Emergence of immunosuppressive LOX-1+ PMN-MDSC in septic shock and severe COVID-19 patients with acute respiratory distress syndrome.

Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of immature myeloid cells with immunosuppressive properties. In cancer patients, the expression of lectin-type oxidized LDL receptor 1 (LOX-1) on granulocytic MDSC identifies a subset of MDSC that retains the most potent immunosuppressive properties. The main objective of the present work was to explore the presence of LOX-1+ MDSC in bacterial and viral sepsis. To this end, whole blood LOX-1+ cells were phenotypically, morphologically, and functionally characterized. They were monitored in 39 coronavirus disease-19 (COVID-19, viral sepsis) and 48 septic shock (bacterial sepsis) patients longitudinally sampled five times over a 3 wk period in intensive care units (ICUs). The phenotype, morphology, and immunosuppressive functions of LOX-1+ cells demonstrated that they were polymorphonuclear MDSC. In patients, we observed the significant emergence of LOX-1+ MDSC in both groups. The peak of LOX-1+ MDSC was 1 wk delayed with respect to ICU admission. In COVID-19, their elevation was more pronounced in patients with acute respiratory distress syndrome. The persistence of these cells may contribute to long lasting immunosuppression leaving the patient unable to efficiently resolve infections.

CAR-HEMATOTOX: a model for CAR T-cell-related hematologic toxicity in relapsed/refractory large B-cell lymphoma.

Hematotoxicity represents a frequent chimeric antigen receptor (CAR) T-cell-related adverse event and remains poorly understood. In this multicenter analysis, we studied patterns of hematopoietic reconstitution and evaluated potential predictive markers in 258 patients receiving axicabtagene ciloleucel (axi-cel) or tisagenlecleucel (tisa-cel) for relapsed/refractory large B-cell lymphoma. We observed profound (absolute neutrophil count [ANC] <100 cells per µL) neutropenia in 72% of patients and prolonged (21 days or longer) neutropenia in 64% of patients. The median duration of severe neutropenia (ANC < 500 cells per µL) was 9 days. We aimed to identify predictive biomarkers of hematotoxicity using the duration of severe neutropenia until day +60 as the primary end point. In the training cohort (n = 58), we observed a significant correlation with baseline thrombocytopenia (r = -0.43; P = .001) and hyperferritinemia (r = 0.54; P < .0001) on univariate and multivariate analysis. Incidence and severity of cytokine-release syndrome, immune effector cell-associated neurotoxicity syndrome, and peak cytokine levels were not associated with the primary end point. We created the CAR-HEMATOTOX model, which included markers associated with hematopoietic reserve (eg, platelet count, hemoglobin, and ANC) and baseline inflammation (eg, C-reactive protein and ferritin). This model was validated in independent cohorts, one from Europe (n = 91) and one from the United States (n = 109) and discriminated patients with severe neutropenia ≥14 days to <14 days (pooled validation: area under the curve, 0.89; sensitivity, 89%; specificity, 68%). A high CAR-HEMATOTOX score resulted in a longer duration of neutropenia (12 vs 5.5 days; P < .001) and a higher incidence of severe thrombocytopenia (87% vs 34%; P < .001) and anemia (96% vs 40%; P < .001). The score implicates bone marrow reserve and inflammation prior to CAR T-cell therapy as key features associated with delayed cytopenia and will be useful for risk-adapted management of hematotoxicity.

Toll-like receptor expression and function differ between splenic marginal zone B cell lymphoma and splenic diffuse red pulp B cell lymphoma.

In splenic marginal zone lymphoma (SMZL), specific and functional Toll-like Receptor (TLR) patterns have been recently described, suggesting their involvement in tumoral proliferation. Splenic diffuse red pulp lymphoma with villous lymphocytes (SDRPL) is close to but distinct from SMZL, justifying here the comparison of TLR patterns and functionality in both entities. Distinct TLR profiles were observed in both lymphoma subtypes. SDRPL B cells showed higher expression of TLR7 and to a lesser degree TLR9, in comparison to SMZL B cells. In both entities, TLR7 and TLR9 pathways appeared functional, as shown by IL-6 production upon TLR7 and TLR9 agonists stimulations. Interestingly, circulating SDRPL, but not SMZL B cells, constitutively expressed CD86. In addition, stimulation with both TLR7 and TLR9 agonists significantly increased CD80 expression in circulating SDRPL but not SMZL B cells. Finally, TLR7 and TLR9 stimulations had no impact on proliferation and apoptosis of SMZL or SDRPL B cells. In conclusion, SMZL and SDRPL may derive from different splenic memory B cells with specific immunological features that can be used as diagnosis markers in the peripheral blood.

