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Simple Summary: Plasmacytoid dendritic cells are the main type I interferon producing cells in humans and are able to modulate innate and adaptive immune responses. Tumor infiltration by plasmacytoid dendritic cells is already described and is associated with poor outcomes in cancers. In hematological diseases, Blastic Plasmacytoid Dendritic Cells Neoplasm (blastic counterpart) is well described, but little is known about tumor infiltration by mature plasmacytoid dendritic cells described in Myeloid Neoplasms (myeloid tumor cells and clonal pDC proliferation). This review provides a comprehensive overview of plasmacytoid dendritic cells in Myeloid Neoplasms. Plasmacytoid dendritic cells (pDC) are the main type I interferon producing cells in humans and are able to modulate innate and adaptive immune responses. Tumor infiltration by plasmacytoid dendritic cells is already well described and is associated with poor outcomes in cancers due to the tolerogenic activity of pDC. In hematological diseases, Blastic Plasmacytoid Dendritic Cells Neoplasm (BPDCN), aggressive leukemia derived from pDCs, is well described, but little is known about tumor infiltration by mature pDC described in Myeloid Neoplasms (MN). Recently, mature pDC proliferation (MPDCP) has been described as a differential diagnosis of BPDCN associated with acute myeloid leukemia (pDC-AML), myelodysplastic syndrome (pDC-MDS) and chronic myelomonocytic leukemia (pDC-CMML). Tumor cells are myeloid blasts and/or mature myeloid cells from related myeloid disorders and pDC derived from a clonal proliferation. The poor prognosis associated with MPDCP requires a better understanding of pDC biology, MN oncogenesis and immune response. This review provides a comprehensive overview about the biological aspects of pDCs, the description of pDC proliferation in MN, and an insight into putative therapies in pDC-AML regarding personalized medicine. [ABSTRACT FROM AUTHOR]

Although purine analogues have significantly improved the outcome of hairy cell leukaemia ( HCL) patients, 30-40% relapse, illustrating the need for minimal residual disease ( MRD) markers that can aid personalized therapeutic management. Diagnostic samples from 34 HCL patients were used to design an 8-colour flow cytometry (8- FC) tube for blood MRD (B/ RD) analysis (188 samples) which was compared to quantitative IGH polymerase chain reaction (Q- PCR) on 83 samples and to qualitative consensus IGH PCR clonality analysis on 165 samples. Despite heterogeneous HCL phenotypes at diagnosis, discrimination from normal B lymphocytes was possible in all cases using a single 8- FC tube, with a robust sensitivity of detection of 10−4, comparable to Q- PCR at this level, but preferable in terms of informativeness, simplicity and cost. B/ RD assessment of 15 patients achieving haematological complete remission after purine analogues was predictive of a clinically significant relapse risk: with a median follow-up of 95 months; only one of the nine patients with reproducible 8- FC B/ RD levels below 10−4 (B/ RDneg) relapsed, compared to 5/6 in the B/ RDpos group ( P = 0·003). These data demonstrate the clinical interest of a robust 8- FC HCL B/ RD strategy that could become a surrogate biomarker for therapeutic stratification and new drug assessment, which should be evaluated prospectively. [ABSTRACT FROM AUTHOR]

The current screening for eligibility of unrelated volunteer marrow donors comprises a complete clinical check-up, a blood CBC and serum protein immunoelectrophoresis. This allows to eliminate acute leukemias, myeloproliferative and myelodysplastic disorders, myelomas and MGUS. To date, the risk of transmission of chronic lymphocytic leukemia (CLL) disease is only evaluated by the clinical evaluation and CBC. We report here the case of a CLL-type MBL disease occurring in a 12-year-old boy after unrelated BMT. Deep biological investigations, as Immunophenotyping, cytogenetic and molecular biology allow us to determine the donor origin of the CLL clone. In 2010, 14.2% donor (105/737) for unrelated hematopoietic stem cell transplantation were over 45y. It is currently estimated (USA) that 1 in 210 men and women will be diagnosed with CLL during their lifetime. Given the long asymptomatic phase of CLL, this raises the case for a detection strategy analog to that used for MGUS and myeloma through serum protein electrophoresis. This case-report, to our knowledge, of a CLL-type MBL unrelated donor-to-recipient transmission through BMT raises ethical and practical questions, such as the proper information about disease transmission risk. The cost-effectiveness of a systematic peripheral blood Immunophenotyping in donors elder than 40y at time of stem cell donation should be evaluated. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented regarding increased level of endothelial-derived and small-size platelet-derived microparticles in psoriasis.

