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.

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.

TIF1γ requires sumoylation to exert its repressive activity on TGFß signaling.

TIF1γ, a new regulator of TGFß signaling, inhibits the Smad4-mediated TGFß response by interaction with Smad2/3 or ubiquitylation of Smad4. We have shown that TIF1γ participates in TGFß signaling as a negative regulator of Smad4 during the TGFß-induced epithelial-to-mesenchymal transition (EMT) in mammary epithelial cells, and during terminal differentiation of mammary alveolar epithelial cells and lactation. We demonstrate here that TIF1γ is sumoylated and interacts with Ubc9, the only known SUMO-conjugating enzyme. Four functional sumoylation sites lie within the middle domain of TIF1γ, the Smad interaction domain. We show that a sumoylation-defective TIF1γ mutant significantly reduces TIF1γ inhibition of Smad complexes and that of the Smad-mediated TGFß transcriptional response. Moreover, chromatin immunoprecipitation experiments indicate that TIF1γ sumoylation is required to limit Smad4 binding on the PAI-1 TGFß target gene promoter. Ectopic expression of TIF1γ in mammary epithelial cells inhibits TGFß-induced EMT, an effect relieved by expression of non-sumoylated TIF1γ. Taken together, our results identify a new TGFß regulatory layer, whereby sumoylation strengthens the TIF1γ repressive action on canonical TGFß signaling.

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.

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.

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.

Senior-COVID-Rea Cohort Study: A Geriatric Prediction Model of 30-day Mortality in Patients Aged over 60 Years in ICU for Severe COVID-19.

The SARS-COV2 pandemic induces tensions on health systems and ethical dilemmas. Practitioners need help tools to define patients not candidate for ICU admission. A multicentre observational study was performed to evaluate the impact of age and geriatric parameters on 30-day mortality in patients aged ≥60 years of age. Patients or next of kin were asked to answer a phone questionnaire assessing geriatric covariates 1 month before ICU admission. Among 290 screened patients, 231 were included between March 7 and May 7, 2020. In univariate, factors associated with lower 30-day survival were: age (per 10 years increase; OR 3.43, [95%CI: 2.13-5.53]), ≥3 CIRS-G grade ≥2 comorbidities (OR 2.49 [95%CI: 1.36-4.56]), impaired ADL, (OR 4.86 [95%CI: 2.44-9.72]), impaired IADL8 (OR 6.33 [95%CI: 3.31-12.10], p<0.001), frailty according to the Fried score (OR 4.33 [95%CI: 2.03-9.24]) or the CFS ≥5 (OR 3.79 [95%CI: 1.76-8.15]), 6-month fall history (OR 3.46 [95%CI: 1.58-7.63]). The final multivariate model included age (per 10 years increase; 2.94 [95%CI:1.78-5.04], p<0.001) and impaired IADL8 (OR 5.69 [95%CI: 2.90-11.47], p<0.001)). Considered as continuous variables, the model led to an AUC of 0.78 [95% CI: 0.72, 0.85]. Age and IADL8 provide independent prognostic factors for 30-day mortality in the considered population. Considering a risk of death exceeding 80% (82.6% [95%CI: 61.2% - 95.0%]), patients aged over 80 years with at least 1 IADL impairment appear as poor candidates for ICU admission.

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.

Prognosis of Old Intensive Care COVID-19 Patients at a Glance: The Senior COVID Study.

OBJECTIVE: Admission in the intensive care unit of the old patient with coronavirus disease 19 raises an ethical question concerning the scarce resources and their short-term mortality. METHODS: Patients aged over 60 from 7 different intensive care units admitted between March 1, 2020 and May 6, 2020, with a diagnosis of coronavirus disease 19 were included in the cohort. Twenty variables were collected during the admission, such as age, severity (Simplified Acute Physiology Score [SAPS] II), several data on physiological status before intensive care unit comorbidities, evaluation of autonomy, frailty, and biological variables. The objective was to model the 30-day mortality with relevant variables, compute their odds ratio associated with their 95% CI, and produce a nomogram to easily estimate and communicate the 30-day mortality. The performance of the model was estimated with the area under the receiving operating curve. RESULTS: We included 231 patients, among them 60 (26.0%) patients have died on the 30th day. The relevant variables selected to explain the 30-day mortality were Instrumental Activities of Daily Living (IADL) score (0.82 [0.71-0.94]), age 1.12 (1.07-1.18), SAPS II 1.05 (1.02-1.08), and dementia 6.22 (1.00-38.58). A nomogram was computed to visually represent the final model. Area under the receiving operating curve was at 0.833 (0.776-0.889). CONCLUSIONS: Age, autonomy, dementia, and severity at admission were important predictive variables for the 30-day mortality status, and the nomogram could help the physician in the decision-making process and the communication with the family.