Septic Shock Shapes B Cell Response toward an Exhausted-like/Immunoregulatory Profile in Patients.

Septic shock is accompanied by the development of immune dysfunctions whose intensity and duration are associated with increased risk of secondary infections and mortality. Although B lymphocytes play a pivotal role in the immune response to infections, no comprehensive exploration of circulating B cell status has been performed during the immunosuppressive phase of septic shock. Thus, our aim was to extensively characterize the phenotype and function of B cells in septic shock, including IL-10 production. Circulating B lymphocyte phenotype and function were evaluated by flow cytometry on fresh whole blood and after ex vivo stimulation in adult septic shock patients sampled at day 1, 3, and 6 after the onset of shock. The circulating B cell number was reduced in septic shock patients, whereas the B cell proportion among total lymphocytes was increased. The remaining circulating B lymphocytes presented with decreased MHC class II expression and increased CD21low CD95high exhausted-like phenotype but showed no change in maturation status. Circulating B cell functions were markedly altered after sepsis with reduced ex vivo activation and proliferation capacities. Finally, B cell response after septic shock was characterized by a clear plasmacytosis and an increased IL-10 production in remaining B cells from patients after ex vivo stimulation. During the sepsis-induced immunosuppression phase, B cell response is altered and is oriented toward an exhausted-like/immunoregulatory profile. Further studies are now needed to confirm the immunoregulatory properties of B lymphocytes and evaluate their role in sepsis-induced immunosuppression.

Exome sequencing identifies recurrent BCOR alterations and the absence of KLF2, TNFAIP3 and MYD88 mutations in splenic diffuse red pulp small B-cell lymphoma.

Splenic diffuse red pulp lymphoma is an indolent small B-cell lymphoma recognized as a provisional entity in the World Health Organization 2008 classification. Its precise relationship to other related splenic B-cell lymphomas with frequent leukemic involvement or other lymphoproliferative disorders remains undetermined. We performed whole-exome sequencing to explore the genetic landscape of ten cases of splenic diffuse red pulp lymphoma using paired tumor and normal samples. A selection of 109 somatic mutations was then evaluated in a cohort including 42 samples of splenic diffuse red pulp lymphoma and compared to those identified in 46 samples of splenic marginal zone lymphoma and eight samples of hairy-cell leukemia. Recurrent mutations or losses in BCOR (the gene encoding the BCL6 corepressor) - frameshift (n=3), nonsense (n=2), splicing site (n=1), and copy number loss (n=4) - were identified in 10/42 samples of splenic diffuse red pulp lymphoma (24%), whereas only one frameshift mutation was identified in 46 cases of splenic marginal zone lymphoma (2%). Inversely, KLF2, TNFAIP3 and MYD88, common mutations in splenic marginal zone lymphoma, were rare (one KLF2 mutant in 42 samples; 2%) or absent (TNFAIP3 and MYD88) in splenic diffuse red pulp lymphoma. These findings define an original genetic profile of splenic diffuse red pulp lymphoma and suggest that the mechanisms of pathogenesis of this lymphoma are distinct from those of splenic marginal zone lymphoma and hairy-cell leukemia.

Splenic diffuse red pulp lymphoma has a distinct pattern of somatic mutations amongst B-cell malignancies.

Splenic Diffuse Red Pulp Lymphoma (SDRPL) has been recently introduced as a provisional entity but differential diagnosis with other splenic lymphomas is needed to be clarified since the therapeutic approaches are distinct. Recently described recurrent mutations or CD180 expression appear useful for differential diagnosis. We completed our previous description in a larger cohort including 53 patients selected on the presence of characteristic villous cells in peripheral blood (PB) and a specific immunophenotype. Immunoglobulin heavy variable (IGHV), BRAF, MYD88, and NOTCH2 mutations were determined and CD180 and BRAF expressions were assessed. Most cases (79%) were IGHV mutated with an overrepresentation of IGHV3-23 (19%) and IGHV4-34 (21%). MYD88 L265P and NOTCH2 mutations were observed in one case each, whereas no BRAF V600E mutation or expression was found. All cases demonstrated a high CD180 expression. Those results strengthen the concept that SDRPL does emerge as a new lymphoma entity distinct from the other splenic lymphomas with circulating lymphocytes.

New Quantitative Method to Identify NPM1 Mutations in Acute Myeloid Leukaemia.

