Along with PaO2/FiO2 ratio and lymphopenia, low HLA-DR monocytes are the only additional parameter that independently predicts the clinical course of undifferentiated SARS-CoV-2 patients in Emergency Departments.

Since 1/3 of patients deteriorate after their admission to the emergency department (ED), assessing the prognosis of COVID-19 patients is of great importance. But to date, only lymphopenia and PaO2/FiO2 (P/F) ratio have been reported as partly predictive of COVID-19 further deterioration and their association has not been evaluated. We asked whether other key biomarkers of SARS-CoV2 immunologic defects - increase in circulating immature granulocytes (IGs), loss of monocyte HLA-DR (mHLA-DR) expression and monocyte differentiation blockade - could also predict further COVID-19 deterioration. A series of 284 consecutive COVID-19 patients with, as sole inclusion criterion of being an adult, were prospectively enrolled at ED admission (D0) of two different hospitals: one for the exploratory (180 patients) and one for the confirmatory cohort (104 patients). Deterioration was assessed over the next seven days. Neither increased IG levels nor monocyte differentiation blockade predicted patient worsening. Among more than 30 clinical, biological and radiological parameters, the value of decreased PaO2/FiO2 (P/F) ratio and lymphopenia for prediction of further COVID-19 deterioration was strongly confirmed and the loss of mHLA-DR was the only additional independent marker. Combined together in a simple OxyLymphoMono score, the three variables perfectly predicted patients who did not worsen and correctly predicted worsening in 59% of cases.By highlighting lymphocyte and monocyte defects as preceding COVID-19 deterioration, these results point on early immunosuppression in COVID-19 deterioration. Combining P/F ratio, lymphopenia and loss of mHLA-DR together in a simple and robust score could offer a pragmatic method for COVID-19 patient stratification.

IGH 3'RR recombination uncovers a non-germinal center imprint and c-MYC-dependent IGH rearrangement in unmutated chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is an incurable indolent non-Hodgkin lymphoma characterized by tumor B cells that weakly express a B-cell receptor. The mutational status of the variable region (IGHV) within the immunoglobulin heavy chain (IGH) locus is an important prognosis indicator and raises the question of the CLL cell of origin. Mutated IGHV gene CLL are genetically imprinted by activation-induced cytidine deaminase (AID). AID is also required for IGH rearrangements: class switch recombination and recombination between switch Mu (Sµ) and the 3' regulatory region (3'RR) (Sµ-3'RRrec). The great majority of CLL B cells being unswitched led us to examine IGH rearrangement blockade in CLL. Our results separated CLL into two groups on the basis of Sµ-3'RRrec counts per sample: Sµ-3'RRrecHigh cases (mostly unmutated CLL) and Sµ-3'RRrecLow cases (mostly mutated CLL), but not based on the class switch recombination junction counts. Sµ-3'RRrec appeared to be ongoing in Sµ-3'RRrecHigh CLL cells and comparison of Sµ-3'RRrec junction structural features pointed to different B-cell origins for both groups. In accordance with IGHV mutational status and PIM1 mutation rate, Sµ-3'RRrecHigh CLL harbor a non-germinal center experienced B-cell imprint while Sµ-3'RRrecLow CLL are from AID-experienced B cells from a secondary lymphoid organ. In addition to the proposals already made concerning the CLL cell of origin, our study highlights that analysis of IGH recombinatory activity can identify CLL cases from different origins. Finally, on-going Sµ-3'RRrec in Sµ-3'RRrecHigh cells appeared to presumably be the consequence of high c-MYC expression, as c-MYC overexpression potentiated IGH rearrangements and Sµ-3'RRrec, even in the absence of AID for the latter.

Hereditary predisposition to malignant myeloid hemopathies: Caution in use of saliva and guideline based on our experience.