The diagnosis of plasmacytoid dendritic cell leukaemia (pDCL) is based on the immunophenotypic profile: CD4+ CD56+ lineageneg CD45RA+/ROneg CD11cneg CD116low CD123+ CD34neg CD36+ HLA-DR+. Several studies have reported pDCL cases that do not express this exact profile or expressing some lineage antigens that could thus be misdiagnosed. This study aimed to validate pDCL-specific markers for diagnosis by flow-cytometry or quantitative reverse transcription polymerase chain reaction on bone marrow samples. Expression of markers previously found in normal pDC was analysed in 16 pDCL, four pDCL presenting an atypical phenotype (apDCL) and 113 non-pDC – lymphoid or myeloid – acute leukaemia. CD123 was expressed at significantly higher levels in pDCL and apDCL. BDCA-2 was expressed on 12/16 pDCL and on 2/4 apDCL, but was never detected in the 113 non-pDC acute leukaemia cases. BDCA-4 expression was found on 13/16 pDCL, but also in 12% of non-pDC acute leukaemia. High levels of LILRA4 and TCL1A transcripts distinguished pDCL and apDCL from all other acute leukaemia (except B-cell acute lymphoblastic leukaemia for TCL1A). We thus propose a diagnosis strategy, scoring first the CD4+ CD56+/− MPOneg cCD3neg cCD79aneg CD11cneg profile and then the CD123high, BDCA-2 and BDCA-4 expression. Atypical pDCL can be also identified this way and non-pDC acute leukaemia excluded: this scoring strategy is useful for diagnosing pDCL and apDCL. [ABSTRACT FROM AUTHOR]

CD4+/CD56+ haematodermic neoplasm or ‘early’ plasmacytoid dendritic cell leukaemia/lymphoma (pDCL) was described as a disease entity in the last World Health Organisation/European Organisation for Research and Treatment of Cancer classification for cutaneous lymphomas. These leukaemia/lymphomas co-express CD4 and CD56 without any other lineage-specific markers and have been identified as arising from plasmacytoid dendritic cells. Despite a fairly homogeneous pattern of markers expressed by most pDCL, numerous distinctive features (e.g. cytological aspects and aberrant marker expression) have been reported. This may be related to the ‘lineage-independent developmental’ programme of dendritic cells, which may be able to develop from either immature or already committed haematopoietic progenitors. This highlights the need for specific validated markers to diagnose such aggressive leukaemia. Here, we propose –among others (e.g. T-cell leukaemia 1) – blood dendritic cell antigen-2 and high levels of CD123 expression as potential markers. In addition, we propose a multidisciplinary approach including several fields of haematology to improve pDCL diagnosis. [ABSTRACT FROM AUTHOR]

Studies the role of dendritic cells (DC) in hematopoietic stem cell transplantation. Potential sources of tolerogenic dendritic cells; Characterization of DC subsets; Interaction of mature DC with infused donor T cells; Modulation of allo-immune responses; Correlation between the host DC and occurrence of graft versus host disease.

