Constitutive activation and oncogenicity are mediated by loss of helical structure at the cytosolic boundary of thrombopoietin receptor mutant dimers.

Dimerization of the thrombopoietin receptor (TpoR) is necessary for receptor activation and downstream signaling through activated Janus kinase 2. We have shown previously that different orientations of the transmembrane (TM) helices within a receptor dimer can lead to different signaling outputs. Here we addressed the structural basis of activation for receptor mutations S505N and W515K that induce myeloproliferative neoplasms. We show using in vivo bone marrow reconstitution experiments that ligand-independent activation of TpoR by TM asparagine (Asn) substitutions is proportional to the proximity of the Asn mutation to the intracellular membrane surface. Solid-state NMR experiments on TM peptides indicate a progressive loss of helical structure in the juxtamembrane (JM) R/KWQFP motif with proximity of Asn substitutions to the cytosolic boundary. Mutational studies in the TpoR cytosolic JM region show that loss of the helical structure in the JM motif by itself can induce activation, but only when localized to a maximum of six amino acids downstream of W515, the helicity of the remaining region until Box 1 being required for receptor function. The constitutive activation of TpoR mutants S505N and W515K can be inhibited by rotation of TM helices within the TpoR dimer, which also restores helicity around W515. Together, these data allow us to develop a general model for activation of TpoR and explain the critical role of the JM W515 residue in the regulation of the activity of the receptor.

Secreted mutant calreticulins as rogue cytokines in myeloproliferative neoplasms.

Mutant calreticulin (CALR) proteins resulting from a -1/+2 frameshifting mutation of the CALR exon 9 carry a novel C-terminal amino acid sequence and drive the development of myeloproliferative neoplasms (MPNs). Mutant CALRs were shown to interact with and activate the thrombopoietin receptor (TpoR/MPL) in the same cell. We report that mutant CALR proteins are secreted and can be found in patient plasma at levels up to 160 ng/mL, with a mean of 25.64 ng/mL. Plasma mutant CALR is found in complex with soluble transferrin receptor 1 (sTFR1) that acts as a carrier protein and increases mutant CALR half-life. Recombinant mutant CALR proteins bound and activated the TpoR in cell lines and primary megakaryocytic progenitors from patients with mutated CALR in which they drive thrombopoietin-independent colony formation. Importantly, the CALR-sTFR1 complex remains functional for TpoR activation. By bioluminescence resonance energy transfer assay, we show that mutant CALR proteins produced in 1 cell can specifically interact in trans with the TpoR on a target cell. In comparison with cells that only carry TpoR, cells that carry both TpoR and mutant CALR are hypersensitive to exogenous mutant CALR proteins and respond to levels of mutant CALR proteins similar to those in patient plasma. This is consistent with CALR-mutated cells that expose TpoR carrying immature N-linked sugars at the cell surface. Thus, secreted mutant CALR proteins will act more specifically on the MPN clone. In conclusion, a chaperone, CALR, can turn into a rogue cytokine through somatic mutation of its encoding gene.

How to choose the right real-time RT-PCR primer sets for the SARS-CoV-2 genome detection?

OBJECTIVES: The SARS-CoV-2 pandemic has created an unprecedented need for rapid large-scale diagnostic testing to prompt clinical and public health interventions. Currently, several quantitative reverse-transcription polymerase chain reaction (RT-qPCR) assays recommended by the World Health Organization are being used by clinical and public health laboratories and typically target regions of the RNA-dependent RNA polymerase (RdRp), envelope (E) and nucleocapsid (N) coding region. However, it is currently unclear if results from different tests are comparable. This study aimed to clarify the clinical performances of the primer/probe sets designed by US CDC and Charité/Berlin to help clinical laboratories in assay selection for SARS-CoV-2 routine detection. METHODS: We compared the clinical performances of the recommended primer/probe sets using one hundred nasopharyngeal swab specimens from patients who were clinically diagnosed with COVID-19. An additional 30 "pre-intervention screening" samples from patients who were not suspected of COVID-19 were also included in this study. We also performed sequence alignment between 31064 European SARS-CoV-2 and variants of concern genomes and the recommended primer/probe sets. RESULTS: The present study demonstrates substantial differences in SARS-CoV-2 RNA detection sensitivity among the primer/probe sets recommended by the World Health Organization especially for low-level viral loads. The alignment of thousands of SARS-CoV-2 sequences reveals that the genetic diversity remains relatively low at the primer/probe binding sites. However, multiple nucleotide mismatches might contribute to false negatives. CONCLUSION: An understanding of the limitations depending on the targeted genes and primer/probe sets may influence the selection of molecular detection assays by clinical laboratories.

