Is Hematopoietic Clonality of Indetermined Potential a Risk Factor for Pulmonary Embolism?

Background Unprovoked pulmonary embolism (uPE) is a severe and frequent condition. Identification of new risk factors is mandatory to identify patients that would benefit from a long-term treatment. Clonal hematopoiesis of indeterminate potential (CHIP) is defined by the acquisition of somatic mutations that drive clonal expansion in the absence of cytopenia. Its prevalence is estimated of 5% in the population above 65 years. Since inflammation and endothelial dysfunction may share a pathophysiological pathway(1), we hypothesized that CHIP, may be a risk factor for uPE. Methods We conducted a pilot retrospective observational study. Patients with iPE between 18 to 65 years old were included. PE was considered as unprovoked, when no transient nor persistant risk factor was present and when thrombophilia testing was negative. We excluded documented atherosclerosis, personal or familial history of VTE and presence of cytopenias. CHIP proportion in uPE patients were analyzed using next generation sequencing of the coding sequence of a custom panel composed by DNMT3A, ASXL1, SF3B1, TET2 and TP 53 . Results Upon 61 patients with uPE consecutively included, a total of 19 somatic mutations were found in 12 patients (20%) IC95% [10 - 20]. 15 mutations were found in DNMT3A gene, 3 in ASXL1 and one in TET2 . There was no diference in terms of age, PE location, DVT presence and risk stratification in CHIP carriers and non carriers. Conclusion We report for the first time, the presence of high rates of CHIP in patients presenting with uPE. Thus, CHIP may be a new risk factor for VTE. These results need to be confirmed in an ongoing prospective case-control study including more patients and using a more diverse gene panel to better determine CHIP incidence in uPE.

[Immortalization of erythroid progenitors for in vitro large-scale red cell production]. / Immortalisation érythrocytaire pour production de globules rouges in vitro.

Population ageing and increase in cancer incidence may lead to a decreased availability of red blood cell units. Thus, finding an alternative source of red blood cells is a highly relevant challenge. The possibility to reproduce in vitro the human erythropoiesis opens a new era, particularly since the improvement in the culture systems allows to produce erythrocytes from induced-Pluripotent Stem Cells (iPSCs), or CD34+ Hematopoietic Stem Cells (HSCs). iPSCs have the advantage of in vitro self-renewal, but lead to poor amplification and maturation defects (high persistence of nucleated erythroid precursors). Erythroid differentiation from HSC allows a far better amplification and adult-like hemoglobin synthesis. But the inability of these progenitors to self-renew in vitro remains a limit in their use as a source of stem cells. A major improvement would consist in immortalizing these erythroid progenitors so that they could expand indefinitively. Inducible transgenesis is the first way to achieve this goal. To date, the best immortalized-cell models involve strong oncogenes induction, such as c-Myc, Bcl-xL, and mostly E6/E7 HPV16 viral oncoproteins. However, the quality of terminal differentiation of erythroid progenitors generated by these oncogenes is not optimal yet and the long-term stability of such systems is unknown. Moreover, viral transgenesis and inducible expression of oncogenes raise important problems in term of safety, since the enucleation rate is not 100% and no nucleated cells having replicative capacities should be present in the final product.

DEK-CAN molecular monitoring of myeloid malignancies could aid therapeutic stratification.

The t(6;9)(p23;q34) is a recurrent chromosomal abnormality observed in 1% of acute myelogenous leukemia (AML), which generates a fusion transcript between DEK and CAN/NUP214 genes. We used a DEK-CAN real-time quantitative (RQ)-PCR strategy to analyze 79 retrospective and prospective samples from 12 patients. Five patients reached DEK-CAN negativity (sensitivity 10(-5)); all underwent early allogeneic hematopoietic stem cell transplantation (median 5.5 months from diagnosis) with some demonstrating molecular positivity at the time of allograft. All four cases in CCR with adequate follow-up (median 18.5 months, range 13--95) demonstrate persistent molecular negativity, whereas all seven patients with persistent DEK-CAN positivity died at a median of 12 months from diagnosis (range 7--27). We conclude that DEK-CAN molecular monitoring by RQ-PCR in t(6;9) malignancies is a useful tool for individual patient management and that molecular negativity is indispensable for survival, but should not be a prerequisite for allografting in this rare, poor prognosis, subset of AML.

Zyxin is up-regulated during megakaryocytic differentiation of human UT-7/c-mpl cells.