Background: Predisposition to myeloid malignancies is a field at the border of hematology and genetics. Knowledge in this domain has so rapidly increased that WHO defined in 2016 the new "Myeloid Neoplasms with Germline Predisposition" category of tumors. High throughput sequencing is frequently performed in tumors either for diagnosis or prognosis, but this approach may identify potential germline variants that have to be confirmed on non-infiltrated tissues. Method: In this study, we systematically compared NGS data from genetic analysis performed on all sample types (bone marrow, blood, saliva, skin fibroblasts and hair follicles) in 29 patients, and 44 of their relatives (blood and saliva). Results: We showed that saliva was usable for relatives, but only for 24% (7/29) of our patients. Most of patients' saliva were either "non-contributive" (14/29 i.e., 48% because clearly or probably infiltrated) or "inconclusive" (8/29 corresponding to 28%). Conclusion: The recommendations for the use of saliva we present here focus on the importance of collecting saliva during remission when possible. Moreover, we propose hair follicles as an alternative to skin biopsy, that remains the gold standard especially in case of allogenic hematopoietic stem cells transplantation. Technological progresses have revolutionized the diagnosis of predisposition to solid or hematological malignancies, and it is very likely that new techniques will help to manage the familial predisposition in the future.

Anti-Proliferative and Pro-Apoptotic vLMW Fucoidan Formulas Decrease PD-L1 Surface Expression in EBV Latency III and DLBCL Tumoral B-Cells by Decreasing Actin Network.

Epstein-Barr virus (EBV) infects 95% of the world's population and persists latently in the body. It immortalizes B-cells and is associated with lymphomas. LCLs (lymphoblastoid cell lines, EBV latency III B-cells) inhibit anti-tumoral T-cell response following PD-L1 overexpression (programmed death-ligand 1 immune checkpoint). Many cancer cells, including some DLBCLs (diffuse large B-cell lymphomas), also overexpress PD-L1. Immunotherapies are based on inhibition of PD-L1/PD-1 interactions but present some dose-dependent toxicities. We aim to find new strategies to improve their efficiency by decreasing PD-L1 expression. Fucoidan, a polysaccharide extracted from brown seaweed, exhibits immunomodulatory and anti-tumor activities depending on its polymerization degree, but data are scarce on lymphoma cells or immune checkpoints. LCLs and DLBCLs cells were treated with native fucoidan (Fucus vesiculosus) or original very-low-molecular-weight fucoidan formulas (vLMW-F). We observed cell proliferation decrease and apoptosis induction increase with vLMW-F and no toxicity on normal B- and T-cells. We highlighted a decrease in transcriptional and PD-L1 surface expression, even more efficient for vLMW than native fucoidan. This can be explained by actin network alteration, suggesting lower fusion of secretory vesicles carrying PD-L1 with the plasma membrane. We propose vLMW-F as potential adjuvants to immunotherapy due to their anti-proliferative and proapoptotic effects and ability to decrease PD-L1 membrane expression.

Demultiplexing Ig repertoires by parallel mRNA/DNA sequencing shows major differential alterations in severe COVID-19.

To understand the fine differential elements that can lead to or prevent acute respiratory distress syndrome (ARDS) in COVID-19 patients, it is crucial to investigate the immune response architecture. We herein dissected the multiple layers of B cell responses by flow cytometry and Ig repertoire analysis from acute phase to recovery. Flow cytometry with FlowSOM analysis showed major changes associated with COVID-19 inflammation such as an increase of double-negative B-cells and ongoing plasma cell differentiation. This paralleled COVID-19-driven expansion of two disconnected B-cell repertoires. Demultiplexing successive DNA and RNA Ig repertoire patterns characterized an early expansion of IgG1 clonotypes with atypically long and uncharged CDR3, the abundance of this inflammatory repertoire being correlated with ARDS and likely pejorative. A superimposed convergent response included convergent anti-SARS-CoV-2 clonotypes. It featured progressively increasing somatic hypermutation together with normal-length or short CDR3 and it persisted until a quiescent memory B-cell stage after recovery.

De novo natural anti-M alloantibody emergence in severe Coronavirus Disease 2019.

The immune response is a key player in the course of SARS-CoV-2 infection, and is often seriously dysfunctional in severe Coronavirus Disease 2019. The hyperinflammatory status has been described to be accompanied by the appearance of autoantibodies. In a lethal COVID-19 infection, we observed the emergence of a de novo natural alloantibody which targeted the M antigen from the MNS blood group on red blood cells (RBC) without evidence of any cross-reaction with SARS-CoV-2 antigens. This IgM lambda alloantibody was unmutated and unswitched. Here, we describe for the first time the emergence of a bystander de novo natural alloantibody against RBCs in a severe COVID-19 patient, highlighting the extra-follicular humoral response reported in these cases.

