Human antibodies in Mexico and Brazil neutralizing tick-borne flaviviruses.

Flaviviruses such as dengue virus (DENV), Zika virus (ZIKV), and yellow fever virus (YFV) are spread by mosquitoes and cause human disease and mortality in tropical areas. In contrast, Powassan virus (POWV), which causes severe neurologic illness, is a flavivirus transmitted by ticks in temperate regions of the Northern hemisphere. We find serologic neutralizing activity against POWV in individuals living in Mexico and Brazil. Monoclonal antibodies P002 and P003, which were derived from a resident of Mexico (where POWV is not reported), neutralize POWV lineage I by recognizing an epitope on the virus envelope domain III (EDIII) that is shared with a broad range of tick- and mosquito-borne flaviviruses. Our findings raise the possibility that POWV, or a flavivirus closely related to it, infects humans in the tropics.

Targeting IRAK4 with Emavusertib in Lymphoma Models with Secondary Resistance to PI3K and BTK Inhibitors.

Inhibitors of phosphatidylinositol 3-kinase (PI3K) and Bruton tyrosine kinase (BTK) represent a recognized option for the treatment of patients affected by indolent B cell lymphomas. However, small molecules as single agents show limited success in their ability in inducing complete responses, with only partial remission achieved in most patients, suggesting the need for combination therapies. IRAK4 is a protein kinase downstream of the Toll-like receptor signaling (TLR), a driver pathway of secondary tumor° resistance in both hematological and solid tumor malignancies. Activation of IRAK4 upon TLRs and IL-1 receptor (IL-1R) stimulation and through the adaptor protein MYD88 initiates a signaling cascade that induces cytokine and survival factor expression mediated by the transcription factor NF-κB. MYD88-L265P encoding mutations occur in diffuse large B-cell lymphomas, in lymphoplasmacytic lymphomas and in few marginal zone lymphomas (MZL). The IRAK4 inhibitor emavusertib (CA-4948) has shown early safety and clinical activity in lymphoma and leukemia patients. In this preclinical study, we assessed emavusertib effectiveness in MZL, both as single agent and in combination with targeted agents, with a particular focus on its capability to overcome resistance to BTK and PI3K inhibitors. We showed that the presence of MYD88 L265P mutation in bona fide MZL cell lines confers sensitivity to the IRAK4 inhibitor emavusertib as single agent. Emavusertib-based combinations improved the sensitivity of MZL cells to BTK and PI3K inhibitors, including cells with a secondary resistance to these agents. Emavusertib exerted its activity via inhibition of NF-κB signaling and induction of apoptosis. Considering the early safety data from clinical trials, our study identifies the IRAK4 inhibitor emavusertib as a novel compound to be explored in trials for patients with MYD88-mutated indolent B cell lymphomas as single agent and as combination partner with BTK or PI3K inhibitors in unselected populations of patients.

Enhancer RNAs (eRNAs) in Cancer: The Jacks of All Trades.

Enhancer RNAs (eRNAs) are non-coding RNAs (ncRNAs) transcribed in enhancer regions. They play an important role in transcriptional regulation, mainly during cellular differentiation. eRNAs are tightly tissue- and cell-type specific and are induced by specific stimuli, activating promoters of target genes in turn. eRNAs usually have a very short half-life but in some cases, once activated, they can be stably expressed and acquire additional functions. Due to their critical role, eRNAs are often dysregulated in cancer and growing number of interactions with chromatin modifiers, transcription factors, and splicing machinery have been described. Enhancer activation and eRNA transcription have particular relevance also in inflammatory response, placing the eRNAs at the interplay between cancer and immune cells. Here, we summarize all the possible molecular mechanisms recently reported in association with eRNAs activity.

HTGQC and shinyHTGQC: an R package and shinyR application for quality controls of HTG EDGE-seq protocols.

Extraction-free HTG EdgeSeq protocols are used to profile sets of genes and measure their expression. Thus, these protocols are frequently used to characterise tumours and their microenvironments. However, although positive and control genes are provided, little indication is given concerning the assessment of the technical success of each sample within the sequencing run. We developed HTGQC, an R package for the quality control of HTG EdgeSeq protocols. Additionally, shinyHTGQC is a shiny application for users without computing knowledge, providing an easy-to-use interface for data quality control and visualisation. Quality checks can be performed on the raw sequencing outputs, and samples are flagged as FAIL or ALERT based on the expression levels of the positive and negative control genes. Availability & Implementation: The code is freely available at https://github.com/LodovicoTerzi/HTGQC (R package) and https://lodovico.shinyapps.io/shinyHTGQC/ (shiny application), including test datasets.