Psoriasis involves TNF- α secretion leading to release of microparticles into the bloodstream. We investigated the effect of TNF blockers on microparticles levels before and after treatment in patients (twenty treated by anti- TNF- α agents and 6 by methotrexate) with severe psoriasis. Plasmatic microparticles were labelled using fluorescent monoclonal antibodies and were analysed using cytometry. Three months later, 70% of patients treated with anti- TNF- α agents achieved a reduction in PASI score of at least 75%. The clinical improvement in patients treated with anti- TNF- α agents was associated with a significant reduction of the mean number of platelet microparticles (2837/ μl vs 1849/ μl, P = 0.02) and of endothelial microparticles (64/ μl vs 22/ μl, P = 0.001). Microparticles are significantly decreased in psoriatic patients successfully treated by anti- TNF- α. Microparticles levels as circulating endothelial cells represent signs of endothelial dysfunction and are elevated in psoriasis. Then, TNF blockade may be effective to reduce cardiovascular risk through the reduction of circulating microparticles. [ABSTRACT FROM AUTHOR]

Production of functional plasmacytoid dendritic cells - targeted chimeric antigen receptor T cells from patients with immune-mediated inflammatory diseases After 15 days of culture, CAR123 expression was stable in both cases (Figure 1a) and CAR123 maintained a good proliferation rate: 244.3 ± 201.4% for patients ( I n i = 23) and 571.6 ± 294.5% for HD ( I n i = 5) ( I P i = 0.0052) (Figure 1b). Https://doi.org/10.1093/bjd/ljad105 Dear Editor, Mainly developed in the field of oncology, chimeric antigen receptor T-cell (CAR-T cell) therapy has been extended more recently to other areas such as infectious diseases, cardiac fibrosis or autoimmune diseases (AIDs).[1] Patients with refractory systemic lupus erythematosus (SLE) treated with CD19-targeted CAR-T cells demonstrated an improvement of clinical symptoms and normalization of laboratory parameters including seroconversion of anti-double-stranded DNA antibodies along with a deep depletion of B cells.[2] Increasing evidence suggests that plasmacytoid dendritic cells (pDCs) are involved in the development of different pDC-driven AIDs and immune-mediated inflammatory diseases (IMIDs), especially through the overproduction of proinflammatory cytokines such as type 1 interferons.[3] It is thus conceivable that elimination of pDCs might be beneficial to improve AID and IMID evolution as was recently demonstrated in a phase II trial involving patients with SLE treated with an antibody-binding of blood dendritic cell antigen 2 (BDCA2).[4] To the best of our knowledge, no CD123-targeted CAR-T cells (CAR123) have yet been developed for AID and IMID treatment. [Extracted from the article]

Background: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Results: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. Conclusions: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients. [ABSTRACT FROM AUTHOR]

Purpose: This study aimed to develop a method to detect ovarian residual disease by multicolor flow cytometry in acute leukemia patients. Methods: We designed an experimental model consisting in adding acute leukemia cells to a cell suspension obtained from healthy ovarian cortex. Leukemic cell detection within the ovarian cell suspension required the development of a specific myeloid antibody panel different from that commonly used for minimal residual disease (MRD) monitoring in bone marrow. The method was then used to detect ovarian residual disease in 11 acute leukemia patients. Results: Multicolor flow cytometry is able to evaluate the presence of viable leukemic cells in the ovarian cortex with good specificity and robust sensitivity of 10−4. We observed a good correlation between multicolor flow cytometry and quantitative polymerase chain reaction results. Ovarian residual disease detection by multicolor flow cytometry was positive in 3 out of 11 acute leukemia patients. Conclusion: Multicolor flow cytometry can potentially be applied to ovarian tissue from all acute leukemia patients and is essential to evaluate the risk of cancer re-seeding before autograft of ovarian tissue in case of acute leukemia. [ABSTRACT FROM AUTHOR]

Simple Summary: We used fresh or thawed Umbilical Cord Blood (UCB) to produce CAR-T cells directed against CD123, and we compared their functionality to Peripheral Blood (PB) CAR-T cells. T cells expressing CD123 CAR, derived from UCB, was exhibited through a high transduction rate, activation status, and cytotoxic potential in vitro as PB derived CAR-T cells. Moreover, we obtained T cells that had a less differentiated profile than the PB-derived T cells. UCB derived CAR-T can significantly control tumor progression in mice models. CAR-T obtained from thawed or fresh UCB gives the same results. Chimeric Antigen Receptor (CAR) therapy has led to great successes in patients with leukemia and lymphoma. Umbilical Cord Blood (UCB), stored in UCB banks, is an attractive source of T cells for CAR-T production. We used a third generation CD123 CAR-T (CD28/4-1BB), which was previously developed using an adult's Peripheral Blood (PB), to test the ability of obtaining CD123 CAR-T from fresh or cryopreserved UCB. We obtained a cell product with a high and stable transduction efficacy, and a poorly differentiated phenotype of CAR-T cells, while retaining high cytotoxic functions in vitro and in vivo. Moreover, CAR-T produced from cryopreserved UCB are as functional as CAR-T produced from fresh UCB. Overall, these data pave the way for the clinical development of UCB-derived CAR-T. UCB CAR-T could be transferred in an autologous manner (after an UCB transplant) to reduce post-transplant relapses, or in an allogeneic setting, thanks to fewer HLA restrictions which ease the requirements for a match between the donor and recipient. [ABSTRACT FROM AUTHOR]

