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•Population-scale genomics (N = 937 939) allowed assembly of a large FVL/PTGM double-heterozygous cohort while mitigating ascertainment bias.•FVL/PTGM DH appeared to be more common than FVL homozygosity and conferred a similar risk of venous thrombosis.

Thrombocytopathy is the most likely diagnosis for patients with easy bruising and/or mucous bleeding and normal platelet counts. However, in a small proportion of these patients, all hemostatic tests are normal and the right diagnosis is actually Ehlers-Danlos syndrome (EDS). EDS is a heterogeneous group of genetic connective tissue disorders, characterized by joint hypermobility, skin and vascular fragility, and tissue friability; the most recent classification describes 13 clinical subtypes of EDS with mutations in 19 different genes (Malfait et al. Am J Med Genet C Semin Med Genet. 2017;175:8). Besides joint hypermobility, cutaneous-mucosal bleeding of variable intensity is observed in the majority of patients. In the vascular subtype of EDS, arterial aneurysms, dissections and ruptures of arteries and arteriovenous fistulas are detected as manifestations of vascular fragility; less frequently, such vascular pathologies can also appear in other types of EDS (Malfait. Matrix Biol 2018;71-72:380).

Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-cell lymphoma with high clinical and biological heterogeneity. Despite current effective immunochemotherapy, up to 40% of patients do not respond or develop refractory disease. DLBCL is characterized by two major cell-of-origin (COO) subtypes, germinal center B cell-like (GCB) and activated B cell-like (ABC), with recent research uncovering additional molecular subgroups based on genomic alterations or tumor microenvironment (TME) features. However, current classifiers use bulk or deconvolution profiling which blurs intra-tumor heterogeneity, hindering the discovery of true functional cell states underlying clinical diversity. Here, we constructed a multimodal single-cell atlas of 62 DLBCL cases, integrating single-cell transcriptomes, immune repertoire and genetic features, to decrypt recurrent B cell states and TME ecosystems while identifying clinically relevant prognostic biomarkers (Fig.1).

Introduction

Introduction:Cutaneous T-cell lymphomas (CTCL) are heterogeneous lymphoproliferative disorders on a spectrum of disease presentation and severity. Around two-thirds of cutaneous T-cell lymphomas can be classified as mycosis fungoides (MF) or Sézary syndrome (SS). While advanced stages of MF and SS are associated with decreased survival and worse outcomes, even early-stage patients can possess a variable course. Numerous deep sequencing studies have fallen short in identifying genetic abnormalities that drive disease pathogenesis and predict prognosis. Large-cell transformation and elevated lactate dehydrogenase levels are associated with worse prognosis in SS; however, such features cannot accurately prognosticate patient survival. There is a need for investigation that may assist in the prognostication of survival in patients with CTCL. Machine learning methods may help to elucidate correlations between clinical and genetic factors to predict disease progression and outcomes.

Previous Fanconi anemia (FA) gene therapy studies have failed to demonstrate engraftment of gene-corrected hematopoietic stem and progenitor cells (HSPCs) from FA patients, either after autologous transplantation or infusion into immunodeficient mice. In this study, we demonstrate that a validated short transduction protocol of G-CSF plus plerixafor-mobilized CD34+cells from FA-A patients with a therapeutic FANCA-lentiviral vector corrects the phenotype of in vitro cultured hematopoietic progenitor cells. Transplantation of transduced FA CD34+cells into immunodeficient mice resulted in reproducible engraftment of myeloid, lymphoid, and CD34+cells. Importantly, a marked increase in the proportion of phenotypically corrected, patient-derived hematopoietic cells was observed after transplantation with respect to the infused CD34+graft, indicating the proliferative advantage of corrected FA-A hematopoietic repopulating cells. Our data demonstrate for the first time that optimized protocols of hematopoietic stem cell collection from FA patients, followed by the short and clinically validated transduction of these cells with a therapeutic lentiviral vector, results in the generation of phenotypically corrected HSPCs capable of repopulating and developing proliferation advantage in immunodeficient mice. Our results suggest that clinical approaches for FA gene therapy similar to those used in this study will facilitate hematopoietic repopulation in FA patients with gene corrected HSPCs, opening new prospects for gene therapy of FA patients.