Epstein-Barr Virus (EBV) Is Mostly Latent and Clonal in Angioimmunoblastic T Cell Lymphoma (AITL).

The Epstein-Barr virus (EBV) is associated with angioimmunoblastic T cell lymphoma (AITL), a peripheral T lymphoma of poor prognosis in at least 90% of cases. The role of EBV in this pathology is unknown. Using next-generation sequencing, we sequenced the entire EBV genome in biopsies from 18 patients with AITL, 16 patients with another EBV-associated lymphoma, and 2 controls. We chose an EBV target capture method, given the high specificity of this technique, followed by a second capture to increase sensitivity. We identified two main viral strains in AITL, one of them associated with the mutations BNRF1 S542N and BZLF1 A206S and with mutations in the EBNA-3 and LMP-2 genes. This strain was characterized in patients with short post-diagnosis survival. The main mutations found during AITL on the most mutated latency or tegument genes were identified and discussed. We showed that the virus was clonal in all the AITL samples, suggesting that it may be involved in this pathology. Additionally, EBV was latent in all the AITL samples; for one sample only, the virus was found to be latent and probably replicative, depending on the cells. These various elements support the role of EBV in AITL.

Alternative c-MYC mRNA Transcripts as an Additional Tool for c-Myc2 and c-MycS Production in BL60 Tumors.

While studying c-Myc protein expression in several Burkitt lymphoma cell lines and in lymph nodes from a mouse model bearing a translocated c-MYC gene from the human BL line IARC-BL60, we surprisingly discovered a complex electrophoretic profile. Indeed, the BL60 cell line carrying the t(8;22) c-MYC translocation exhibits a simple pattern, with a single c-Myc2 isoform. Analysis of the c-MYC transcripts expressed by tumor lymph nodes in the mouse λc-MYC (Avy/a) showed for the first time five transcripts that are associated with t(8;22) c-MYC translocation. The five transcripts were correlated with the production of c-Myc2 and c-MycS, and loss of c-Myc1. The contribution of these transcripts to the oncogenic activation of the t(8;22) c-MYC is discussed.

Flow cytometry detection of CD138 expression continuum between monotypic B and plasma cells is associated with both high IgM peak levels and MYD88 mutation and contributes to diagnosis of Waldenström macroglobulinemia.

BACKGROUND: Differential diagnosis of Waldenström macroglobulinemia (WM) with other indolent B-cell malignancies is still a challenge. Here, we propose an original and simple analysis of routine flow cytometry (FCM) unraveling the characteristic ongoing plasma cell (PC) differentiation of WM tumor B-cells. METHODS: FCM analysis of both B-cells and PC was performed on a series of 77 patients with IgM peak. MYD88 and CXCR4 mutations were studied using an allele-specific PCR and by high throughput sequencing. RESULTS: Twenty seven (35%), 46 (58%) and 4 (5%) patients were classified as WM, IgM monoclonal gammopathy of undetermined significance (MGUS) or other B-NHL respectively. MYD88 mutation was found in 25/27 WM (93%) and in 29/46 MGUS (63%). Using FCM, monotypic B-cells were found in 27/27 WM (100%) and 34/46 MGUS (74%). Monotypic CD138pos/CD38pos PCs were detected in 23/27 WM (85%) and 25/46 MGUS (54%). Highlighting the ongoing PC differentiation of WM tumor B-cells by FCM, we evidenced a CD138 expression continuum between monotypic B-cells and PCs. This pattern remained absent in control samples and was significantly associated with higher IgM peaks (p = 6.10-5 ) and MYD88 mutations (p = 10-3 ) in both WM and MGUS cases. CONCLUSIONS: FCM exploration of both B-cells and PC led to identify a CD138 expression continuum as an objective marker of ongoing PC differentiation of WM tumor cells and was strongly associated with increased IgM peak levels and MYD88 mutations. This approach could contribute to place FCM at the forefront of WM diagnosis.