Microparticles (MPs) are known to be increased in various malignancies and are involved in tumor invasion, angiogenesis, coagulation, and metastasis. We investigated the plasma levels of annexin-V MPs (AV+MPs), platelet-derived MPs (PMPs), and endothelial-derived MPs (EMPs) in patients with melanoma (n=129) and in healthy controls (n=49). A functional coagulation test STA Procoag-PPL measuring the clotting time was performed on samples containing MPs to evaluate their procoagulant potential. The plasma levels of PMPs, EMPs, and AV+MPs were significantly higher, and the clotting time-PPL was significantly lower in melanoma patients than in healthy controls. The plasma levels of PMPs, EMPs, and AV+MPs were higher in stage IV than in the other stages of melanoma, but with no significant difference. In addition, we observed an inverse correlation between PMPs, AV+MPs, and clotting times. Our data suggest that MPs are involved in the progression of melanoma and may be associated to melanoma-associated thrombogenesis. [ABSTRACT FROM AUTHOR]

Background: Cryopreservation of ovarian tissue is a fertility-preservation option for women before gonadotoxic treatments. However, cryopreserved ovarian tissue transplantation must be performed with caution in women with malignancies that may metastasize to the ovaries. For this purpose, detecting minimal residual disease (MRD) in the ovarian cortex using sensitive methods is a crucial step. We developed an automated ovarian tissue dissociation method to obtain ovarian cell suspensions. Results: We assessed MRD by multicolor flow cytometry (MFC) in cryopreserved ovarian cortex of 15 leukemia patients: 6 with B-cell acute lymphoblastic leukemia (B-ALL), 2 with T-cell acute lymphoblastic leukemia (T-ALL) and 7 with acute myeloid leukemia (AML). Ovarian MRD was positive in 5 of the 15 leukemia patients (one T-ALL and 4 AML). No B-ALL patient was positive by MFC. Quantitative reverse-transcribed polymerase chain reaction was performed when a molecular marker was available, and confirmed the MFC results for 3 patients tested. Xenografts into immunodeficient mice were also performed with ovarian cortical tissue from 10 leukemia patients, with no evidence of leukemic cells after the 6-month grafting period. Conclusions: In conclusion, this is the first study using MFC to detect MRD in ovarian cortical tissue from acute leukemia patients. MFC has been accepted in clinical practice for its ease of use, the large number of parameters available simultaneously, and high throughput analysis. We demonstrate here that MFC is a reliable method to detect MRD in cryopreserved ovarian tissue, with a view to controlling the oncological risk before ovarian tissue transplantation in leukemia patients. [ABSTRACT FROM AUTHOR]

1 Pre-treatment TEP SCAN (a) and after 3 months of treatment (b) A PET scan was performed after the third cycle of association showing a complete metabolic remission (Fig. 1:CAS:528:DC%2BD1MXmvFyjur0%3D. 10.1111/j.1365-2141.2009.07679.x 2 Garnache-Ottou F, Vidal C, Biichlé S, Renosi F, Poret E, Pagadoy M. How should we diagnose and treat blastic plasmacytoid dendritic cell neoplasm patients? Recent papers [[6]-[8]] showed that venetoclax combined with decitabine or 5-Aza in treatment-naive elderly patients with AML was effective and well tolerated, leading this association to become a standard of treatment in this indication. [Extracted from the article]

