Jérémie Riou

Clinical and biological characterization of MPN patients harboring two driver mutations, a French intergroup of myeloproliferative neoplasms (FIM) study

To the Editor: Mutations in “driver” genes (JAK2, MPL, or CALR) are found in the vast majority of Philadelphia-negative myeloproliferative neoplasms (MPN): JAK2V617F or exon 12 mutations in polycythemia vera (PV), JAK2V617F, CALR exon 9, or MPL exon 10 mutations in essential thrombocythemia (ET) or primary myelofibrosis (PMF). In the latter, phenotypic presentation and evolutive course vary according to the mutation, eg, higher platelet counts in CALR mutated ET patients and higher hemoglobin, white blood cell counts and higher risk of thrombosis in JAK2-mutated ET. Driver mutations were initially described as mutually exclusive, but can co-exist in rare “doubly mutated” (DM) patients. We have previously shown that double mutations were more frequently encountered in patients with low (<5%) JAK2V617F allelic burdens. However, due to the limited reports of DM patients, it is unclear how co-occurrence of driver mutations affects the presentation or evolution of MPN. In order to confirm DM frequency in a multicentric fashion, two additional cohorts of JAK2-mutated MPN were screened for CALR mutations: 134 JAK2V617F-mutated ET from the University Hospital of Cr eteil (France) and a cohort of 31 MPN with a low JAK2V617F allele burden (<2%) from the University Hospital of Nantes (France). Five of 49 patients with a JAK2V617F allele burden <5% also had a CALR mutation (10.2%), similar to our previous findings (14.3%) versus none of the 119 patients with JAK2V617F>5%. This confirms the relatively high frequency of DM in patients with low JAK2V617F burden. In addition, clinical and biological data were gathered on 21 DM patients discovered fortuitously in 4 other French centers. Overall, 47 cases of DM patients (Supporting Information Figure 1) were analyzed, of which 28 harbored a JAK2V617F allele burden equal to or below 1% (60%) while only 2 (5%) had a JAK2V617F allele burden above 5%. Among the 47 DM patients, 40 were diagnosed with ET, 5 with PMF, 1 with PV, and 1 with MDS/MPN-RS-T. Thirty-two patients (68%) presented with JAK2V617F and CALR mutations, 11 (23%) with JAK2V617F and MPL mutations, 2 with CALR and MPL mutations, 1 PV patient had JAK2V617F and JAK2 exon 12 mutations, and 1 patient had an association of two different CALR mutations. In order to know whether a double mutation impacts the clinical presentation or the evolutive course, the 40 DM patients with ET were compared to a control cohort comprising 577 “classical” ET patients from OBENE cohort, NCT02897297. Patients’ characteristics were compared using Fisher exact test for categorical variables as well as Mann and Whitney and Kruskal–Wallis (followed by Dunn tests for multiples comparisons) tests for continuous variables. For multiple tests, P-values were adjusted with Hochberg’s method. Survival estimates were obtained with the Kaplan–Meier method, and statistical analyses were performed with Log-rank test and Cox model. DM-ET patients were more often males (M/F sex ratio 1.4 [23/17] vs. 0.68 [233/344], P 5 .045) and significantly older than control ET patients (median: 72 vs. 61 years old, P< .001), especially compared to JAK2-singly mutated (SM) (63 years old, P 5 .006), CALR-SM (59 years old, P< .001) or triple-negative (56 years old, P< .001) ET patients. For clinical comparisons, the 37 DM-ET patients with a JAK2V617F mutation (29 with JAK2-CALR and 8 with JAK2-MPL) were compared to ageand sex-matched ET patients (Supporting Information Table 1): each CALR-JAK2V617F DM patient was matched with 3 JAK2V617F and 2 CALR; MPL-JAK2V617F DM with 3 JAK2V617F and 1 MPL-SM ET patients. At diagnosis, DM-ET and CALR or MPL-SM matched control patients had similar hemoglobin and platelet counts, but lower hemoglobin and higher platelet counts than JAK2-SM controls (Figure 1A,B). Leukocyte counts of JAK2-CALR DM-ET patients were intermediate between those of CALR and JAK2-mutated controls, without statistically significant difference (Figure 1C). Splenomegaly at diagnosis was observed in 17% (4/24) of JAK2-CALR DM-ET patients and none of the 6 JAK2-MPL DM-ET for whom this data was available. These frequencies were not significantly different from those observed in respective JAK2, CALR, or MPL SM control groups (Supporting Information Table 1). A history of thrombosis before or at diagnosis was found in 3/16 (19%) of JAK2-CALR DM-ET, which is equivalent to the proportion observed in CALR mutated controls (21%), but lower than that in JAK2 mutated controls (41%), without reaching statistical significance (P5 .27). The JAK2V617F allele burden was much lower in DM-ET patients than in matched controls (median 1% vs. 26%, P< .001, Supporting Information Figure 2A). The relatively frequent discovery of low JAK2V617F burdens in DM patients might be explained by a preexisting clonal hematopoiesis of indeterminate potential (CHIP), followed by the acquisition of a second oncogenic event (CALR or MPL mutation) leading to the MPN phenotype. This would be supported by

Spontaneous nano-emulsification: Process optimization and modeling for the prediction of the nanoemulsion’s size and polydispersity

The aim of the present study was to optimize the size and polydispersity of a lipid nanoemulsion as a function of the oil (Labrafac® WL1349), surfactant (Kolliphor® HS 15) and cosurfactant (Span® 80) phase composition and temperature. The nanoemulsions were prepared using a low-energy self-emulsification method. The Z-average diameter and the polydispersity index (PDI) were modeled with mixture experiments. Nanoemulsions from 20nm to 120nm with PDI<0.2 were obtained at the three different tested temperatures (30°C, 50°C and 90°C). The nanoemulsion size was able to be controlled with the oil, surfactant and cosurfactant concentrations. Interestingly, the smallest PDIs were obtained at 30°C, and the cosurfactant concentration was able to be adjusted to optimize the formulation and to obtain nanoemulsions in the 20-120nm range with a PDI smaller than 0.14. These nanoemulsions have shown a good stability at 4°C in storage conditions and at 37°C in diluted conditions.