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.

Deep Multi-Instance Learning Using Multi-Modal Data for Diagnosis of Lymphocytosis.

We investigate the use of recent advances in deep learning and propose an end-to-end trainable multi-instance convolutional neural network within a mixture-of-experts formulation that combines information from two types of data-images and clinical attributes-for the diagnosis of lymphocytosis. The convolutional network learns to extract meaningful features from images of blood cells using an embedding level approach and aggregates them. Moreover, the mixture-of-experts model combines information from these images as well as clinical attributes to form an end-to-end trainable pipeline for diagnosis of lymphocytosis. Our results demonstrate that even the convolutional network by itself is able to discover meaningful associations between the images and the diagnosis, indicating the presence of important unexploited information in the images. The mixture-of-experts formulation is shown to be more robust while maintaining performance via. a repeatability study to assess the effect of variability in data acquisition on the predictions. The proposed methods are compared with different methods from literature based both on conventional handcrafted features and machine learning, and on recent deep learning models based on attention mechanisms. Our method reports a balanced accuracy of [Formula: see text] and outperfroms the handcrafted feature-based and attention-based approaches as well that of biologists which scored [Formula: see text], [Formula: see text] and [Formula: see text] respectively. These results give insights on the potentials of the applicability of the proposed method in clinical practice. Our code and datasets can be found at https://github.com/msahasrabudhe/lymphoMIL.

Risk factors associated with day-30 mortality in patients over 60 years old admitted in ICU for severe COVID-19: the Senior-COVID-Rea Multicentre Survey protocol.

INTRODUCTION: With the spread of COVID-19 epidemic, health plans must be adapted continuously. There is an urgent need to define the best care courses of patients with COVID-19, especially in intensive care units (ICUs), according to their individualised benefit/risk ratio. Since older age is associated with poorer short-term and long-term outcomes, prediction models are needed, that may assist clinicians in their ICU admission decision. Senior-COVID-Rea was designed to evaluate, in patients over 60 years old admitted in ICU for severe COVID-19 disease, the impact of age and geriatric and paraclinical parameters on their mortality 30 days after ICU admission. METHODS AND ANALYSIS: This is a multicentre survey protocol to be conducted in seven hospitals of the Auvergne-Rhône-Alpes region, France. All patients over 60 years old admitted in ICU for severe COVID-19 infection (or their legally acceptable representative) will be proposed to enter the study and to fill in a questionnaire regarding their functional and nutritional parameters 1 month before COVID-19 infection. Paraclinical parameters at ICU admission will be collected: lymphocytes and neutrophils counts, high-fluorescent lymphoid cells and immature granulocytes percentages (Sysmex data), D-dimers, C-reactive protein, lactate dehydrogenase (LDH), creatinine, CT scan for lung extension rate as well as clinical resuscitation scores, and the delay between the first signs of infection and ICU admission. The primary outcome will be the overall survival at day 30 post-ICU admission. The analysis of factors predicting mortality at day 30 will be carried out using univariate and multivariate logistic regressions. Multivariate logistic regression will consider up to 15 factors.The ambition of this trial, which takes into account the different approaches of geriatric vulnerability, is to define the respective abilities of different operational criteria of frailty to predict patients' outcomes. ETHICS AND DISSEMINATION: The study protocol was ethically approved. The results of the primary and secondary objectives will be published in peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04422340.

Flagging performance of Sysmex XN-10 haematology analyser for malaria detection.

AIM: The aim was to assess the flagging performance of Sysmex XN-10 haematology analyser for malaria detection through the parasitic red blood cell ('pRBC') alarm. METHODS: We retrospectively studied 584 blood samples performed on the Sysmex XN-10 analyser that were tested for malaria. Sensitivity, specificity, positive and negative predictive values, and prevalence were established for the pRBC alarm. RESULTS: Sensitivity, specificity, and positive and negative predictive values for the pRBC flag were 7.8%, 100%, 100% and 87.7%, respectively. The prevalence of pRBC flag of 0.026% in the overall population was significantly different from the prevalence of 1.027% in the population tested for malaria. CONCLUSIONS: Considering the excellent specificity and the low prevalence of the flag in the overall population, we suggest, in case of the presence of pRBC flag, the implementation of a rapid review of the blood smear looking for Plasmodium, mostly if the patient had fever and had not been tested for malaria.

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.

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.