Introduction: Monocytosis is a frequent trigger for blood smear review in a routine hematology laboratory whereas chronic myelomonocytic leukemia (CMML) is infrequent and arises mostly in elderly patients. In order to define the best workflow for monocytosis, we studied three diagnostic approaches: the classical morphology approach (blood smear review), the flow cytometry assay (quantification of monocyte subsets as described by Selimoglu‐Buet et al in 2015), and the "mono‐dysplasia‐score" also referred to as "Monoscore (as described by our team in 2018 using the structural parameters of the Sysmex XN™ analyzers). Methods: Studying a multicentric cohort of 196 nonclonal monocytoses and CMML patients aged over 50 years, we compared the diagnostic performance of the three approaches alone and in combination to propose a diagnostic decision tree. Results: In patients presenting with additional criteria for slide review to monocytosis (37% of our cohort), we propose to sequentially combine morphology, Monoscore, and flow cytometry. On the contrary, for patients with isolated monocytosis (63%), slide review is not mandatory and we suggest performing flow cytometry depending on the Monoscore value. Using the proposed algorithm, 98% of CMML patients would have been correctly identified, slide review rate drastically reduced, and flow cytometry would have been carried out in 44% of patients. Conclusion: We have shown that implementation of Monoscore is a useful input filter to significantly reduce slide reviews without losing sensitivity and that flow cytometry is a performant technique in the second step of the diagnostic workup of CMML. [ABSTRACT FROM AUTHOR]

According to WHO recommendations, diagnosis of chronic myelomonocytic leukemia (CMML) beforehand requires microscopic examination of peripheral blood to identify dysplasia and/or blasts when monocytes are greater or equal to 1.0 × 109/L and 10% of leucocytes. We analyzed parameters derived from SysmexTM XN analyzers to improve the management of microscopic examination for monocytosis. We analyzed results of the complete blood count and the positioning and dispersion parameters of polymorphonuclear neutrophils and monocytes in 61 patients presenting with CMML and 635 control patients presenting with a reactive monocytosis. We used logistic regression and multivariate analysis to define a score for smear review. Three parameters were selected: neutrophil/monocyte ratio, structural neutrophil dispersion (Ne-WX) and monocyte absolute value. We established an equation in which the threshold of 0.160 guided microscopic examination in the search for CMML abnormalities with a sensitivity of 0.967 and a specificity of 0.978 in the learning cohort (696 samples) and 0.923 and 0.936 in the validation cohort (1809 samples) respectively. We created a score for microscopic smear examination of patients presenting with a monocytosis greater or equal to 1.0 × 109/L and 10% of leucocytes, improving efficiency in laboratory routine practice. [ABSTRACT FROM AUTHOR]

Background: Expression of SRY [sex-determining region Y]-box11 (SOX11) is specific to mantle cell lymphoma (MCL) and contributes, in conjunction with immunoglobulin variable heavy chain gene mutation status, to the identification of two forms of this disease. Methods: The aim of this report was firstly, to design an easy and suitable RT-qPCR method to quantify SOX11 mRNA expression in mantle cell lymphoma and other B cell malignancies with the proper reference gene; secondly, to define the best threshold of relative quantity of SOX11 mRNA in order to reach the best compromise between sensitivity and specificity. Results: For best discrimination of MCL and non-MCL groups we determined an area under the curve (AUC) of 0.9750 and a threshold of 1.76 with 100% sensitivity and 88% specificity. AUC and threshold values of respectively 0.91/1.346 [87% sensitivity, 80% specificity] and 0.9525/1.7120 [100% sensitivity, 88% specificity] for GAPDH and RPLP0 respectively denote that the RPLP0 reference gene alone is sufficient for PCR housekeeping gene. Conclusion: This work describes an RT-qPCR assay for SOX11 expression in order to better characterize MCL at diagnosis. Further studies on larger cohorts are needed to evaluate this molecular tool, especially for the follow-up of minimal residual disease. [ABSTRACT FROM AUTHOR]