Somatic mutations in the NPM1 gene, which encodes for nucleophosmin, have been reported to be the most frequent genetic abnormalities found in acute myeloid leukaemia (AML). Their identification and quantification remain crucial for the patients' residual disease monitoring. We investigated a new method that could represent a novel reliable alternative to sequencing for its identification. This method was based on high-resolution melting analysis in order to detect mutated patients and on an allele-specific oligonucleotide real-time quantitative polymerase chain reaction (ASO-RQ-PCR) for the identification and quantification of the transcripts carrying NPM1 mutations (NPM1m). Few patients carrying known NPM1m enabled us to set up a table with the different primers' ΔCT values, identifying a profile for each mutation type. We then analysed a series of 337 AML patients' samples for NPM1 mutational status characterization and confirmed the ASO-RQ-PCR results by direct sequencing. We identified some mutations in 86 samples, and the results were fully correlated in 100% of the 36 sequenced samples. We also detected other rare NPM1m in two samples, that we confirmed by direct sequencing. This highly specific method provides a novel quick, useful, and costless tool, easy to use in routine practice.

Immunoarchitectural patterns in splenic marginal zone lymphoma: correlations with chromosomal aberrations, IGHV mutations, and survival. A study of 76 cases.

AIMS: To describe 76 cases of splenic marginal zone lymphoma (SMZL), including correlations with clinical and other characteristics. METHODS AND RESULTS: Patients were predominantly female, with a median age of 62 years. The main clinical presentation was splenomegaly, except for eight cases presenting with evolution of autoimmune disorders or spontaneous splenic rupture. White pulp infiltration was nodular, with a monophasic (42%) or biphasic (53%) pattern, and associated diffuse or nodular infiltration of the red pulp, except for four cases which had atrophic white pulp. Plasmacytic differentiation and the MYD88 L265P mutation were observed in 18% and 5% of the cases, respectively. Histological progression was considered in cases with a significant association of large cells with Ki67 > 30% and macronodular architecture (P = 0.001). Other significant correlations were found between del7q (44%) and del6q (17%) (P = 0.018), IGHV1-2*04 segment usage (35%) (P = 0.001) and unmutated IGHV (39%) (P = 0.019), and between CD5 expression (27%) and higher lymphocytosis (P = 0.002). Patients requiring intensive chemotherapy after splenectomy because of clinical and/or histological progression had significantly shorter overall survival (P = 0.012). CONCLUSIONS: We report the histological spectrum of SMZL, and discuss the differential diagnosis and requirement for molecular and cytogenetic analysis in atypical cases.

High DNA methyltransferase DNMT3B levels: a poor prognostic marker in acute myeloid leukemia.

It has been recently shown that DNA methyl transferase overexpression is correlated with unfavourable prognosis in human malignancies while methylation deregulation remains a hallmark that defines acute myeloid leukemia (AML). The oncogenic transcription factor EVI1 is involved in methylation deregulation and its overexpression plays a major role for predicting an adverse outcome. Moreover, the identification of DNMT3A mutations in AML patients has recently been described as a poor prognostic indicator. In order to clarify relationship between these key actors in methylation mechanisms and their potential impact on patient outcomes, we analysed 195 de novo AML patients for the expression of DNMT3A, 3B (and its non-catalytic variant 3B(NC)) and their correlations with the outcome and the expression of other common prognostic genetic biomarkers (EVI1, NPM1, FLT3ITD/TKD and MLL) in adult AML. The overexpression of DNMT3B/3B(NC) is (i) significantly correlated with a shorter overall survival, and (ii) inversely significantly correlated with event-free survival and DNMT3A expression level. Moreover, multivariate analysis showed that a high expression level of DNMT3B/3B(NC) is statistically a significant independent poor prognostic indicator. This study represents the first report showing that the overexpression of DNMT3B/3B(NC) is an independent predictor of poor survival in AML. Its quantification should be implemented to the genetic profile used to stratify patients for therapeutical strategies and should be useful to identify patients who may benefit from therapy based on demethylating agents.

In non-follicular lymphoproliferative disorders, IGH/BCL2-fusion is not restricted to chronic lymphocytic leukaemia.