Biological variation data and analytical specification goal estimates of the thrombin generation assay with and without thrombomodulin in healthy individuals.

BACKGROUND: Evaluation of an individual's thrombin-generating capacity enables a global assessment of the coagulation cascade and is therefore thought to better reflect the clotting function of blood. However, the lack of standardization still hampers the use in routine clinical practice. METHODS: Nineteen healthy subjects were sampled once a week for 5 consecutive weeks. Thrombin generation assay (TGA) was performed in duplicate by calibrated automated thrombogram (CAT) on platelet poor plasma with and without thrombomodulin. After exclusion of outliers, a nested analysis of variance (ANOVA) was performed to evaluate the biological variability (BV) results. Analytical variation (CVA ), within-individual variation (CVI ), between-individual variation (CVG ), index of individuality (II), and reference change value (RCV) were calculated. RESULTS: All parameters taken together, the CVA, CVI , and CVG without TM, ranged from 2.8% to 6.5%, from 4.1% to 13.3% and from 10.4% to 28.4%, respectively. For TG with TM, CVI and CVG were higher and ranged from 5.0% to 18.1% and from 14.9% to 35.3%, respectively. For endogenous thrombin potential (ETP), a CVI of 4.1% and CVG of 10.4% were obtained without addition of thrombomodulin (TM). With addition of TM, both CVI and CVG were higher: 14.0% and 34.8%, respectively. The II was low and the RCV ranged from 17.2% to 50.4%. CONCLUSION: CAT parameters are highly individualized and population-based reference values could be called into question. The assessment of BV and RCV for thrombin generation assays could optimize interpretation of serial patient results and guide setting of analytical specification goals.

B-acute Lymphoblastic Leukemia With Hypereosinophilia Associated With Severe Cardiac Complications: A Clinical Case.

Hypereosinophilia (HE) is rare but often secondary to a nonhematologic disease such as allergic disorders and parasitic infections. HE can also be associated with hematologic malignancies and be the result of a clonal proliferation or reactive to another hematologic condition. Association of HE with acute lymphoblastic leukemia (ALL) is rare in children. We reported a case of a teenager presented with HE secondary to B-ALL who experienced severe cardiac complications with severe absolute eosinophil count. We compared his clinical evolution with other published cases and we reported 2 mutations linked to B-ALL never described before in this context.

A biological profile for diagnosis and outcome of COVID-19 patients.

Objectives As severe acute respiratory syndrome coronavirus 2 (SARS-Cov-2) pandemic is increasing its victims on a global scale with recurring outbreaks, it remains of outmost importance to rapidly identify people requiring an intensive care unit (ICU) hospitalization. The aim of this study was to identify Coronavirus Disease 2019 (COVID-19) biomarkers, to investigate their correlation with disease severity and to evaluate their usefulness for follow-up. Methods Fifty patients diagnosed with SARS-Cov-2 were included in March 2020. Clinical and biological data were collected at admission, during hospitalization and one month after discharge. Patients were divided into two severity groups: non-ICU (28) and ICU and/or death (22) to stratify the risk. Results Blood parameters in COVID-19 patients at admission showed increased C-reactive protein (CRP) (100%), ferritin (92%), lactate dehydrogenase (LDH) (80%), white blood cell (WBC) count (26%) with lymphopenia (52%) and eosinopenia (98%). There were significant differences in levels of CRP, ferritin, D-dimers, fibrinogen, lymphocyte count, neutrophil count and neutrophil-to-lymphocyte ratio (NLR) among the two severity groups. Mapping of biomarker's kinetics distinguished early and late parameters. CRP, ferritin, LDH, lymphopenia and eosinopenia were present upon admission with a peak at the first week. Late biomarkers such as anemia, neutrophilia and elevated liver biomarkers appeared after one week with a peak at three weeks of hospitalization. Conclusions We confirmed that high-values of CRP, NLR, D-dimers, ferritin as well as lymphopenia and eosinopenia were consistently found and are good markers for risk stratification. Kinetics of these biomarkers correlate well with COVID-19 severity. Close monitoring of early and late biomarkers is crucial in the management of critical patients to avoid preventable deaths.