Genetic and phenotypic attributes of splenic marginal zone lymphoma.

Splenic marginal zone B-cell lymphoma (SMZL) is a heterogeneous clinico-biological entity. The clinical course is variable, multiple genes are mutated with no unifying mechanism, and essential regulatory pathways and surrounding microenvironments are diverse. We sought to clarify the heterogeneity of SMZL by resolving different subgroups and their underlying genomic abnormalities, pathway signatures, and microenvironment compositions to uncover biomarkers and therapeutic vulnerabilities. We studied 303 SMZL spleen samples collected through the IELSG46 multicenter international study (NCT02945319) by using a multiplatform approach. We carried out genetic and phenotypic analyses, defined self-organized signatures, validated the findings in independent primary tumor metadata and in genetically modified mouse models, and determined correlations with outcome data. We identified 2 prominent genetic clusters in SMZL, termed NNK (58% of cases, harboring NF-κB, NOTCH, and KLF2 modules) and DMT (32% of cases, with DNA-damage response, MAPK, and TLR modules). Genetic aberrations in multiple genes as well as cytogenetic and immunogenetic features distinguished NNK- from DMT-SMZLs. These genetic clusters not only have distinct underpinning biology, as judged by differences in gene-expression signatures, but also different outcomes, with inferior survival in NNK-SMZLs. Digital cytometry and in situ profiling segregated 2 basic types of SMZL immune microenvironments termed immune-suppressive SMZL (50% of cases, associated with inflammatory cells and immune checkpoint activation) and immune-silent SMZL (50% of cases, associated with an immune-excluded phenotype) with distinct mutational and clinical connotations. In summary, we propose a nosology of SMZL that can implement its classification and also aid in the development of rationally targeted treatments.

Characterization of GECPAR, a noncoding RNA that regulates the transcriptional program of diffuse large B-cell lymphoma.

Enhancers are regulatory regions of DNA, which play a key role in cell-type specific differentiation and development. Most active enhancers are transcribed into enhancer RNA (eRNA) that can regulate transcription of target genes by means of in cis as well as in trans action. eRNA stabilize contacts between distal genomic regions and mediate the interaction of DNA with master transcription factors. Here, we characterized an enhancer eRNA, GECPAR (germinal center proliferative adapter RNA), which is specifically transcribed in normal and neoplastic germinal center B cells from the super-enhancer of POU2AF1, a key regulatory gene of the germinal center reaction. Using diffuse large B-cell lymphoma cell line models, we demonstrated the tumor suppressor activity of GECPAR, which is mediated via its transcriptional regulation of proliferation and differentiation genes, particularly MYC and the Wnt pathway.

Broad and potent neutralizing human antibodies to tick-borne flaviviruses protect mice from disease.

Tick-borne encephalitis virus (TBEV) is an emerging human pathogen that causes potentially fatal disease with no specific treatment. Mouse monoclonal antibodies are protective against TBEV, but little is known about the human antibody response to infection. Here, we report on the human neutralizing antibody response to TBEV in a cohort of infected and vaccinated individuals. Expanded clones of memory B cells expressed closely related anti-envelope domain III (EDIII) antibodies in both groups of volunteers. However, the most potent neutralizing antibodies, with IC50s below 1 ng/ml, were found only in individuals who recovered from natural infection. These antibodies also neutralized other tick-borne flaviviruses, including Langat, louping ill, Omsk hemorrhagic fever, Kyasanur forest disease, and Powassan viruses. Structural analysis revealed a conserved epitope near the lateral ridge of EDIII adjoining the EDI-EDIII hinge region. Prophylactic or early therapeutic antibody administration was effective at low doses in mice that were lethally infected with TBEV.

Targeting CD205 with the antibody drug conjugate MEN1309/OBT076 is an active new therapeutic strategy in lymphoma models.

Antibody drug conjugates represent an important class of anti-cancer drugs in both solid tumors and hematological cancers. Here, we report preclinical data on the anti-tumor activity of the first-in-class antibody drug conjugate MEN1309/OBT076 targeting CD205. The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination and validation experiments on in vivo models. CD205 was first shown frequently expressed in lymphomas, leukemias and multiple myeloma by immunohistochemistry on tissue microarrays. Anti-tumor activity of MEN1309/OBT076 as single agent was then shown across 42 B-cell lymphoma cell lines with a median IC50 of 200 pM and induction of apoptosis in 25/42 (59.5%) of the cases. The activity appeared highly correlated with its target expression. After in vivo validation as the single agent, the antibody drug conjugate synergized with the BCL2 inhibitor venetoclax, and the anti-CD20 monoclonal antibody rituximab. The first-in-class antibody drug targeting CD205, MEN1309/OBT076, demonstrated strong pre-clinical anti-tumor activity in lymphoma, warranting further investigations as a single agent and in combination.

