First, a cohort of 103 JAK2 V617F-negative ET cases diagnosed between 1980 and 2013 at several Belgian hospitals was collected and analyzed for CALR and MPL mutations. We also collected a control cohort of 57 JAK2 V617F-positive ET patients diagnosed between 1987 and 2009 in the University Hospitals Leuven. The median follow-up of the whole cohort of 160 patients was 8 years (range, 1-34 years). Hematologic parameters (platelet counts, erythrocyte counts, leukocyte counts, hemoglobin, and hematocrit) at diagnosis were retrieved as was information on cardiovascular events and complications (arterial thrombosis, and venous events). During follow-up, progression to myelofibrosis, progression to acute myeloid leukemia, need for cytoreductive treatment, and presence of splenomegaly were recorded.

Supplementary data are available at Bioinformatics online.

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Introduction: The JAK2 p.V617F, MPL p.W515K/L and CALR indels occur in a mutually exclusive pattern in 80-90% of cases with Essential Thrombocythemia (ET), but the driver mutations are unknown in the remaining 10-20%. In this study we aimed to identify driver mutations in the latter group of triple negative (TN) ET by exome sequencing of 10 such cases. Results: We found 27 somatic variants, including indels, in 6 out of 10 TN ET patients (range: 1-10 mutations/case; mean: 2,7 mutations/case), none of which were recurrent. In one case, we found a MPL c.610T>C (p.S204P) mutation, which is located in the extracellular domain of the MPLreceptor. By Sanger sequencing of MPL exon 4 in 20 additional TN ET cases, an additional patient with the MPL S204P mutation was identified. Moreover, this mutation was previously reported in one case with idiopathic myelofibrosis1. In order to study the effect of this mutation on the function of MPL, we produced stable Ba/F3 cell lines expressing MPL S204P, MPL W515K or MPL WT, and assessed the dependence of their growth on exogenous thrombopoietin (TPO). Only MPL W515K transduced Ba/F3 cells proliferated in the absence of TPO, but growth of MPL S204P Ba/F3 and of MPL WT Ba/F3 could be rescued by exogenous TPO, indicating the proper surface expression and the functionality of the transduced receptors. The levels of phospho-JAK2 and phospho-STAT5 were low in cytokine-deprived MPL S204P cells but increased upon TPO stimulation. In contrast, phospho-JAK2 and phospho-STAT5 were detectable in MPL W515K transduced Ba/F3 in the absence of cytokines as assessed by Western blotting. Culture of MPL S204P transduced Ba/F3 in the presence of TPO over a range of concentrations (0,01-10 ng/ml) yielded growth curves comparable with MPL WT transduced Ba/F3. Using flow cytometry, we also explored cell surface marker expression on peripheral blood platelets from the two MPL S204P ET patients. Data were compared with healthy donors or ET patients with JAK2 or CALR mutations. MPL S204P ET platelets displayed higher expression of CD61 than platelets from healthy donors or from JAK2 or CALR mutated ET (p<0,01). In addition, there was a trend for higher expression of KIT, CD36 and CD42b on platelets from the MPL S204P ET cases. Moreover, following platelet activation through the protease activated receptor 1, the degranulation response of platelets from MPL S204P ET was decreased in comparison with JAK2 or CALR mutated ET. Conclusion: The MPL S204P mutation is a recurrent mutation in TN ET, with a frequency of 7% (2/30) in this series, but this mutation does not induce TPO-independent growth nor increased TPO-sensitivity in Ba/F3 cells. However, preliminary phenotypic and functional evidence supports the notion that MPL S204P platelets display specific characteristics as compared with JAK2 or CALR mutated ET. The mechanisms by which the MPL S204P mutation influences megakaryopoiesis and platelet function remain to be elucidated. 1. Williams DM, et al. Phenotypic variations and new mutations in JAK2 V617F-negative polycythemia vera, erythrocytosis, and idiopathic myelofibrosis. Exp Hematol 2007; 35: 1641. Disclosures Graux: Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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Background Essential thrombocythemia (ET) is a myeloproliferative neoplasm featured by a sustained elevation of platelet count and a tendency for thrombosis and hemorrhage. Cytogenetic abnormalities are rare in ET. The most common molecular abnormality in ET is JAK2 V617F, found in approximately 50% of ET cases followed by MPL W515K/L, found in about 10% of cases. The molecular cause of the remaining ET cases is still largely unknown. As such, in a substantial fraction of ET cases, the underlying molecular cause is yet to be discovered. In a recent study by Hou et al., single cells derived from an ET JAK2 V617F-negative ET patient were sequenced using a method based on exome sequencing. Eight genes were identified as possible candidate drivers. However, their recurrence rate in ET was not established. Aims To establish the recurrence rate in JAK2 V617F-negative and MPL W515K/L-Negative ET of potential candidate driver mutations, as identified by Hou et al. Methods and Results We studied unfractionated blood or bone marrow samples from a series of 64 cases of JAK2 V617F-negative and MPL W515K/L-negative ET. In this series, we used PCR and Sanger sequencing to detect the following mutations: SESN2 P87S, TOP1MT S479L, ST13 Q349*, and DNAJC17 A292P, as they exhibited the highest scores in the study of Hou et al. In addition, we included NTRK1 N323S, a mutant tyrosine kinase. None of the mutations reported by Hou et al. was detected in our patients. However, we identified a novel acquired heterozygous mutation in TOP1MT (c.1400 A>G, p.N467S) which is predicted to be damaging by polyphen 2. This mutation was not detected in the germline DNA from the buccal swab of the patient. TOP1MT is a mitochondrial topoisomerase encoded by the genomic DNA. It is a type IB enzyme, which sustains the appropriate conformation of DNA during replication, transcription, recombination, and repair. This mutation might affect the interaction of TOP1MT with the DNA molecule as suggested by the results of in silico analysis from I-Tasser. p.N467S mutation causes the gain of a helix and the loss of a β strand which are in close proximity to the bound DNA molecule. We screened exon 11 of TOP1MT gene in 38 additional JAK2 V617F-negative MPL W515K/L-negative ET cases, but did not find any additional cases. Conclusions In this series of 102 cases of JAK2 V617F-negative and MPL W515K/L-negative ET, only one case was identified with a mutation of TOP1MT. Mutations of SESN2, ST13, DNAJC17, or NTRK1, four other candidate driver genes as identified by Hou et al., could not be identified in a series of 64 cases. The functional role of TOP1MT in the pathogenesis of ET remains to be established. The absence of the mutations, as proposed by Hou et al., in our cohort raises questions about their role as potential driver mutations in JAK2 V617F-negative and MPL W515K/L-negative ET. The quest for the full complement of driver mutations in ET therefore remains open. Reference Hou, Y., et al., Single-cell exome sequencing and monoclonal evolution of a JAK2-negative myeloproliferative neoplasm. Cell, 2012. 148(5): p. 873-85. Disclosures: No relevant conflicts of interest to declare.