Short telomeres increase the risk of severe COVID-19.

Telomeres are non-coding DNA sequences that protect chromosome ends and shorten with age. Short telomere length (TL) is associated with chronic diseases and immunosenescence. The main risk factor for mortality of coronavirus disease 2019 (COVID-19) is older age, but outcome is very heterogeneous among individuals of the same age group. Therefore, we hypothesized that TL influences COVID-19-related outcomes. In a prospective study, we measured TL by Flow-FISH in 70 hospitalized COVID-19 patients and compared TL distribution with our reference cohort of 491 healthy volunteers. We also correlated TL with baseline clinical and biological parameters. We stained autopsy lung tissue from six non-survivor COVID-19 patients to detect senescence-associated ß-galactosidase activity, a marker of cellular aging. We found a significantly higher proportion of patients with short telomeres (<10th percentile) in the COVID-19 patients as compared to the reference cohort (P<0.001). Short telomeres were associated with a higher risk of critical disease, defined as admission to intensive care unit (ICU) or death without ICU. TL was negatively correlated with C-reactive protein and neutrophil-to-lymphocyte ratio. Finally, lung tissue from patients with very short telomeres exhibit signs of senescence in structural and immune cells. Our results suggest that TL influences the severity of the disease.

Two-site evaluation of a new workflow for the detection of malignant cells on the Sysmex XN-1000 body fluid analyzer.

INTRODUCTION: The presence of high fluorescent cells (HF-BF) on the Sysmex XN-1000 hematology analyzers has gained interest regarding the prediction of malignant cells in body fluids, but lacks sensitivity. We aimed to increase this sensitivity by combining HF-BF value, automated results, and clinical information. METHODS: We evaluated a new workflow for the management of body fluids in the hematology laboratory, including the HF-BF criterion and clinical information. In two laboratories, 1623 serous fluids were retrospectively analyzed on the XN-1000 BF mode. All samples were morphologically screened for malignant cells. Optimal HF-BF cutoffs were determined to predict their presence. Thereafter, the added value of clinical information was evaluated. Other reflex testing rules (eosinophilic count >5% and presence of the WBC Abnormal Scattergram flag) were also used to refine our workflow. RESULTS: Optimal HF-BF cutoffs in the two hematology centers were 108 and 45 cells/µL, yielding a sensitivity/specificity of 66.7/93.6% and 86.8/66.6% for malignant cell detection. When adding clinical information, sensitivity/specificity evolved to 100.0/68.9% and 100.0%/not determined. Of 104 samples containing malignant cells, 97 had positive clinical information; the remainder had a HF-BF > cutoff. CONCLUSION: Combining clinical information and HF-BF reached 100% sensitivity for malignant cell detection in body fluid analysis. Lack of robustness of the optimal HF-BF cutoff deserves the use of local cutoffs. Rapid automated results reporting from the XN-1000 BF mode are also feasible in clinical practice. Prospective evaluation of the workflow is needed before its implementation in clinical practice.

MPL mutations in essential thrombocythemia uncover a common path of activation with eltrombopag dependent on W491.

Mutations in the MPL gene encoding the human thrombopoietin receptor (TpoR) drive sporadic and familial essential thrombocythemias (ETs). We identified 2 ET patients harboring double mutations in cis in MPL, namely, L498W-H499C and H499Y-S505N. Using biochemical and signaling assays along with partial saturation mutagenesis, we showed that L498W is an activating mutation potentiated by H499C and that H499C and H499Y enhance the activity of the canonical S505N mutation. L498W and H499C can activate a truncated TpoR mutant, which lacks the extracellular domain, indicating these mutations act on the transmembrane (TM) cytosolic domain. Using a protein complementation assay, we showed that L498W and H499C strongly drive dimerization of TpoR. Activation by tryptophan substitution is exquisitely specific for position 498. Using structure-guided mutagenesis, we identified upstream amino acid W491 as a key residue required for activation by L498W or canonical activating mutations such as S505N and W515K, as well as by eltrombopag. Structural data point to a common dimerization and activation path for TpoR via its TM domain that is shared between the small-molecule agonist eltrombopag and canonical and novel activating TpoR mutations that all depend on W491, a potentially accessible extracellular residue that could become a target for therapeutic intervention.

