Preparedness and activities of the anti-SARS-CoV-2 convalescent plasma bank in the Veneto region (Italy): An organizational model for future emergencies.

BACKGROUND: Convalescent plasma (CP) has been used in the past in various pandemics, in particular in H1N1, SARS and MERS infections. In Spring 2020, when ongoing the SARS-CoV-2 pandemics, the Veneto Region (V-R) has proposed setting-up an anti-SARS-CoV-2 CP (CCP) Bank, with the aim of preparing a supply of CCP immediately available in case of subsequest epidemic waves. MATERIALS AND METHODS: Key-points to be developed for a quick set-up of the V-R CCP Bank have been recruitment of donors recovered from COVID-19 infection, laboratory analysis for the biological qualification of the CCP units, including titre of neutralizing antibodies and reduction of pathogens, according to National Blood Centre (CNS) Directives, adaptation of the V-R Information Technology systems and cost analysis. Some activities, including diagnostic and viral inactivation processes, have been centralized in 2 or 3 sites. Laboratory analysis upon preliminary admission of the donor included all tests required by the Italian laws and the CNS directives. RESULTS: From April to August 2020, 3,298 people have contacted the V-R Blood Transfusion Services: of these, 1,632 have been evaluated and examined as first time donors and those found to be suitable have carried out 955 donations, from which 2,626 therapeutic fractions have been obtained, at a cost around 215,00 Euro. Since October 2020, the number of COVID-19 inpatients has had a surge with a heavy hospital overload. Moreover, the high request of CCP therapy by clinicians has been just as unexpected, showing a wide therapeutic use. CONCLUSIONS: The organizational model here presented, which has allowed the rapid collection of a large amount of CCP, could be useful when facing new pandemic outbreaks, especially in low and middle income countries, with generally acceptable costs.

Cortactin, another player in the Lyn signaling pathway, is over-expressed and alternatively spliced in leukemic cells from patients with B-cell chronic lymphocytic leukemia.

Cortactin, an actin binding protein and Lyn substrate, is up-regulated in several cancers and its level is associated with increased cell migration, metastasis and poor prognosis. The identification that the Src kinase Lyn and its substrate HS1 are over-expressed in B-cell chronic lymphocytic leukemia and involved in resistance to chemotherapy and poor prognosis, prompted us to investigate the role of cortactin, an HS1 homolog, in the pathogenesis and progression of this disorder. In this study, we observed that cortactin is over-expressed in leukemic cells of patients (1.10 ± 0.12) with respect to normal B lymphocytes (0.19 ± 0.06; P=0.0065). Fifty-three percent of our patients expressed the WT mRNA and p80/85 protein isoforms, usually lacking in normal B lymphocytes which express the SV1 variant and the p70/75 protein isoforms. Moreover, we found an association of the cortactin overexpression and negative prognostic factors, including ZAP-70 (P<0.01), CD38 (P<0.01) and somatic hypermutations in the immunoglobulin heavy-chain variable region (P<0.01). Our results show that patients with B-cell chronic lymphocytic leukemia express high levels of cortactin with a particular overexpression of the WT isoform that is lacking in normal B cells, and a correlation to poor prognosis, suggesting that this protein could be relevant in the pathogenesis and aggressiveness of the disease.

Bendamustine in chronic lymphocytic leukemia: outcome according to different clinical and biological prognostic factors in the everyday clinical practice.

Bendamustine proved to be effective for the treatment of chronic lymphocytic leukemia (CLL). However, the relationship between its activity with clinico-biological prognosticators has been addressed only in few studies. We retrospectively evaluated the efficacy of bendamustine, in a real-life contest, on 142 patients, median age 70 years, median number of previous regimens 2 (0-8, 13% previously untreated). Bendamustine was administered for a median number of 4 cycles, in 84% of cases with rituximab. Overall (ORR) and complete response (CRR) rates were 68 and 16.5%, respectively. Multivariate analysis demonstrated a relationship between ORR and number of prior treatments (OR 0.25, 95% CI 0.08-0.71; P = 0.009), del(17p) (OR 0.10, 95% CI 0.03-0.32; P < 0.001) and concomitant rituximab (OR 4.37, 95% CI 1.12-17.04; P = 0.033). The estimated 1- and 2-years overall survival (OS) and progression free survival (PFS) rates were 76, 61, 51, and 26%, respectively. Previous sensitivity to fludarabine (HR 0.36, 95% CI 0.16-0.82), response to bendamustine (HR 0.21, 95% CI 0.10-0.45), and del(17p) (HR 2.18, 95% CI 1.002-4.74) had a prognostic significance in multivariate analysis for PFS, while the number of previous therapies (HR 3.48, 95% CI 1.29-9.38; P = 0.014), concomitant use of rituximab (HR 0.32, 95% CI 0.11-0.93) and response to bendamustine (HR 0.22, 95% CI 0.07-0.66) were significant for OS. Side effects included grade 3-4 neutropenia, infections, thrombocytopenia and anemia which occurred in 40, 14, 14, and 10% of patients, respectively. These results confirm the activity and safety of bendamustine and rituximab combination even in patients with unfavorable clinical and biological features excluding del(17p).

Peculiar whole blood rotation thromboelastometry (Rotem) profile in 40 sideropenic anaemia patients.