Circulating tumor DNA reveals genetics, clonal evolution, and residual disease in classical Hodgkin lymphoma.

The rarity of neoplastic cells in the biopsy imposes major technical hurdles that have so far limited genomic studies in classical Hodgkin lymphoma (cHL). By using a highly sensitive and robust deep next-generation sequencing approach for circulating tumor DNA (ctDNA), we aimed to identify the genetics of cHL in different clinical phases, as well as its modifications on treatment. The analysis was based on specimens collected from 80 newly diagnosed and 32 refractory patients with cHL, including longitudinal samples collected under ABVD (adriamycin, bleomycin, vinblastine, dacarbazine) chemotherapy and longitudinal samples from relapsing patients treated with chemotherapy and immunotherapy. ctDNA mirrored Hodgkin and Reed-Sternberg cell genetics, thus establishing ctDNA as an easily accessible source of tumor DNA for cHL genotyping. By identifying STAT6 as the most frequently mutated gene in ∼40% of cases, we refined the current knowledge of cHL genetics. Longitudinal ctDNA profiling identified treatment-dependent patterns of clonal evolution in patients relapsing after chemotherapy and patients maintained in partial remission under immunotherapy. By measuring ctDNA changes during therapy, we propose ctDNA as a radiation-free tool to track residual disease that may integrate positron emission tomography imaging for the early identification of chemorefractory patients with cHL. Collectively, our results provide the proof of concept that ctDNA may serve as a novel precision medicine biomarker in cHL.

PQR309 Is a Novel Dual PI3K/mTOR Inhibitor with Preclinical Antitumor Activity in Lymphomas as a Single Agent and in Combination Therapy.

Purpose: Activation of the PI3K/mTOR signaling pathway is recurrent in different lymphoma types, and pharmacologic inhibition of the PI3K/mTOR pathway has shown activity in lymphoma patients. Here, we extensively characterized the in vitro and in vivo activity and the mechanism of action of PQR309 (bimiralisib), a novel oral selective dual PI3K/mTOR inhibitor under clinical evaluation, in preclinical lymphoma models.Experimental Design: This study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination, validation experiments on in vivo models and primary cells, proteomics and gene-expression profiling, and comparison with other signaling inhibitors.Results: PQR309 had in vitro antilymphoma activity as single agent and in combination with venetoclax, panobinostat, ibrutinib, lenalidomide, ARV-825, marizomib, and rituximab. Sensitivity to PQR309 was associated with specific baseline gene-expression features, such as high expression of transcripts coding for the BCR pathway. Combining proteomics and RNA profiling, we identified the different contribution of PQR309-induced protein phosphorylation and gene expression changes to the drug mechanism of action. Gene-expression signatures induced by PQR309 and by other signaling inhibitors largely overlapped. PQR309 showed activity in cells with primary or secondary resistance to idelalisib.Conclusions: On the basis of these results, PQR309 appeared as a novel and promising compound that is worth developing in the lymphoma setting. Clin Cancer Res; 24(1); 120-9. ©2017 AACR.

An improved method to compute the solute and water derangements of hyperglycaemia.

Evaluation and treatment of hyperglycaemic hyponatremia, being quantitatively inaccurate, is open to new advancements. We herein describe the improvement of previous calculations of glucose appearance (G(A)), solute and solvent changes. From G(A) we derive the predicted plasma sodium concentration (PNa(G)), assuming no change in total body water (TBW), but only water shift from cells to the extracellular space (ECV). This assumption is validated by the respective solute ratios (PCl/PNa) unchanged from normal values, as well as the ratios between actual and normal solute concentrations (PNa(1)/PNa(0), PCl(1)/PCl(0)), identical for all solutes. When the assumption is met, G(A) can be exactly calculated. When the ratios are different from normal, they indicate the presence of a mixed abnormality due to a loss either of sodium, or sodium and water. These are estimated by computing the difference between PNa(G) and the actual PNa measured (PNa(1)). PNa(1) approximately equal PNa(G) if TBW and Na are unchanged, PNa(1) < PNa(G) in the presence of prevalent Na depletion, PNa(1) > PNa(G )when volume depletion prevails. In the first circumstance the ECV expansion is exactly established by appropriate mathematical formulas, in the latter conditions either Na or volume depletion are empirically estimated with algebric expressions. These equations were validated on computer-simulated models, and applied to 49 subjects with plasma glucose concentration >15 mM/L. G(A) and PNa(G) were computed, and, with the same formulas used in computer-simulated experiments, we calculated water and Na deficits. The PNa measured after correction of hyperglycaemia was correctly predicted (R(2) = 0.63, P < 0.0001). This method provides a firm ground to select the correct equation to accurately estimate the initial conditions of hyperosmolar hyperglycaemia, significantly improving its quantitative correction.