Adult haemophagocytic lymphohistiocytosis: a Review.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening disorder characterized by hyperimmune response. The mortality is high despite progress being made in the diagnosis and treatment of the disease. AIM: This review aimed to update knowledge on adult HLH pathophysiology, identifiy the numerous causes, and help clinicians make early diagnosis and initiate treatment. DESIGN AND METHODS: Using Embase, we searched relevant articles published from January 1, 2010 to October 31, 2019, with the MESH term « hemophagocytic lymphohistiocytosis; macrophagic activation syndrome, adult ¼. RESULTS: The mean age at presentation is about 50 years, with a male predominance. The most frequent disease associations are haematological diseases, viral or bacterial infections, and autoimmune diseases. The pathophysiologic mechanism is probably the combination of inherited genetic mutations and extrinsic triggers. The mortality rate is 26.5% to 74.8%. H-score is more efficient than HLH-2004 criteria to identify HLH, with diagnostic sensitivity and specificity 90% and 79%, respectively.18F-FDG PET/CT is potentially useful for detecting underlying disease and the extent of secondary HLH. Disease-specific treatment should be given as soon as possible. Treatment with corticosteroids combined or not with etoposide is the mainstay of treatment. Monoclonal antibodies and JAK pathway inhibitors show promise of being effective. CONCLUSION: In adult HLH, infectious diseases, autoimmune disease and malignancy should be suspected so that disease-specific treatment can be given promptly. Treatment with corticosteroids combined or not with etoposide is the mainstay of treatment, but new therapies show promise of being effective.

Knock-in of murine Calr del52 induces essential thrombocythemia with slow-rising dominance in mice and reveals key role of Calr exon 9 in cardiac development.

Frameshifting mutations (-1/+2) of the calreticulin (CALR) gene are responsible for the development of essential thrombocythemia (ET) and primary myelofibrosis (PMF). The mutant CALR proteins activate the thrombopoietin receptor (TpoR) inducing cytokine-independent megakaryocyte progenitor proliferation. Here, we generated via CRISPR/Cas9 technology two knock-in mouse models that are heterozygous for a type-I murine Calr mutation. These mice exhibit an ET phenotype with elevated circulating platelets compared with wild-type controls, consistent with our previous results showing that murine CALR mutants activate TpoR. We also show that the mutant CALR proteins can be detected in plasma. The phenotype of Calr del52 is transplantable, and the Calr mutated hematopoietic cells have a slow-rising advantage over wild-type hematopoiesis. Importantly, a homozygous state of a type-1 Calr mutation is lethal at a late embryonic development stage, showing narrowed ventricular myocardium walls, similar to the murine Calr knockout phenotype, pointing to the C terminus of CALR as crucial for heart development.

Can the 72-hour rule based on "Blast/Abn Lymph" flag on Sysmex XN-10 optimize the workflow in hematology laboratory?

Despite the continuing improvement of automated blood cell counters, confirmation by blood smear examination remains the gold standard in case of anomalies. With a constant goal of standardisation, different experts committees (e.g. the French-speaking cellular hematology group (Groupe francophone d'hématologie cellulaire, GFHC and the ISLH International society for laboratory hematology) recently published criteria for microscopic analysis of blood smears. Cornet et al. evaluated the application of those criteria and propose to suppress any review for 72 hours when a "Blast/Abn lymph" flag is triggered for a sample with no abnormal cell on the microscopic review. The aims of our study were to retrospectively evaluate whether this 72-hour rule adequately operates and whether it is possible to extend the arbitrary 72-hour timeframe to 96h and 144h. To achieve this goal, 40,688 blood samples were collected from three French-speaking hospitals. 1,548 samples presented an isolated "Blast/Abn lymph" flag. Only 221 samples presented the application of the 72-hour rule at least once for our study period. We were able to extend this rule to 144 hours for 10 samples of them. All blood smears for which the rule was applied were verified and there was no abnormal cell on smears at 72 and 144 hours. In conclusion, the 72-hour rule derived from the GFHC's criteria is secure and reduces the slide review rate and thus the production costs and the turnaround time of hemogram results. Further investigations could confirm that its extension to 144 hours is also adequate.

