Florence Pasquier

A Senescence-Like Cell-Cycle Arrest Occurs During Megakaryocytic Maturation: Implications for Physiological and Pathological Megakaryocytic Proliferation

During normal megakaryocyte development, in response to thrombopoetin, mature cells enter a senescence-like state in which they shed platelets; this state, characterized by cell cycle arrest, is defective in malignant megakaryocytes.

Presence of atypical thrombopoietin receptor (MPL) mutations in triple negative essential thrombocythemia patients.

Mutations in signaling molecules of the cytokine receptor axis play a central role in myeloproliferative neoplasm (MPN) pathogenesis. Polycythemia vera is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. Here, we studied the mutational profile of 17 ET patients negative for JAK2V617F, MPLW515K/L, and CALR mutations, using whole-exome sequencing and next-generation sequencing (NGS) targeted on JAK2 and MPL. We found several signaling mutations including JAK2V617F at very low allele frequency, 1 homozygous SH2B3 mutation, 1 MPLS505N, 1 MPLW515R, and 2 MPLS204P mutations. In the remaining patients, 4 presented a clonal and 7 a polyclonal hematopoiesis, suggesting that certain triple-negative ETs are not MPNs. NGS on 26 additional triple-negative ETs detected only 1 MPLY591N mutation. Functional studies on MPLS204P and MPLY591N revealed that they are weak gain-of-function mutants increasing MPL signaling and conferring either TPO hypersensitivity or independence to expressing cells, but with a low efficiency. Further studies should be performed to precisely determine the frequency of MPLS204 and MPLY591 mutants in a bigger cohort of MPN.

Myeloproliferative Neoplasms: JAK2 Signaling Pathway as a Central Target for Therapy

The discovery of the JAK2V617F mutation followed by the discovery of other genetic abnormalities allowed important progress in the understanding of the pathogenesis and management of myeloproliferative neoplasms (MPN)s. Classical Breakpoint cluster region-Abelson (BCR-ABL)-negative neoplasms include 3 main disorders: essential thrombocythemia (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). Genomic studies have shown that these disorders are more heterogeneous than previously thought with 3 main entities corresponding to different gene mutations: the JAK2 disorder, essentially due to JAK2V617F mutation, which includes nearly all PVs and a majority of ETs and PMFs with a continuum between these diseases and the myeloproliferative leukemia (MPL) and calreticulin (CALR) disorders, which include a fraction of ET and PMF. All of these mutations lead to a JAK2 constitutive activation. Murine models either with JAK2V617F or MPLW515L, but also with JAK2 or MPL germ line mutations found in hereditary thrombocytosis, have demonstrated that they are drivers of myeloproliferation. However, the myeloproliferative driver mutation is still unknown in approximately 15% of ET and PMF, but appears to also target the JAK/Signal Transducer and Activator of Transcription (STAT) pathway. However, other mutations in genes involved in epigenetics or splicing also can be present and can predate or follow mutations in signaling. They are involved either in clonal dominance or in phenotypic changes, more particularly in PMF. They can be associated with leukemic progression and might have an important prognostic value such as additional sex comb-like 1 mutations. Despite this heterogeneity, it is tempting to target JAK2 and its signaling for therapy. However in PMF, Adenosine Tri-Phosphate (ATP)-competitive JAK2 inhibitors have shown their interest, but also their important limitations. Thus, other approaches are required, which are discussed in this review.

JAK2 and MPL protein levels determine TPO-induced megakaryocyte proliferation vs differentiation

Megakaryopoiesis is a 2-step differentiation process, regulated by thrombopoietin (TPO), on binding to its cognate receptor myeloproliferative leukemia (MPL). This receptor associates with intracytoplasmic tyrosine kinases, essentially janus kinase 2 (JAK2), which regulates MPL stability and cell-surface expression, and mediates TPO-induced signal transduction. We demonstrate that JAK2 and MPL mediate TPO-induced proliferation arrest and megakaryocytic differentiation of the human megakaryoblastic leukemia cell line UT7-MPL. A decrease in JAK2 or MPL protein expression, and JAK2 chemical inhibition, suppress this antiproliferative action of TPO. The expression of JAK2 and MPL, which progressively increases along normal human megakaryopoiesis, is decreased in platelets of patients diagnosed with JAK2- or MPL-mutated essential thrombocytemia and primary myelofibrosis, 2 myeloproliferative neoplasms in which megakaryocytes (MKs) proliferate excessively. Finally, low doses of JAK2 chemical inhibitors are shown to induce a paradoxical increase in MK production, both in vitro and in vivo. We propose that JAK2 and MPL expression levels regulate megakaryocytic proliferation vs differentiation in both normal and pathological conditions, and that JAK2 chemical inhibitors could promote a paradoxical thrombocytosis when used at suboptimal doses.

Clinical efficacy of second generation tyrosine kinase inhibitor and 5-azacytidine combination in chronic myelogenous leukaemia in myeloid blast crisis

Even in the tyrosine kinase inhibitors era, the prognosis of patients with chronic myeloid leukaemia in myeloid blast crisis remains dismal with few patients surviving longer than 6 months. Here we report the cases of 5 patients treated with the combination of 5-azacytidine and tyrosine kinase inhibitors for myeloid blast crisis CML. All patients achieved a complete haematological response including two with a complete cytogenetic and major molecular response. Two patients underwent an allogeneic stem cell transplantation. One died from relapse 34 months from diagnosis. The second is alive and free from disease at 11 months from diagnosis. The other 3 patients are still in complete haematological response after 15, 24 and 33 months of follow-up. These results suggest that the combination has a significant activity in myeloid blast crisis and may increase survival.