Estimating excess glucose, sodium and water deficits in non-ketotic hyperglycaemia.

BACKGROUND: The treatment of solute addition, Na and water losses in hyperglycaemic hyponatraemia is guided by clinical judgement rather than by a quantitative assessment. METHODS: We devised an iteration method to compute glucose appearance (G(A)) within the extracellular space, to obtain the PNa (plasma sodium concentration) expected by glucose addition only (PNa(G)). The difference between this and the actual measurement (PNa(1)) was used to compute the attending Na and/or volume depletion, and the PNa expected during correction. The equations were validated on computer-built models, where the electrolyte derangements were simulated, generating true values of plasma glucose (P(G)) and Na concentrations, from which surfeit and deficits were back-calculated with our formulas. We also computed G(A) and PNa(G) on 43 patients who were stratified into a group with normal hydration (PNa(1) = PNa(G)), one with prevalent Na depletion (PNa(1) < PNa(G)), and one with prevalent volume depletion (PNa(1) > PNa(G)). The volume conditions established by our computations were compared by logistic regression analysis with those assessed from clinical laboratory data. RESULTS: The computer simulations demonstrated that the method gave exact results when only one variable changed, clinically useful estimates in the presence of mixed volume and sodium deficits. There was a strongly significant concordance between the clinical and the quantitative method (P < 0.001). The latter predicted the PNa measured after correction of hyperglycaemia (P < 0.001). CONCLUSION: This new method more accurately computes the initial conditions, resulting in a useful stratification of patients which improves the quantitative evaluation and treatment of hyperosmolar coma.

Primitive hematopoietic stem cells shows a polyclonal pattern in myelodysplastic syndromes.

BACKGROUND AND OBJECTIVES: Clonal hematopoiesis is the hallmark of myelodysplastic syndromes, but the role played by pluripotent stem cells and progenitor cells in these disorders remains unclear. DESIGN AND METHODS: Eight female patients with myelodysplastic syndrome were studied. X-chromosome inactivation patterns were analyzed in peripheral blood granulocytes, T-lymphocytes, single colonies originating from bone marrow progenitors and pluripotent stem cells, using the human androgen receptor locus polymorphism assay. RESULTS: Granulocytes and progenitor cells were monoclonal in 7/8 cases. Immature stem cells showed a non-clonal pattern of X-inactivation and were detectable at diagnosis in the presence of clonal hematopoiesis. T-lymphocyte clonality was heterogeneous. INTERPRETATION AND CONCLUSIONS: In myelodysplastic syndromes, hematopoiesis may be dominated by a neoplastic clone by virtue of its biological advantage over a residual polyclonal, probably still normal, population of immature stem cells still able to grow in vitro.

Human herpesvirus type 8-associated primary lymphomatous effusion in an elderly HIV-negative patient: clinical and molecular characterization.

Primary lymphomatous effusions are defined as lymphomas presenting in the serous body cavities in the absence of clinically identifiable tumor masses. Recently, a peculiar type of primary lymphomatous effusion associated with tumor clone infection by human herpesvirus type 8 (HHV-8) and preferentially arising in HIV-positive patients has been described and termed as primary effusion lymphoma (PEL). This report describes a case of PEL which has developed in a HIV-negative, 92-year-old man with longstanding Mediterranean Kaposi's sarcoma, a disease also associated with HHV-8 infection. The patient presented with pleural and ascitic effusions in the absence of solid masses within the lungs, mediastinum, thoracic wall or abdominal cavity. The effusions consisted of malignant lymphocytes with morphologic features bridging immunoblastic and anaplastic cells. Immunophenotypic studies revealed that the lymphoma population expressed an antigenic profile consistent with PEL, i.e. the absence of common B- and T-cell markers (non-B, non-T phenotype) coupled to CD138 positivity. Molecular analysis demonstrated infection of the tumor clone by HHV-8 as well as monoclonally rearranged immunoglobulin genes, consistent with a B-cell histogenesis of the lymphoma. In addition, this PEL case harbored PAX-5 gene mutations, which have been recently demonstrated as a key feature of the proto-oncogene hypermutation process involved in the pathogenesis of some lymphoma types. Following two courses of etoposide and prednisone, a partial remission was achieved. The patient died of liver failure 3 months after the diagnosis of PEL. Overall, this case report illustrates the need for an integrated diagnostic approach based on clinical features, morphology, immunophenotype, and molecular genetics to primary lymphomatous effusions.