Calreticulin mutants as oncogenic rogue chaperones for TpoR and traffic-defective pathogenic TpoR mutants.

Calreticulin (CALR) +1 frameshift mutations in exon 9 are prevalent in myeloproliferative neoplasms. Mutant CALRs possess a new C-terminal sequence rich in positively charged amino acids, leading to activation of the thrombopoietin receptor (TpoR/MPL). We show that the new sequence endows the mutant CALR with rogue chaperone activity, stabilizing a dimeric state and transporting TpoR and mutants thereof to the cell surface in states that would not pass quality control; this function is absolutely required for oncogenic transformation. Mutant CALRs determine traffic via the secretory pathway of partially immature TpoR, as they protect N117-linked glycans from further processing in the Golgi apparatus. A number of engineered or disease-associated TpoRs such as TpoR/MPL R102P, which causes congenital thrombocytopenia, are rescued for traffic and function by mutant CALRs, which can also overcome endoplasmic reticulum retention signals on TpoR. In addition to requiring N-glycosylation of TpoR, mutant CALRs require a hydrophobic patch located in the extracellular domain of TpoR to induce TpoR thermal stability and initial intracellular activation, whereas full activation requires cell surface localization of TpoR. Thus, mutant CALRs are rogue chaperones for TpoR and traffic-defective TpoR mutants, a function required for the oncogenic effects.

Failure to eliminate a phosphorylated glucose analog leads to neutropenia in patients with G6PT and G6PC3 deficiency.

Neutropenia represents an important problem in patients with genetic deficiency in either the glucose-6-phosphate transporter of the endoplasmic reticulum (G6PT/SLC37A4) or G6PC3, an endoplasmic reticulum phosphatase homologous to glucose-6-phosphatase. While affected granulocytes show reduced glucose utilization, the underlying mechanism is unknown and causal therapies are lacking. Using a combination of enzymological, cell-culture, and in vivo approaches, we demonstrate that G6PT and G6PC3 collaborate to destroy 1,5-anhydroglucitol-6-phosphate (1,5AG6P), a close structural analog of glucose-6-phosphate and an inhibitor of low-KM hexokinases, which catalyze the first step in glycolysis in most tissues. We show that 1,5AG6P is made by phosphorylation of 1,5-anhydroglucitol, a compound normally present in human plasma, by side activities of ADP-glucokinase and low-KM hexokinases. Granulocytes from patients deficient in G6PC3 or G6PT accumulate 1,5AG6P to concentrations (∼3 mM) that strongly inhibit hexokinase activity. In a model of G6PC3-deficient mouse neutrophils, physiological concentrations of 1,5-anhydroglucitol caused massive accumulation of 1,5AG6P, a decrease in glucose utilization, and cell death. Treating G6PC3-deficient mice with an inhibitor of the kidney glucose transporter SGLT2 to lower their blood level of 1,5-anhydroglucitol restored a normal neutrophil count, while administration of 1,5-anhydroglucitol had the opposite effect. In conclusion, we show that the neutropenia in patients with G6PC3 or G6PT mutations is a metabolite-repair deficiency, caused by a failure to eliminate the nonclassical metabolite 1,5AG6P.

Characterization of two new high-grade B-cell lymphoma cell lines with MYC and BCL2 rearrangements that are suitable for in vitro drug sensitivity studies.

High-grade B-cell lymphomas with MYC and BCL2 or BCL6 rearrangements are highly aggressive B-cell lymphomas called double-hit lymphomas (HGBL-DH). They are particularly refractory to standard treatments and carry a poor prognosis. Fragments of resected tumoral lymph nodes from two HGBL-DH patients were put in culture. Continuously proliferating cells were characterized and compared with the original tumors. In both cases, the proliferating cells and the tumor displayed MYC and BCL2 rearrangements. Both cell lines (called LB5848-LYMP and LB5871-LYMP) presented a high proliferation rate and were maintained in culture for more than one year. Upon injection in immunodeficient mice, LB5848-LYMP gave rise to lymphoid tumors. In vitro treatment of these cell lines with a BCL2-inhibitory drug (ABT-199) selectively stopped their proliferation. These new cell lines represent valuable tools for studying HGBL-DH and for the in vitro testing of candidate therapies targeting HGBL-DH. LB5848-LYMP is also suitable for similar experiments in vivo.