Impact of Infection Status and Cyclosporine on Voriconazole Pharmacokinetics in an Experimental Model of Cerebral Scedosporiosis

Cerebral Scedosporium infections usually occur in lung transplant recipients as well as in immunocompetent patients in the context of near drowning. Voriconazole is the first-line treatment. The diffusion of voriconazole through the blood-brain barrier in the context of cerebral infection and cyclosporine administration is crucial and remains a matter of debate. To address this issue, the pharmacokinetics of voriconazole was assessed in the plasma, cerebrospinal fluid (CSF), and brain in an experimental model of cerebral scedosporiosis in rats receiving or not receiving cyclosporine. A single dose of voriconazole (30 mg/kg, i.v.) was administered to six groups of rats randomized according to the infection status and the cyclosporine dosing regimen (no cyclosporine, a single dose, or three doses; 15 mg/kg each). Voriconazole concentrations in plasma, CSF, and brain samples were quantified using ultra-performance liquid chromatography–tandem mass spectrometry and high-performance liquid chromatography UV methods and were documented up to 48 hours after administration. Pharmacokinetic parameters were estimated using a noncompartmental approach. Voriconazole pharmacokinetic profiles were similar for plasma, CSF, and brain in all groups studied. The voriconazole Cmax and area under the curve (AUC) (AUC0 ≥ 48 hours) values were significantly higher in plasma than in CSF [CSF/plasma ratio, median (range) = 0.5 (0.39–0.55) for AUC0 ≥ 48 hours and 0.47 (0.35 and 0.75) for Cmax]. Cyclosporine administration was significantly associated with an increase in voriconazole exposure in the plasma, CSF, and brain. In the plasma, but not in the brain, an interaction between the infection and cyclosporine administration reduced the positive impact of cyclosporine on voriconazole exposure. Together, these results emphasize the impact of cyclosporine on brain voriconazole exposure.

Positive impact of molecular analysis on prognostic scores in essential thrombocythemia: a single center prospective cohort experience

Classical Philadelphia-negative myeloproliferative syndromes (MPN) are characterized by the presence of driver mutations (JAK2, CALR or MPL) and comprise polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF). The prognosis of ET is highly variable from one patient to another and is related to two major risks of complications: an increased risk for thrombosis (the most frequent) and a risk of hematological transformation (the most severe) into secondary myelofibrosis or acute leukemia. Different prognostic scoring systems based on clinical (age, history of thrombosis) and biological factors (leukocyte count, driver mutation) have been developed in ET. Since the milestone study from Vannucchi et al., additional mutations have been clearly demonstrated as important prognostic factors in PMF but, to date, few data are available in ET. In this study, we analyzed prognostic factors and validated scoring systems in a single center cohort of ET patients, and we evaluated the additive impact of NGS analysis. The cohort consisted of 190 consecutive ET patients diagnosed at the Henri Mondor hospital between January 2000 and December 2016 (patients’ clinical and biologic characteristics at diagnosis are summarized in Online Supplementary Table S1). All patients meet the ET WHO 2008 criteria for diagnosis. With a median follow up of 6.4 years, thrombosis occurred in 18 cases (9.4%) and transformation into myelofibrosis, myelodysplastic syndrome or acute myeloid leukemia was observed in 11 cases (5.7%). A total of 16 deaths (8.4%) were observed during the follow up. Driver molecular mutations were as follow: JAK2V617F (allelic burden over 0.5%) in 116 patients (61.1 %), CALR mutations in 27 patients (14.2%, of which 15 are del52 (type 1), 8 ins5 (type 2) and 4 other mutations inducing a similar frameshift (details in Online Supplementary Table S2)) and MPLW515K/Lmutation in 4 patients (2.1%). Triple negative patients represented 22.6% of the cohort (n= 43). CALR mutations were previously associated with a favorable prognosis in ET. In this study we failed to draw a conclusion about the prognostic impact of these mutations, probably due to the small number of CALR mutated cases.

Développement et validation d’une méthode de dosage des traces de détergents inactivants totaux du prion

OBJECTIVES In this study, a novel analytical method to quantify prion inactivating detergent in rinsing waters coming from the washer-disinfector of a hospital sterilization unit has been developed. The final aim was to obtain an easy and functional method in a routine hospital process which does not need the cleaning product manufacturer services. METHODS An ICP-MS method based on the potassium dosage of the washer-disinfectors rinsing waters was developed. Potassium hydroxide is present on the composition of the three prion inactivating detergent currently on the French market. The detergent used in this study was the Actanios LDI(®) (Anios laboratories). A Passing and Bablok regression compares concentrations measured with this developed method and with the HPLC-UV manufacturer method. RESULTS According to results obtained, the developed method is easy to use in a routine hospital process. The Passing and Bablok regression showed that there is no statistical difference between the two analytical methods during the second rinsing step. Besides, both methods were linear on the third rinsing step, with a 1.5ppm difference between the concentrations measured for each method. CONCLUSIONS This study shows that the ICP-MS method developed is nonspecific for the detergent, but specific for the potassium element which is present in all prion inactivating detergent currently on the French market. This method should be functional for all the prion inactivating detergent containing potassium, if the sensibility of the method is sufficient when the potassium concentration is very low in the prion inactivating detergent formulation.