Monitoring antibiotic-resistant enterobacteria faecal levels is helpful in predicting antibiotic susceptibility of bacteraemia isolates in patients with haematological malignancies.

Delay of active antimicrobial therapy in haematological patients with Gram-negative bacilli bacteraemia during profound neutropenia exposes them to increased morbidity and mortality. The digestive tract is the main source of enterobacteria causing bacteraemia in these patients. We thus evaluated the usefulness of broad-spectrum beta-lactam resistant enterobacteria (BSBL-RE) faecal shedding assessment in forecasting the susceptibility to BSBLs of the strains isolated from blood cultures. From 2002 to 2011, neutropenic haematological patients with bacteraemia caused by enterobacteria who had a stool culture during the previous 7u200adays were retrospectively included. BSBL-RE intestinal carriers were compared with non-carriers in terms of clinical and microbiological criteria. One hundred and four patients were included and 16 of them (15.4u200a%) were BSBL-RE carriers. Multivariate analysis showed that BSBL-RE carriage was independently associated with BSBL-RE identified in blood cultures (Pu2009<u20090.001) and the use of carbapenems as empirical treatment of the bacteraemia (Pu2009=u20090.008). Sensitivity, specificity, and the positive and negative predictive values of the test were 80u200a%, 91u200a%, 50u200a% and 98u200a%, respectively. Among the carriers, those with the highest level of BSBL-RE carriage were also those with the highest risk of bacteraemia due to BSBL-RE (Pu2009<u20090.001). Close monitoring of BSBL-RE intestinal carriage may help to choose the most appropriate initial antimicrobial treatment for neutropenic haematological patients with bacteraemia.

Hyperinsulinemic hypoglycemia without insulinoma: Think of activating glucokinase mutation

Congenital hyperinsulinism (CHI) is a rare condition that can lead to persistent severe hypoglycemia, mostly discovered in neonates and infants, exceptionally in adulthood [1,2]. The present report describes a case of CHI discovered in adult, mimicking insulinoma but without radiological abnormalities, leading to the identification of a novel Glucokinase (GCK) activating mutation. A 25-year-old woman without significant medical history was referred for uneasiness with a random blood glucose measurement of 45 mg/dL and HbA1c of 4.2% (n < 6.5). She had noticed fluctuating episodes of mid-morning tiredness, hunger, headaches relieved by food, and night sweats since childhood. These symptoms occurred both during fasting and after eating. She was not taking any medication. The physical examination was normal. Her BMI was 26.2 kg/m and stable. An oral glucose tolerance test (OGTT) revealed hyperinsulinaemic hypoglycemia 120 min post-stimulation (glucose 37 mg/dL, insulin 30 mIU/L [normal: 2.5–10.4], C-peptide 6.19 ng/mL [normal: 0.95–2.30]). A fasting test (table SI) induced an episode of uneasiness after 32 h. Of note was that the glucose level remained between 43 and 57 mg/dL without any complaint, and no lower values. Biological investigations eliminated iatrogenic, metabolic, adrenal or pituitary causes of hypoglycemia. Abdominal CT scan, pancreatic MRI, endoscopic ultrasound, octreoscan scintigraphy and fluorine-18-6-fluoro-L-dopa PET scan, revealed no insulinoma. Continuous glucose monitoring (CGM) (figure 1A) showed a low mean glucose, especially during the night, reproducible day-to-day, but with no severe hypoglycaemia. A CHI Next-Generation-Sequencing (NGS) analysis revealed a heterozygous mutation (c.538A > G; p.Asn180Asp) of the GCK gene, never reported in literature. Bioinformatics prediction systems (SIFT, Polyphen) argued for a pathogenicity of this variant.

New pathogenic mechanisms induced by germline erythropoietin receptor mutations in primary erythrocytosis

Primary familial and congenital polycythemia is characterized by erythropoietin hypersensitivity of erythroid progenitors due to germline nonsense or frameshift mutations in the erythropoietin receptor gene. All mutations so far described lead to the truncation of the C-terminal receptor sequence that contains negative regulatory domains. Their removal is presented as sufficient to cause the erythropoietin hypersensitivity phenotype. Here we provide evidence for a new mechanism whereby the presence of novel sequences generated by frameshift mutations is required for the phenotype rather than just extensive truncation resulting from nonsense mutations. We show that the erythropoietin hypersensitivity induced by a new erythropoietin receptor mutant, p.Gln434Profs*11, could not be explained by the loss of negative signaling and of the internalization domains, but rather by the appearance of a new C-terminal tail. The latter, by increasing erythropoietin receptor dimerization, stability and cell-surface localization, causes pre-activation of erythropoietin receptor and JAK2, constitutive signaling and hypersensitivity to erythropoietin. Similar results were obtained with another mutant, p.Pro438Metfs*6, which shares the same last five amino acid residues (MDTVP) with erythropoietin receptor p.Gln434Profs*11, confirming the involvement of the new peptide sequence in the erythropoietin hypersensitivity phenotype. These results suggest a new mechanism that might be common to erythropoietin receptor frameshift mutations. In summary, we show that primary familial and congenital polycythemia is more complex than expected since distinct mechanisms are involved in the erythropoietin hypersensitivity phenotype, according to the type of erythropoietin receptor mutation.

Dissociated responses to newer antimyeloma drugs identify a subset of refractory patients with an extremely poor prognosis

We describe the striking and unexpected evolution of 10 patients with de novo multiple myeloma treated with novel-agent based induction therapy, who displayed a dissociated evolution characterised by an apparent good response contrasting with the concomitant development of aggressive non-secreting plasmocytomas immediately before, or just after, autologous stem cell transplantation. Patients did not respond to salvage therapies. Eight of them died from progression less than 12 months after diagnosis. This unusual evolution in the era of novel agents warrants further scrutiny.