Background: The procoagulant and proinflammatory microparticles (MPs) released during storage of packed red blood cells (pRBCs) can potentially modify transfusion benefits. A robust method to quantify MPs in pRBCs is needed to evaluate their impact in clinical trials.Study Design and Methods: The objective was to validate the preanalytic conditions required to prepare pRBC supernatant as well as a method to quantify and evaluate MP variations over 42 days of pRBC storage.A flow cytometry method with size-calibrated beads was developed and fully validated. Quantification of MPs in pRBCs (n = 109) was assessed during short-term (7 days) and long-term (42 days) storage at 4°C, during short-term storage (8 hours) at room temperature, and after 2 years frozen.Results: Repeatability, reproducibility, and linearity of the quantification method were validated, and variations during conservation are presented. There was high variability in RBC (erythrocyte) MP (ERMP) and platelet MP (PMP) levels between RBC units, depending on the filter used for leukocyte reduction. During the 42 days of storage at 4°C, significant increases in ERMPs and PMPs occurred (from 58 to 138 ERMPs/µL from Day 2 to Day 42; p = 0.0002; and from 326 to 771 PMPs/µL from Day 2 to Day 42; p = 0.00026).Conclusion: We use a robust method to confirm that ERMPs and PMPs are present to various degrees in pRBCs and that storage for 42 days significantly increases their generation. This method is robust enough to allow MP quantification in pRBCs and is adapted to evaluate the clinical impact of transfused MPs in prospective clinical trials. [ABSTRACT FROM AUTHOR]

Blastic plasmacytoid dendritic cell (PDC) neoplasm (BPDCN) is an aggressive hematological malignancy with a poor prognosis that derives from PDCs. No consensus for optimal treatment modalities is available today and the full characterization of this leukemia is still emerging. We identified here a BPDCN-specific transcriptomic profile when compared with those of acute myeloid leukemia and T-acute lymphoblastic leukemia, as well as the transcriptomic signature of primary PDCs. This BPDCN gene signature identified a dysregulation of genes involved in cholesterol homeostasis, some of them being liver X receptor (LXR) target genes. LXR agonist treatment of primary BPDCN cells and BPDCN cell lines restored LXR target gene expression and increased cholesterol efflux via the upregulation of adenosine triphosphate-binding cassette (ABC) transporters, ABCA1 and ABCG1. LXR agonist treatment was responsible for limiting BPDCN cell proliferation and inducing intrinsic apoptotic cell death. LXR activation in BPDCN cells was shown to interfere with 3 signaling pathways associated with leukemic cell survival, namely: NF-κB activation, as well as Akt and STAT5 phosphorylation in response to the BPDCN growth/survival factor interleukin-3. These effects were increased by the stimulation of cholesterol efflux through a lipid acceptor, the apolipoprotein A1. In vivo experiments using a mouse model of BPDCN cell xenograft revealed a decrease of leukemic cell infiltration and BPDCN-induced cytopenia associated with increased survival after LXR agonist treatment. This demonstrates that cholesterol homeostasis is modified in BPDCN and can be normalized by treatment with LXR agonists which can be proposed as a new therapeutic approach. [ABSTRACT FROM AUTHOR]

Background: CD20 is a B cell lineage-specific marker expressed by normal and leukemic B cells and targeted by several antibody immunotherapies. We have previously shown that the protein from a CD20 mRNA splice variant (D393-CD20) is expressed at various levels in leukemic B cells or lymphoma B cells but not in resting, sorted B cells from the peripheral blood of healthy donors. Results: Western blot (WB) analysis of B malignancy primary samples showed additional CD20 signals. Deep molecular PCR analysis revealed four new sequences corresponding to in-frame CD20 splice variants (D657-CD20, D618-CD20, D480-CD20, and D177-CD20) matching the length of WB signals. We demonstrated that the cell spliceosome machinery can process ex vivo D480-, D657-, and D618-CD20 transcript variants by involving canonical sites associated with cryptic splice sites. Results of specific and quantitative RT-PCR assays showed that these CD20 splice variants are differentially expressed in B malignancies. Moreover, Epstein-Barr virus (EBV) transformation modified the CD20 splicing profile and mainly increased the D393-CD20 variant transcripts. Finally, investigation of three cohorts of chronic lymphocytic leukemia (CLL) patients showed that the total CD20 splice variant expression was higher in a stage B and C sample collection compared to routinely collected CLL samples or relapsed refractory stage A, B, or C CLL. Conclusion: The involvement of these newly discovered alternative CD20 transcript variants in EBV transformation makes them interesting molecular indicators, as does their association with oncogenesis rather than non-oncogenic B cell diseases, differential expression in B cell malignancies, and correlation with CLL stage and some predictive CLL markers. This potential should be investigated in further studies. [ABSTRACT FROM AUTHOR]