Previous Fanconi anemia (FA) gene therapy studies have failed to demonstrate engraftment of gene-corrected hematopoietic stem and progenitor cells (HSPCs) from FA patients, either after autologous transplantation or infusion into immunodeficient mice. In this study, we demonstrate that a validated short transduction protocol of G-CSF plus plerixafor-mobilized CD34+ cells from FA-A patients with a therapeutic FANCA-lentiviral vector corrects the phenotype of in vitro cultured hematopoietic progenitor cells. Transplantation of transduced FA CD34+ cells into immunodeficient mice resulted in reproducible engraftment of myeloid, lymphoid, and CD34+ cells. Importantly, a marked increase in the proportion of phenotypically corrected, patient-derived hematopoietic cells was observed after transplantation with respect to the infused CD34+ graft, indicating the proliferative advantage of corrected FA-A hematopoietic repopulating cells. Our data demonstrate for the first time that optimized protocols of hematopoietic stem cell collection from FA patients, followed by the short and clinically validated transduction of these cells with a therapeutic lentiviral vector, results in the generation of phenotypically corrected HSPCs capable of repopulating and developing proliferation advantage in immunodeficient mice. Our results suggest that clinical approaches for FA gene therapy similar to those used in this study will facilitate hematopoietic repopulation in FA patients with gene corrected HSPCs, opening new prospects for gene therapy of FA patients.

Abnormalities of platelet size are one of the distinguishing features of inherited thrombocytopenias (ITs), and evaluation of blood films is recommended as an essential step for differential diagnosis of these disorders. Nevertheless, what we presently know about this subject is derived mainly from anecdotal evidence. To improve knowledge in this field, we evaluated platelet size on blood films obtained from 376 patients with all 19 forms of IT identified so far and found that these conditions differ not only in mean platelet diameter, but also in platelet diameter distribution width and the percentage of platelets with increased or reduced diameters. On the basis of these findings, we propose a new classification of ITs according to platelet size. It distinguishes forms with giant platelets, with large platelets, with normal or slightly increased platelet size, and with normal or slightly decreased platelet size. We also measured platelet diameters in 87 patients with immune thrombocytopenia and identified cutoff values for mean platelet diameter and the percentage of platelets with increased or reduced size that have good diagnostic accuracy in differentiating ITs with giant platelets and with normal or slightly decreased platelet size from immune thrombocytopenia and all other forms of IT.

Abnormalities of platelet size are one of the distinguishing features of inherited thrombocytopenias (ITs), and evaluation of blood films is recommended as an essential step for differential diagnosis of these disorders. Nevertheless, what we presently know about this subject is derived mainly from anecdotal evidence. To improve knowledge in this field, we evaluated platelet size on blood films obtained from 376 patients with all 19 forms of IT identified so far and found that these conditions differ not only in mean platelet diameter, but also in platelet diameter distribution width and the percentage of platelets with increased or reduced diameters. On the basis of these findings, we propose a new classification of ITs according to platelet size. It distinguishes forms with giant platelets, with large platelets, with normal or slightly increased platelet size, and with normal or slightly decreased platelet size. We also measured platelet diameters in 87 patients with immune thrombocytopenia and identified cutoff values for mean platelet diameter and the percentage of platelets with increased or reduced size that have good diagnostic accuracy in differentiating ITs with giant platelets and with normal or slightly decreased platelet size from immune thrombocytopenia and all other forms of IT.

Fanconi anemia (FA) is a rare bone marrow failure disorder with defective DNA interstrand crosslink repair. Still, there are FA patients without mutations in any of the 15 genes individually underlying the disease. A candidate protein for those patients, FA nuclease 1 (FAN1), whose gene is located at chromosome 15q13.3, is recruited to stalled replication forks by binding to monoubiquitinated FANCD2 and is required for interstrand crosslink repair, suggesting that mutation of FAN1may cause FA. Here we studied clinical, cellular, and genetic features in 4 patients carrying a homozygous 15q13.3 micro-deletion, including FAN1and 6 additional genes. Biallelic deletion of the entire FAN1gene was confirmed by failure of 3′- and 5′-PCR amplification. Western blot analysis failed to show FAN1 protein in the patients' cell lines. Chromosome fragility was normal in all 4 FAN1-deficient patients, although their cells showed mild sensitivity to mitomycin C in terms of cell survival and G2phase arrest, dissimilar in degree to FA cells. Clinically, there were no symptoms pointing the way to FA. Our results suggest that FAN1 has a minor role in interstrand crosslink repair compared with true FA genes and exclude FAN1 as a novel FA gene.