The alternative RelB NF-κB subunit is a novel critical player in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most frequent lymphoid malignancy affecting adults. The NF-κB transcription factor family is activated by 2 main pathways, the canonical and the alternative NF-κB activation pathway, with different functions. The alternative NF-κB pathway leads to activation of the transcriptionally active RelB NF-κB subunit. Alternative NF-κB activation status and its role in DLBCL pathogenesis remain undefined. Here, we reveal a frequent activation of RelB in a large cohort of DLBCL patients and cell lines, independently of their activated B-cell-like or germinal center B-cell-like subtype. RelB activity defines a new subset of patients with DLBCL and a peculiar gene expression profile and mutational pattern. Importantly, RelB activation does not correlate with the MCD genetic subtype, enriched for activated B-cell-like tumors carrying MYD88L265P and CD79B mutations that cooperatively activate canonical NF-κB, thus indicating that current genetic tools to evaluate NF-κB activity in DLBCL do not provide information on the alternative NF-κB activation. Furthermore, the newly defined RelB-positive subgroup of patients with DLBCL exhibits a dismal outcome after immunochemotherapy. Functional studies revealed that RelB confers DLBCL cell resistance to DNA damage-induced apoptosis in response to doxorubicin, a genotoxic agent used in the front-line treatment of DLBCL. We also show that RelB positivity is associated with high expression of cellular inhibitor of apoptosis protein 2 (cIAP2). Altogether, RelB activation can be used to refine the prognostic stratification of DLBCL and may contribute to subvert the therapeutic DNA damage response in a segment of patients with DLBCL.

CD20 expression, TrkB activation and functional activity of diffuse large B cell lymphoma-derived small extracellular vesicles.

BACKGROUND: Small extracellular vesicles (sEVs) including exosomes, carrying the CD20, could be involved in immunotherapy resistance in diffuse large B cell lymphoma (DLBCL). We have reported endogenous brain-derived neurotrophic factor/TrkB (tropomyosin-related kinase B) survival axis in DLBCL. Here, we performed a comparative study of sEV production by germinal centre B cell (GCB) and activated B cell (ABC)-DLBCL cell lines, and analysed TrkB activation on this process. METHODS: GCB (SUDHL4 and SUDHL6) and ABC (OCI-LY3, OCI-LY10 and U2932) cell lines were used. sEVs were characterised using nanoparticle tracking analysis technology and western blot. CD20 content was also analysed by enzyme-linked immunoassay, and complement-dependent cytotoxicity of rituximab was investigated. 7,8-Dihydroxyflavone (7,8-DHF) was used as a TrkB agonist. In vivo role of sEVs was evaluated in a xenograft model. RESULTS: sEVs production varied significantly between DLBCL cells, independently of subtype. CD20 level was consistent with that of parental cells. Higher CD20 expression was found in sEVs after TrkB activation, with a trend in increasing their concentration. sEVs determined in vitro and in vivo protection from rituximab, which seemed CD20 level-dependent; the protection was enhanced when sEVs were produced by 7,8-DHF-treated cells. CONCLUSIONS: DLBCL-derived sEVs have the differential capacity to interfere with immunotherapy, which could be enhanced by growth factors like neurotrophins. Evaluating the sEV CD20 level could be useful for disease monitoring.

Immature granulocytes can help the diagnosis of pulmonary bacterial infections in patients with severe COVID-19 pneumonia.

During COVID-19, immature granulocyte (IG) concentration is heterogeneous with higher concentrations than those found in bacterial sepsis. We investigated the relationship between IG levels at ICU admission and on days 7 (± 2) and 15 (± 2) and associated pulmonary bacterial infections in intensive care unit (ICU) patients hospitalized for an acute respiratory distress syndrome (ARDS) related to SARS-CoV-2. Patients with associated pulmonary bacterial infection had a peak of IGs. IG thresholds of 18% or 2 G/L allowed discriminating patients with ventilator associated pneumonia with 100% sensitivity and specificity. Our study supports that IGs could help identifying pulmonary bacterial infections in this population.

Genetically Engineered Mouse Models Support a Major Role of Immune Checkpoint-Dependent Immunosurveillance Escape in B-Cell Lymphomas.