Submicron-sized extra-cellular vesicles generated by budding from the external cell membranes, microparticles (MPs) are important actors in transfusion as well as in other medical specialties. After briefly positioning their role in the characterization of labile blood products, this technically oriented chapter aims to review practical points that need to be considered when trying to use flow cytometry for the analysis, characterization and absolute counting of MP subsets. Subjects of active discussions relative to instrumentation will include the choice of the trigger parameter, possible standardization approaches requiring instrument quality-control, origin and control of non-specific background and of coincidence artifacts, choice of the type of electronic signals, optimal sheath fluid and sample speed. Questions related to reagents will cover target antigens and receptors, multi-color reagents, negative controls, enumeration of MPs and limiting artifacts due to unexpected (micro-) coagulation of plasma samples. Newly detected problems are generating innovative solutions and flow cytometry will continue to remain the technology of choice for the analysis of MPs, in the domain of transfusion as well as in many diverse specialties. [ABSTRACT FROM AUTHOR]

We investigated the plasma levels of PMPs in patients with 45 stage III and 45 stage IV melanoma. PMPs were characterised by flow cytometry and their thrombogenic activity. We also investigated the link between PMPs circulating levels and tumor burden. The circulating levels of PMPs were significantly higher in stage IV (8500 μL−1) than in patients with stage III (2041 μL−1) melanoma ( P=.0001). We calculated a highly specific (93.3%) and predictive (91.7%) cut-off value (5311 μL−1) allowing the distinction between high-risk stage III and metastatic stage IV melanoma. The thrombogenic activity of PMPs was significantly higher in patients with stage IV melanoma (clotting time: 40.7 second vs 65 second, P=.0001). There was no significant association between the radiological tumoral syndrome and the plasma level of PMPs. Our data suggest the role of PMPs in metastatic progression of melanoma. [ABSTRACT FROM AUTHOR]

Hairy cell leukaemia (HCL) is a rare haematological malignancy, with approximately 175 new incident cases in France. Diagnosis is based on a careful examination of the blood smear and immunophenotyping of the tumour cells, with a panel of four markers being used specifically to screen for hairy cells (CD11c, CD25, CD103 and CD123). In 2011, the V600E mutation of the BRAF gene in exon 15 was identified in HCL; being present in HCL, it is absent in the variant form of HCL (HCL-v) and in splenic red pulp lymphoma (SRPL), two entities related to HCL. The management of patients with HCL has changed in recent years. A poorer response to purine nucleoside analogues (PNAs) is observed in patients with more marked leukocytosis, bulky splenomegaly, an unmutated immunoglobulin variable heavy chain ( IgVH) gene profile, use of VH4-34 or with TP53 mutations. We present the recommendations of a group of 11 experts belonging to a number of French hospitals. This group met in November 2013 to examine the criteria for managing patients with HCL. The ideas and proposals of the group are based on a critical analysis of the recommendations already published in the literature and on an analysis of the practices of clinical haematology departments with experience in managing these patients. The first-line treatment uses purine analogues: cladribine or pentostatin. The role of BRAF inhibitors, whether or not combined with MEK inhibitors, is discussed. The panel of French experts proposed recommendations to manage patients with HCL, which can be used in a daily practice. [ABSTRACT FROM AUTHOR]