<b><i>Aim:</i></b> The aim of the study was to assess the acceptance of patients with opioid use disorder (OUD) to switching their opioid dependence treatment (ODT) for a prolonged-release buprenorphine (PRB) injection according to their prior ODT (buprenorphine/naloxone [B/N] or methadone). <b><i>Methods:</i></b> This was an observational, retrospective/cross-sectional, multicentre study of adult patients diagnosed with OUD on ODT. Data collected from diaries were analysed to know their interest and opinion on PRB. Questions with fixed response options were included, and several Likert scales were used. <b><i>Results:</i></b> A total of 98 patients were enrolled (B/N: 50.0%, methadone: 50.0%). The mean age was 46.9 ± 8.43 years and 79.6% were males. PRB was similarly perceived by both groups in most variables analysed, receiving a mean score of 7.2/10 (B/N: 7.4, methadone: 7.0; <i>p</i> = 0.520), and approximately 65% of patients said they were willing to switch to PRB (B/N: 63.3%, methadone: 65.3%; <i>p</i> = 0.833). Of these, a higher percentage in the B/N group considered that switching would be easy/very easy (B/N: 90.3%, methadone: 46.9%; <i>p</i> &#x3c; 0.001) and that they would start PRB when available (B/N: 64.5%, methadone: 34.3%; <i>p</i> = 0.005). More than 90% would prefer the monthly injection (B/N: 93.6%, methadone: 100%; <i>p</i> = 0.514). One-third of patients in both groups were unsure/would not switch their ODT to PRB (B/N: 36.7%, methadone: 34.7%; <i>p</i> = 0.833). The main reason was administration by injection. <b><i>Conclusion:</i></b> Two-thirds of patients would switch their treatment for PRB, and most patients on B/N considered that switching would be easy. PRB could be a suitable alternative for OUD management.

Motivation: Predict whether a mutation is deleterious based on the custom 3D model of a protein. Methods:We have developed modict, a mutation prediction tool which is based on per residue rmsd (root mean square deviation) values of superimposed 3D protein models. Our mathematical algorithm was tested for 42 described mutations in multiple genes including renin, beta-tubulin, biotinidase, sphingomyelin phosphodiesterase-1, phenylalanine hydroxylase and medium chain Acyl-Coa dehydrogenase. Moreover, modict scores corresponded to experimentally verified residual enzyme activities in mutated biotinidase, phenylalanine hydroxylase and medium chain Acyl-CoA dehydrogenase. Several commercially available prediction algorithms were tested and results were compared. The modict perl package and the manual can be downloaded from https://github.com/MODICT/MODICT. Conclusion:We show here that modict is capable tool for mutation effect prediction at the protein level, using superimposed 3D protein models instead of sequence based algorithms used by polyphen and sift.