These last 20 years, research on immune tumor microenvironment led to identify some critical recurrent mechanisms used in cancer to escape immune response. Through immune checkpoints, which are cell surface molecules involved in the immune system control, it is now established that tumor cells are able to shutdown the immune response. Due to the complexity and heterogeneity of Non Hodgkin B-cell Lymphomas (NHBLs), it is difficult to understand the precise mechanisms of immune escape and to explain the mitigated effect of immune checkpoints blockade for their treatment. Because genetically engineered mouse models are very reliable tools to improve our understanding of molecular mechanisms involved in B-cell transformation and, at the same time, can be useful preclinical models to predict immune response, we reviewed hereafter some of these models that highlight the immune escape mechanisms of NHBLs and open perspectives on future therapies.

c-Rel Is the Pivotal NF-κB Subunit in Germinal Center Diffuse Large B-Cell Lymphoma: A LYSA Study.

Relationships between c-Rel and GCB-DLBCLs remain unclear. We found that strong c-Rel DNA-binding activity was mostly found in GCBs on two independent series of 48 DLBCLs and 66 DLBCLs, the latter issued from the GHEDI series. c-Rel DNA-binding activity was associated with increased REL mRNA expression. Extending the study to the whole GHEDI and Lenz DLBCL published series of 202 and 233 cases, it was found that the c-Rel gene expression profile (GEP) overlapped partially (12%) but only with the GCB GEP and not with the GEP of ABC-DLBCLs. Cases with both overexpression of REL mRNA and c-Rel GEP were defined as those having a c-Rel signature. These cases were GCBs in 88 and 83% of the GHEDI or Lenz's DLBCL series respectively. The c-Rel signature was also associated with various recurrent GCB-DLBCL genetic events, including REL gains, BCL2 translocation, MEF2B, EZH2, CREBBP, and TNFRSF14 mutations and with the EZB GCB genetic subtype. By CGH array, the c-Rel signature was specifically correlated with 2p15-16.1 amplification that includes XPO1, BCL11A, and USP34 and with the 22q11.22 deletion that covers IGLL5 and PRAME. The total number of gene copy number aberrations, so-called genomic imbalance complexity, was decreased in cases with the c-Rel signature. These cases exhibited a better overall survival. Functionally, overexpression of c-Rel induced its constitutive nuclear localization and protected cells against apoptosis while its repression tended to increase cell death. These results show that, clinically and biologically, c-Rel is the pivotal NF-κB subunit in the GCB-DLBCL subgroup. Functionally, c-Rel overexpression could directly promote DLBCL tumorigenesis without need for further activation signals.

Comparative immune profiling of acute respiratory distress syndrome patients with or without SARS-CoV-2 infection.

Acute respiratory distress syndrome (ARDS) is the main complication of coronavirus disease 2019 (COVID-19), requiring admission to the intensive care unit (ICU). Despite extensive immune profiling of COVID-19 patients, to what extent COVID-19-associated ARDS differs from other causes of ARDS remains unknown. To address this question, here, we build 3 cohorts of patients categorized in COVID-19-ARDS+, COVID-19+ARDS+, and COVID-19+ARDS-, and compare, by high-dimensional mass cytometry, their immune landscape. A cell signature associating S100A9/calprotectin-producing CD169+ monocytes, plasmablasts, and Th1 cells is found in COVID-19+ARDS+, unlike COVID-19-ARDS+ patients. Moreover, this signature is essentially shared with COVID-19+ARDS- patients, suggesting that severe COVID-19 patients, whether or not they experience ARDS, display similar immune profiles. We show an increase in CD14+HLA-DRlow and CD14lowCD16+ monocytes correlating to the occurrence of adverse events during the ICU stay. We demonstrate that COVID-19-associated ARDS displays a specific immune profile and may benefit from personalized therapy in addition to standard ARDS management.

Continuous MYD88 Activation Is Associated With Expansion and Then Transformation of IgM Differentiating Plasma Cells.