The Thrombopoietin Receptor: Structural Basis of Traffic and Activation by Ligand, Mutations, Agonists, and Mutated Calreticulin.

A well-functioning hematopoietic system requires a certain robustness and flexibility to maintain appropriate quantities of functional mature blood cells, such as red blood cells and platelets. This review focuses on the cytokine receptor that plays a significant role in thrombopoiesis: the receptor for thrombopoietin (TPO-R; also known as MPL). Here, we survey the work to date to understand how this receptor functions at a molecular level throughout its lifecycle, from traffic to the cell surface, dimerization and binding cognate cytokine via its extracellular domain, through to its subsequent activation of associated Janus kinases and initiation of downstream signaling pathways, as well as the regulation of these processes. Atomic level resolution structures of TPO-R have remained elusive. The identification of disease-causing mutations in the receptor has, however, offered some insight into structure and function relationships, as has artificial means of receptor activation, through TPO mimetics, transmembrane-targeting receptor agonists, and engineering in dimerization domains. More recently, a novel activation mechanism was identified whereby mutated forms of calreticulin form complexes with TPO-R via its extracellular N-glycosylated domain. Such complexes traffic pathologically in the cell and persistently activate JAK2, downstream signal transducers and activators of transcription (STATs), and other pathways. This pathologic TPO-R activation is associated with a large fraction of human myeloproliferative neoplasms.

What is the normal value of the neutrophil-to-lymphocyte ratio?

BACKGROUND: Neutrophil-to-lymphocyte ratio (NLR) has proven its prognostic value in cardiovascular diseases, infections, inflammatory diseases and in several types of cancers. However, no cut-off has been proposed on the basis of reference values coming from healthy population. METHODS: Routine blood samples were obtained (n = 413) from workers (age: median 38, range: 21-66 years) involved in a health care prevention program, to determine means, standard deviations (SDs), 95% confidence intervals (95% CI), percentiles P2.5 and P97.5. A second independent sample of healthy volunteers is compared (n = 29). RESULTS: The mean NLR is 1.65 [±1.96 SD: 0.78-3.53] (95% CI [0.75-0.81] and [3.40-3.66]). In the second cohort (healthy control), the NLR values are in the same range, whichever the used analyzer. No NLR assessed in the validation series is out of the proposed limits. CONCLUSIONS: We have identified that the normal NLR values in an adult, non-geriatric, population in good health are between 0.78 and 3.53. These data will help to define the normal values of the NLR.

An incomplete trafficking defect to the cell-surface leads to paradoxical thrombocytosis for human and murine MPL P106L.

The mechanisms behind the hereditary thrombocytosis induced by the thrombopoietin (THPO) receptor MPL P106L mutant remain unknown. A complete trafficking defect to the cell surface has been reported, suggesting either weak constitutive activity or nonconventional THPO-dependent mechanisms. Here, we report that the thrombocytosis phenotype induced by MPL P106L belongs to the paradoxical group, where low MPL levels on platelets and mature megakaryocytes (MKs) lead to high serum THPO levels, whereas weak but not absent MPL cell-surface localization in earlier MK progenitors allows response to THPO by signaling and amplification of the platelet lineage. MK progenitors from patients showed no spontaneous growth and responded to THPO, and MKs expressed MPL on their cell surface at low levels, whereas their platelets did not respond to THPO. Transduction of MPL P106L in CD34+ cells showed that this receptor was more efficiently localized at the cell surface on immature than on mature MKs, explaining a proliferative response to THPO of immature cells and a defect in THPO clearance in mature cells. In a retroviral mouse model performed in Mpl-/- mice, MPL P106L could induce a thrombocytosis phenotype with high circulating THPO levels. Furthermore, we could select THPO-dependent cell lines with more cell-surface MPL P106L localization that was detected by flow cytometry and [125I]-THPO binding. Altogether, these results demonstrate that MPL P106L is a receptor with an incomplete defect in trafficking, which induces a low but not absent localization of the receptor on cell surface and a response to THPO in immature MK cells.