed for further validation by protein analysis of microarray gene expression suggested from less differentiated hematopoietic progenitor cells. Folate receptor 3 (FOLR3), constitutively secreted in hematopoietic tissues, has increased protein levels in granulocytes of JAK2V617F homozygous PV as well as mRNA levels in hematopoietic progenitor cells of patients with PV. The enzyme matrix metallopeptidase 9 (MMP9), involved in IL-8-induced mobilization of hematopoietic progenitor cells from bone marrow, also has significantly increased protein levels in granulocytes of PV patients with increased JAK2 mutation allele burden. In addition, Ras-related C3 botulinum toxin substrate 2 (RAC2) protein level, essential for erythropoiesis, is increased specifically in PV granulocytes with JAK2V617F homozygosity. Moreover, RAC2 gene expression is significantly increased in hematopoietic progenitor cells of PV, with no changes in its granulocytes. Although, like PV, RAC2 gene expression was also increased in ET and PMF hematopoietic progenitor cells compared to healthy individuals, in granulocytes of ET and PMF patients with JAK2 mutation RAC2 protein levels were decreased, contrary to the elevated level in PV. Furthermore, inconsistent with JAK2V617F homozygous PV patients, granulocytes of ET and PMF with the JAK2 mutation exhibit FOLR3 protein at levels lower than the ET and PMF with no JAK2 mutation. Investigating the extent to which these genes participate in the complex molecular and cellular mechanisms of MPN will likely lead to new insights of malignancy development. In conclusion, molecular profiling of hematopoietic progenitor cells and granulocytes of MPN patients revealed gene expression patterns that are beyond their recognized function in disease pathogenesis and can be related to patients' clinical characteristics with imminent prognostic relevance.
No relevant conflicts of interest to declare.
Abstract Gliomas are extremely aggressive and lethal forms of brain cancer. Unlike many other cancer types, glioma cells rarely metastasize. They spread throughout the brain and invasiveness of ...glioma cells is a major cause of therapeutic failure. In plant ecosystem, biodiversity acts locally as a barrier to ecological invasion. By analogy, we hypothesize that the low cell diversity of differentiated tissues, a counterpart of their functional specificity, opens the way to local cancer cell invasion. Seeding the brain tumor microenvironment with heterogeneous cell populations could be a mean to limit cancer cell invasion by enhancing cell biodiversity.
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