Activating mutations of MYD88 (MYD88L265P being the far most frequent) are found in most cases of Waldenström macroglobulinemia (WM) as well as in various aggressive B-cell lymphoma entities with features of plasma cell (PC) differentiation, such as activated B-cell type diffuse large B-cell lymphoma (DLBCL). To understand how MYD88 activation exerts its transformation potential, we developed a new mouse model in which the MYD88L252P protein, the murine ortholog of human MYD88L265P, is continuously expressed in CD19 positive B-cells together with the Yellow Fluorescent Protein (Myd88L252P mice). In bone marrow, IgM B and plasma cells were expanded with a CD138 expression continuum from IgMhigh CD138low to IgMlow CD138high cells and the progressive loss of the B220 marker. Serum protein electrophoresis (SPE) longitudinal analysis of 40 Myd88L252P mice (16 to 56 weeks old) demonstrated that ageing was first associated with serum polyclonal hyper gammaglobulinemia (hyper Ig) and followed by a monoclonal immunoglobulin (Ig) peak related to a progressive increase in IgM serum levels. All Myd88L252P mice exhibited spleen enlargement which was directly correlated with the SPE profile and was maximal for monoclonal Ig peaks. Myd88L252P mice exhibited very early increased IgM PC differentiation. Most likely due to an early increase in the Ki67 proliferation index, IgM lymphoplasmacytic (LP) and plasma cells continuously expanded with age being first associated with hyper Ig and then with monoclonal Ig peak. This peak was consistently associated with a spleen LP-like B-cell lymphoma. Clonal expression of both membrane and secreted µ chain isoforms was demonstrated at the mRNA level by high throughput sequencing. The Myd88L252P tumor transcriptomic signature identified both proliferation and canonical NF-κB p65/RelA activation. Comparison with MYD88L265P WM showed that Myd88L252P tumors also shared the typical lymphoplasmacytic transcriptomic signature of WM bone marrow purified tumor B-cells. Altogether these results demonstrate for the first time that continuous MYD88 activation is specifically associated with clonal transformation of differentiating IgM B-cells. Since MYD88L252P targets the IgM PC differentiation continuum, it provides an interesting preclinical model for development of new therapeutic approaches to both WM and aggressive MYD88 associated DLBCLs.

Transcriptomic and genomic heterogeneity in blastic plasmacytoid dendritic cell neoplasms: from ontogeny to oncogenesis.

Oncogenesis and ontogeny of blastic plasmacytoid dendritic cell neoplasm (BPDCN) remain uncertain, between canonical plasmacytoid dendritic cells (pDCs) and AXL+ SIGLEC6+ DCs (AS-DCs). We compared 12 BPDCN to 164 acute leukemia by Affymetrix HG-U133 Plus 2.0 arrays: BPDCN were closer to B-cell acute lymphoblastic leukemia (ALL), with enrichment in pDC, B-cell signatures, vesicular transport, deubiquitination pathways, and AS-DC signatures, but only in some cases. Importantly, 1 T-cell ALL clustered with BPDCN, with compatible morphology, immunophenotype (cCD3+ sCD3- CD123+ cTCL1+ CD304+), and genetics. Many oncogenetic pathways are deregulated in BPDCN compared with normal pDC, such as cell-cycle kinases, and importantly, the transcription factor SOX4, involved in B ontogeny, pDC ontogeny, and cancer cell invasion. High-throughput sequencing (HaloPlex) showed myeloid mutations (TET2, 62%; ASXL1, 46%; ZRSR2, 31%) associated with lymphoid mutations (IKZF1), whereas single-nucleotide polymorphism (SNP) array (Affymetrix SNP array 6.0) revealed frequent losses (mean: 9 per patient) involving key hematological oncogenes (RB1, IKZF1/2/3, ETV6, NR3C1, CDKN2A/B, TP53) and immune response genes (IFNGR, TGFB, CLEC4C, IFNA cluster). Various markers suggest an AS-DC origin, but not in all patients, and some of these abnormalities are related to the leukemogenesis process, such as the 9p deletion, leading to decreased expression of genes encoding type I interferons. In addition, the AS-DC profile is only found in a subgroup of patients. Overall, the cellular ontogenic origin of BPDCN remains to be characterized, and these results highlight the heterogeneity of BPDCN, with a risk of a diagnostic trap.

Comprehensive Epstein-Barr Virus Transcriptome by RNA-Sequencing in Angioimmunoblastic T Cell Lymphoma (AITL) and Other Lymphomas.