The ROtation ThromboElastoMetry analyser (ROTEM, Pentapharm, Munich, Germany) is useful for studying whole blood (WB) clot formation and lysis. Reduction of haematocrit (HCT) has been reported to influence traditional thromboelastography parameters without compromising "in vitro" blood coagulation. We performed this case-control study to evaluate ROTEM profiles in sideropenic anaemia patients with different degrees of reduction of HCT levels. Forty consecutively referred patients with sideropenic anaemia were enrolled. A group of 40 healthy age and gender matched subjects acted as a control. The influence of HCT on ROTEM was assessed in the study population and in a model of artificially reconstituted blood with modified HCT values. Cases presented significantly increased levels of maximum clot firmness (MCF) as compared to controls (p < 0.001) mimicking a sort of "hypercoagulable profile". However, thrombin generation tests failed to detect an increase in thrombin generation in cases as compared to controls. A statistically significant inverse linear correlation between HCT and MCF (p < 0.0001) was found. In addition, ROTEM profiles following "in vitro" manipulation of HCT confirmed the inverse linear correlation between HCT and MCF found in the study population. In conclusion, the increased clot firmness found by ROTEM in anaemic patients is likely to be related to the method in itself rather than representing a marker of hypercoagulability "in vivo". Since ROTEM is widely used by anaesthesiologists when deciding the optimisation of products supplementation during surgery, attention should be paid in the case of anaemic patients taking depending on the peculiar thrombo-elastography profile found.

Cross-talk between chronic lymphocytic leukemia (CLL) tumor B cells and mesenchymal stromal cells (MSCs): implications for neoplastic cell survival.

Leukemic cells from Chronic Lymphocytic Leukemia (CLL) patients interact with stromal cells of the surrounding microenvironment. Mesenchymal Stromal Cells (MSCs) represent the main population in CLL marrow stroma, which may play a key role for disease support and progression. In this study we evaluated whether MSCs influence in vitro CLL cell survival. MSCs were isolated from the bone marrow of 46 CLL patients and were characterized by flow cytometry analysis. Following co-culture of MSCs and leukemic B cells, we demonstrated that MSCs were able to improve leukemic B cell viability, this latter being differently dependent from the signals coming from MSCs. In addition, we found that the co-culture of MSCs with leukemic B cells induced an increased production of IL-8, CCL4, CCL11, and CXCL10 chemokines.As far as drug resistance is concerned, MSCs counteract the cytotoxic effect of Fludarabine/Cyclophosphamide administration in vivo, whereas they do not protect CLL cells from the apoptosis induced by the kinase inhibitors Bafetinib and Ibrutinib. The evidence that leukemic clones are conditioned by environmental stimuli suggest new putative targets for therapy in CLL patients.

Integrated CLL Scoring System, a New and Simple Index to Predict Time to Treatment and Overall Survival in Patients With Chronic Lymphocytic Leukemia.

INTRODUCTION: Several prognostic factors have been identified to predict the outcome of patients with chronic lymphocytic leukemia (CLL), but only a few studies analyzed more markers together. PATIENTS AND METHODS: Taking advantage of a population of 608 patients, we identified the strongest prognostic markers of survival and, subsequently, in a cohort of 212 patients we integrated data of cytogenetic lesions, IGHV mutational status, and CD38 expression in a new and easy scoring system we called the integrated CLL scoring system (ICSS). ICSS defines 3 groups of risk: (1) low risk (patients with 13q(-) or normal fluorescence in-situ hybridization analysis results, mutated IGHV, and CD38) (2) high risk (all 11q(-) or 17p(-) patients and/or all unmutated IGHV and CD38(+) patients); and (3) intermediate risk (all remaining patients). RESULTS: Using only these 3 already available prognostic factors, we were able to properly redefine patients and better predict the clinical course of the disease. CONCLUSION: ICSS could become a useful tool for CLL patients' management.

Endothelial, platelet, and tissue factor-bearing microparticles in cancer patients with and without venous thromboembolism.

BACKGROUND: Cancer is a prothrombotic state, with an increased prevalence of venous thromboembolism (VTE). Microparticles (MPs) are sub-micron-sized vesicles derived from activated or apoptotic cells that may play a role in VTE, although evidence of this association is still limited. OBJECTIVES: To evaluate the hypothesis that elevated numbers of endothelial (EMPs), platelets (PMPs), and Tissue Factor-bearing MPs (TF(+)MPs) in plasma may contribute to cancer-associated thrombosis. PATIENTS/METHODS: EMPs, PMPs and TF(+)MPs plasma levels were measured in 90 consecutive patients (cases) referred to our Department (30 with a first episode of unprovoked VTE; 30 with active cancer; 30 with a diagnosis of acute VTE associated with active cancer), and in a group of 90 healthy subjects (controls). MPs analyses were performed by flow-cytometry (Cytomics FC500). RESULTS: Cases showed statistically significant higher (mean ± SD) circulating EMPs and PMPs plasma levels (920 ± 341 and 1221 ± 413 MP/µL, respectively) than controls (299 ± 102 and 495 ± 241 MP/µL; p<0.005). Moreover cancer patients (with and without VTE) showed higher (mean ± SD) TF(+)MPs (927 ± 415 MPs/µL) than controls (204 ± 112 MPs/µL; p<0.001). The subgroup of cancer patients plus VTE showed statistically significant higher TF(+)MPs plasma levels (1019 ± 656 MPs/µL) than cancer patients without VTE (755 ± 391 MPs/µL, p = 0.002). Multivariate analysis failed to show a significant association between elevated TF(+)MPs and VTE in cancer patients. CONCLUSIONS: Our results suggest that MPs might be an important intermediate in the cascade of cellular injury and vascular dysfunctions underlying the process of thrombosis, particularly in cancer. Further clinical investigations are needed to confirm the precise role of MPs in predicting hypercoagulable state in patients with cancer.