The translocation t(14;18) and its t(2;18) and t(18,22) variants, which involve the BCL2 genetic hallmark for follicular lymphoma (FL), have been reported in several cases of chronic B-cell lymphoproliferative disease (CLPD) and frequently in chronic lymphocytic leukaemia (CLL). We describe here the clinical, morphological, immunological, cytogenetic and molecular findings from 37 cases of t(14;18)-positive CLPD, identified from our series of non-FL B-cell neoplasms (n=993) that were routinely analysed in peripheral blood by conventional cytogenetics analyses. The FL diagnosis was excluded by morphology and immunology (the samples were CD10 negative in all cases). The BCL2 translocations were observed in 22 CLL cases, including 7 monoclonal B-cell lymphocytosis (MBL) cases re-classified according to the new International Workshop on CLL criteria, six small lymphocytic lymphoma (SLL) cases, 1 splenic marginal zone lymphoma (SMZL) case and eight cases of unclassifiable CLPD with overlapping CLL/MZL features. In the CLL cases, the IGH/BCL2 fusion was remarkably associated with trisomy 12 (13/22) and mutated IGHV status (20/21) and did not affect the outcome. Moreover, most of these CLLs harboured a low mutation load of BCL6 gene and unmutated FAS (CD95) loci, which points to a post-germinal-centre cellular origin.

CD10 and ICOS expression by multiparametric flow cytometry in angioimmunoblastic T-cell lymphoma.

Angioimmunoblastic T-cell lymphoma is immunologically defined by the expression of CD10 and the follicular helper T cell (T(FH)) markers such as CXCL13, programmed death-1 (PD-1) and inducible T-cell costimulator (ICOS). This T(FH) profile has been mainly reported by immunohistochemistry. Here, using multiparametric flow cytometry, the relevance of ICOS and PD-1 to angioimmunoblastic T-cell lymphoma diagnosis was evaluated in lymph node (n=15) as well as in peripheral blood (n=13) among a series of 28 angioimmunoblastic T-cell lymphoma cases, in addition to the CD10 expression (available in 26 lymph node and 15 peripheral blood specimens). In this series, CD10 expression was present in 23/26 (88%) lymph node and in 12/15 (80%) peripheral blood cases and ICOS in 13/15 (87%) lymph node and in 6/13 (47%) peripheral blood cases, whereas neither significant CD10 nor ICOS T cells were identified in the control group (lymph nodes with reactive hyperplasia=10, peripheral blood of healthy donors=15). PD-1 expression was less informative as observed in both angioimmunoblastic T-cell lymphoma and control cases. The multiparametric approach allowed us to confirm the frequent blood dissemination in angioimmunoblastic T-cell lymphoma and to show that circulating neoplastic T cells correspond more often to a CD10-positive subset than to an ICOS-positive subset. Consequently, if ICOS constitutes an additional feature for the diagnosis of angioimmunoblastic T-cell lymphoma, it appears less sensitive than CD10 expression for the detection of circulating neoplastic T cells.

Telomeric damage in early stage of chronic lymphocytic leukemia correlates with shelterin dysregulation.

Cells of B-cell chronic lymphocytic leukemia (B-CLL) are characterized by short telomeres despite a low proliferative index. Because telomere length has been reported to be a valuable prognosis criteria, there is a great interest in a deep understanding of the origin and consequences of telomere dysfunction in this pathology. Cases of chromosome fusion involving extremely short telomeres have been reported at advanced stage. In the present study, we address the question of the existence of early telomere dysfunction during the B-CLL time course. In a series restricted to 23 newly diagnosed Binet stage A CLL patients compared with 12 healthy donors, we found a significant increase in recruitment of DNA-damage factors to telomeres showing telomere dysfunction in the early stage of the disease. Remarkably, the presence of dysfunctional telomeres did not correlate with telomere shortening or chromatin marks deregulation but with a down-regulation of 2 shelterin genes: ACD (coding for TPP1; P = .0464) and TINF2 (coding for TIN2; P = .0177). We propose that telomeric deprotection in the early step of CLL is not merely the consequence of telomere shortening but also of shelterin alteration.

Emergence of therapy-unrelated CML on a background of BCR-ABL-negative JAK2V617F-positive chronic idiopathic myelofibrosis.

We report the emergence of a chronic myeloid leukaemia (CML) during the course of a JAK2V617F-positive chronic idiopathic myelofibrosis (CIMF) in the absence of any myelosuppressive treatment. Although a response to imatinib was observed, the underlying myelofibrosis persisted after treatment and hydroxyurea was finally added to control the persistent thrombocytosis. Such rare patients with co-existing BCR-ABL translocation and JAK2V617F mutation must be identified in view of the possibility of targeted therapies. Moreover, the detection of BCR-ABL translocation appears to be crucial especially in the case of treated CIMF with an atypical course to identify CML before acute transformation.