The Epstein-Barr virus (EBV) is associated with angioimmunoblastic T cell lymphoma (AITL) in more than 80% of cases. Few studies have focused on this association and it is not clear now what role the virus plays in this pathology. We used next-generation sequencing (NGS) to study EBV transcriptome in 14 AITLs compared to 21 other lymphoma samples and 11 cell lines including 4 lymphoblastoid cell lines (LCLs). Viral transcripts were recovered using capture probes and sequencing was performed on Illumina. Bam-HI A rightward transcripts (BARTs) were the most latency transcripts expressed in AITLs, suggesting they may play a role in this pathology. Thus, BARTs, already described as highly expressed in carcinoma cells, are also very present in AITLs and other lymphomas. They were poorly expressed in cell lines other than LCLs. AITLs showed a latency IIc, with BNLF2a gene expression. For most AITLs, BCRF1, which encodes a homologous protein of human interleukin 10, vIL-10, was in addition expressed. This co-expression can contribute to immune escape and survival of infected cells. Considering these results, it can be assumed that EBV plays a pathogenic role in AITLs.

Plasmacytoid dendritic cells proliferation associated with acute myeloid leukemia: phenotype profile and mutation landscape

Neoplasms involving plasmacytoid Dendritic Cells (pDCs) include Blastic pDC Neoplasms (BPDCN) and other pDC proliferations, where pDCs are associated with myeloid malignancies: most frequently Chronic MyeloMonocytic Leukemia (CMML) but also Acute Myeloid Leukemia (AML), hereafter named pDC-AML. We aimed to determine the reactive or neoplastic origin of pDCs in pDC-AML, and their link with the CD34+ blasts, monocytes or conventional DCs (cDCs) associated in the same sample, by phenotypic and molecular analyses (targeted NGS, 70 genes). We compared 15 pDC-AML at diagnosis with 21 BPDCN and 11 normal pDCs from healthy donors. CD45low CD34+ blasts were found in all cases (10-80% of medullar cells), associated with pDCs (4-36%), monocytes in 14 cases (1-10%) and cDCs (2 cases, 4.8-19%). pDCs in pDC-AML harbor a clearly different phenotype from BPDCN: CD4+ CD56- in 100% of cases, most frequently CD303+, CD304+ and CD34+; lower expression of cTCL1 and CD123 with isolated lymphoid markers (CD22/CD7/CD5) in some cases, suggesting a pre-pDC stage. In all cases, pDCs, monocytes and cDC are neoplastic since they harbor the same mutations as CD34+ blasts. RUNX1 is the most commonly mutated gene: detected in all AML with minimal differentiation (M0-AML) but not in the other cases. Despite low number of cases, the systematic association between M0-AML, RUNX1 mutations and an excess of pDC is puzzling. Further evaluation in a larger cohort is required to confirm RUNX1 mutations in pDC-AML with minimal differentiation and to investigate whether it represents a proliferation of blasts with macrophage and DC progenitor potential.

A reduced panel of eight genes (ATM, SF3B1, NOTCH1, BIRC3, XPO1, MYD88, TNFAIP3, and TP53) as an estimator of the tumor mutational burden in chronic lymphocytic leukemia.

INTRODUCTION: Mutational complexity or tumor mutational burden (TMB) influences the course of chronic lymphocytic leukemia (CLL). However, this information is not routinely used because TMB is usually obtained from whole genome or exome, or from large gene panel high-throughput sequencing. METHODS: Here, we used the C-Harrel concordance index to determine the minimum panel of genes for which mutations predict treatment-free survival (TFS) as well as large resequencing panels. RESULTS: An eight gene estimator was defined encompassing ATM, SF3B1, NOTCH1, BIRC3, XPO1, MYD88, TNFAIP3, and TP53. TMB estimated from either a large panel of genes or the eight gene estimator was increased in treated patients or in those with a short TFS (<2 years), unmutated IGHV gene or with an unfavorable karyotype. Being an independent prognostic parameter, any mutation in the eight gene estimator predicted a shorter TFS better than Binet stage and IGHV mutational status among patients with an apparently non-progressive disease (TFS >6 months). Strikingly, the eight gene estimator was also highly informative for patients with Binet stage A CLL or with a good prognosis karyotype. CONCLUSION: These results suggest that the eight gene estimator, that is easily achievable by high-throughput resequencing, brings robust and valuable information that predicts evolution of untreated patients at diagnosis better than any other parameter.