To characterize genes involved in megakaryocytic commitment, we compared expression profiles of bipotent cells (UT-7/c-mpl) with those of the same cells induced to differentiate towards megakaryopoiesis in the presence of TPO. Using cDNA arrays, we showed that 12 out of 2260 genes changed their expression level after 6h of TPO stimulation. One of these genes encodes for zyxin, a cytoskeleton protein component. Zyxin is up-regulated at the mRNA and protein levels in UT-7/c-mpl cells in response to TPO confirming the reliability of the cDNA array technology. Similarly, when CD34 positive cells were induced to differentiate into megakaryocytes, zyxin mRNA was accumulated. Furthermore, when megakaryocytes were allowed to spread on fibrinogen, formation of stress fibers and lamellipodia was induced and zyxin was localized at the picks of actin stress fibers. These results suggest an important role for zyxin during megakaryocytic differentiation and more precisely in the regulation of the integrin mediated adhesion process in megakaryocytes.

Targeting the kinesin Eg5 to monitor siRNA transfection in mammalian cells.

RNA interference, the inhibition of gene expression by double-stranded RNA, provides a powerful tool for functional studies once the sequence of a gene is known. In most mammalian cells, only short molecules can be used because long ones induce the interferon pathway. With the identification of a proper target sequence, the penetration of the oligonucleotides constitutes the most serious limitation in the application of this technique. Here we show that a small interfering RNA (siRNA) targeting the mRNA of the kinesin Eg5 induces a rapid mitotic arrest and provides a convenient assay for the optimization of siRNA transfection. Thus, dose responses can be established for different transfection techniques, highlighting the great differences in response to transfection techniques of various cell types. We report that the calcium phosphate precipitation technique can be an efficient and cost-effective alternative to Oligofectamine in some adherent cells, while electroporation can be efficient for some cells growing in suspension such as hematopoietic cells and some adherent cells. Significantly, the optimal parameters for the electroporation of siRNA differ from those for plasmids, allowing the use of milder conditions that induce less cell toxicity. In summary, a single siRNA leading to an easily assayed phenotype can be used to monitor the transfection of siRNA into any type of proliferating cells of both human and murine origin.

[Determination of total plasma homocysteine and other aminothiols by liquid chromatography coupled to the detection by fluorescence]. / Dosage de l'homocystéine plasmatique totale et autres aminothiols par chromatographie liquide couplée à la détection par fluorescence.

Plasma concentrations of homocysteine, cysteine, cysteinylglycine and glutathione were measured using a HPLC technique with fluorescence detection of the derivatives obtained with 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide (ABD-F). Blood was drawn into chilled EDTA-evacuated tubes. After centrifugation at 4 degrees C without delay, plasma samples were kept frozen at -20 degrees C until analysis. Reduction of protein bound aminothiols and disulfides standards was achieved with tri-n-butylphosphine. N-acetylcysteine was used as internal standard. After protein precipitation, derivatization was carried out at pH 8.0 and 50 degrees C for 20 min. Stability of ABD-thiols was ensured for at least 5 days by lowering pH to 2. Derivatives were separated by isocratic elution on a Waters mu Bondapak C18 column (10 microns, 3.9 x 300 mm) with 0.1 M phosphate buffer pH 3.2 containing 10% acetonitrile. Excitation and emission wavelengths were 385 and 515 nm. Retention times were 4.9, 5.8, 7.3, 9.9 and 20.1 min respectively for cysteine, cysteinylglycine, homocysteine, glutathione and N-acetylcysteine. Peaks were quantified by comparison to a standard curve prepared by plotting peak height versus the different levels of known standard solutions after normalization with internal standard. Between-run CVs varied from 5 to 8.5%. The detection limit was < 0.5 mumol/l for homocysteine and glutathione. In plasma samples from healthy subjects, concentration of homocysteine was higher in men than in women (11.0 +/- 2.9 versus 9.2 +/- 2.7 mumol/l, p < 0.01). These values are similar to those obtained with other widely used methods.

[About 500 carcinomas of the skin of the face (author's transl)]. / Cancers cutanés de la face

After having studied etiological and histological cases of 500 malignant growth of the face's skin the authors think that: -- carcinomas of lips and ears are chiefly men's cancers and above all squamous cell-tumors. Ear-tumors beeing serious. For the other parts of the face, cancers are more frequently met in females than in males. Squamous cell-tumors are perhaps more frequent than usually said; -- as for treatment the authors had rather use surgery with immediate plastic reconstruction.