This is the first prospective study of treatment of patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN), an aggressive hematologic malignancy derived from plasmacytoid dendritic cells that typically involves the skin and rapidly progresses to a leukemia phase. Despite being initially responsive to intensive combination chemotherapy, most patients relapse and succumb to their disease. Because BPDCN blasts overexpress the interleukin-3 receptor (IL3R), the activity of SL-401, diptheria toxin (DT)388IL3 composed of the catalytic and translocation domains of DT fused to IL3, was evaluated in BPDCN patients in a phase 1 -2 study. Eleven patients were treated with a single course of SL-401 at 12.5 μg/kg intravenously over 15 minutes daily for up to 5 doses; 3 patients who had initial responses to SL-401 received a second course in relapse. The most common adverse events including fever, chills, hypotension, edema, hypoalbuminemia, thrombocytopenia, and transaminasemia were transient. Seven of 9 evaluable (78%) BPDCN patients had major responses including 5 complete responses and 2 partial responses after a single course of SL-401. The median duration of responses was 5 months (range, 1-20+ months). Further studies of SL-401 in BPDCN including those involving multiple sequential courses, alternate schedules, and combinations with other therapeutics and warranted. This trial is registered at clinicaltrials.gov as #NCT00397579. [ABSTRACT FROM AUTHOR]

Highlights: [•] GALIG is a cell death-related gene nested within the human galectin-3 locus. [•] GALIG pro-apoptotic function is counteracted by the anti-apoptotic Mcl-1. [•] During spontaneous neutrophil apoptosis, GALIG mRNAs are up-regulated. [•] In Acute Myeloid Leukemia cell samples, GALIG mRNAs are highly down-regulated. [ABSTRACT FROM AUTHOR]

STUDY QUESTION How can leukemic cells be detected in cryopreserved ovarian tissue? SUMMARY ANSWER Multicolor flow cytometry (FCM) is useful to evaluate the presence of viable leukemic cells in the ovarian cortex with a high specificity and a robust sensitivity. WHAT IS KNOWN ALREADY Storing ovarian tissue is an option to preserve fertility before gonadotoxic radiotherapy or chemotherapy treatments. However, transplantation of cryopreserved ovarian cortex to women cured of leukemia is currently not possible due to the risk of cancer re-seeding. STUDY DESIGN, SIZE, DURATION We developed an automated ovarian cortex dissociation technique and we used eight-color FCM to identify leukemic cells with a series of dilutions added to ovarian single cell suspensions obtained from healthy cortex. PARTICIPANTS/MATERIALS, SETTINGS, METHODS Healthy ovarian cortex originated from women between 23 and 39 years of age undergoing laparoscopic ovarian drilling for polycystic ovary syndrome. Blood or bone marrow cells were collected in acute lymphoblastic leukemia (ALL) patients at diagnosis. MAIN RESULTS AND THE ROLE OF CHANCE The tissue dissociation technique yield was 1.83 ± 1.49 × 106 viable nucleated cells per 100 mg of ovarian cortex. No cell exhibiting a leukemic phenotype was present in the normal ovarian cortex. Added leukemic cells were detected using their leukemia-associated phenotype up to a dilution of 10–4. When specific gene rearrangements were present, they were detected by real-time quantitative PCR at the same dilution. The ovarian cortex from two leukemia patients was then used, respectively, as positive and negative controls. LIMITATIONS, REASONS FOR CAUTION Making available minimal residual disease (MRD) detection techniques (multicolor FCM, PCR and xenograft), that can be used either alone or together, is essential to add a fail-safe oncological dimension to pre-autograft monitoring. WIDER IMPLICATIONS OF THE FINDINGS This approach can be performed on fresh ovarian tissue during cryopreservation or on frozen/thawed tissue before reimplantation and it is currently the only available technique in cases of ALL where no molecular markers are identified. This new perspective should lead to studies on ovarian tissue from leukemia patients, for whom the presence of MRD should be established before autograft. STUDY FUNDINGS/COMPETING INTEREST(S) The study was supported by the BioMedicine Agency, the Committee of the League against Cancer, the Besançon University Hospital, DGOS/INSERM/INCa and the regional Council of Franche-Comté. There were no conflicts of interest to declare. [ABSTRACT FROM AUTHOR]