Fanconi anemia (FA) is a rare bone marrow failure disorder with defective DNA interstrand crosslink repair. Still, there are FA patients without mutations in any of the 15 genes individually underlying the disease. A candidate protein for those patients, FA nuclease 1 (FAN1), whose gene is located at chromosome 15q13.3, is recruited to stalled replication forks by binding to monoubiquitinated FANCD2 and is required for interstrand crosslink repair, suggesting that mutation of FAN1 may cause FA. Here we studied clinical, cellular, and genetic features in 4 patients carrying a homozygous 15q13.3 micro-deletion, including FAN1 and 6 additional genes. Biallelic deletion of the entire FAN1 gene was confirmed by failure of 3′- and 5′-PCR amplification. Western blot analysis failed to show FAN1 protein in the patients' cell lines. Chromosome fragility was normal in all 4 FAN1-deficient patients, although their cells showed mild sensitivity to mitomycin C in terms of cell survival and G2 phase arrest, dissimilar in degree to FA cells. Clinically, there were no symptoms pointing the way to FA. Our results suggest that FAN1 has a minor role in interstrand crosslink repair compared with true FA genes and exclude FAN1 as a novel FA gene.

Until recently, thrombocytopenia 2 (THC2) was considered an exceedingly rare form of autosomal dominant thrombocytopenia and only 2 families were known. However, we recently identified mutations in the 5′-untranslated region of the ANKRD26gene in 9 THC2 families. Here we report on 12 additional pedigrees with ANKRD26mutations, 6 of which are new. Because THC2 affected 21 of the 210 families in our database, it has to be considered one of the less rare forms of inherited thrombocytopenia. Analysis of all 21 families with ANKRD26mutations identified to date revealed that thrombocytopenia and bleeding tendency were usually mild. Nearly all patients had no platelet macrocytosis, and this characteristic distinguishes THC2 from most other forms of inherited thrombocytopenia. In the majority of cases, platelets were deficient in glycoprotein Ia and α-granules, whereas in vitro platelet aggregation was normal. Bone marrow examination and serum thrombopoietin levels suggested that thrombocytopenia was derived from dysmegakaryopoiesis. Unexplained high values of hemoglobin and leukocytes were observed in a few cases. An unexpected finding that warrants further investigation was a high incidence of acute leukemia. Given the scarcity of distinctive characteristics, the ANKRD26-related thrombocytopenia has to be taken into consideration in the differential diagnosis of isolated thrombocytopenias.

Until recently, thrombocytopenia 2 (THC2) was considered an exceedingly rare form of autosomal dominant thrombocytopenia and only 2 families were known. However, we recently identified mutations in the 5′-untranslated region of the ANKRD26 gene in 9 THC2 families. Here we report on 12 additional pedigrees with ANKRD26 mutations, 6 of which are new. Because THC2 affected 21 of the 210 families in our database, it has to be considered one of the less rare forms of inherited thrombocytopenia. Analysis of all 21 families with ANKRD26 mutations identified to date revealed that thrombocytopenia and bleeding tendency were usually mild. Nearly all patients had no platelet macrocytosis, and this characteristic distinguishes THC2 from most other forms of inherited thrombocytopenia. In the majority of cases, platelets were deficient in glycoprotein Ia and α-granules, whereas in vitro platelet aggregation was normal. Bone marrow examination and serum thrombopoietin levels suggested that thrombocytopenia was derived from dysmegakaryopoiesis. Unexplained high values of hemoglobin and leukocytes were observed in a few cases. An unexpected finding that warrants further investigation was a high incidence of acute leukemia. Given the scarcity of distinctive characteristics, the ANKRD26-related thrombocytopenia has to be taken into consideration in the differential diagnosis of isolated thrombocytopenias.

Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCAmutations, we determined a FANCAmutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCAmutation is not the result of a mutational “hot-spot” but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCAand 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCAmutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations.

Fanconi anemia is characterized by congenital abnormalities, bone marrow failure, and cancer predisposition. To investigate the origin, functional role, and clinical impact of FANCA mutations, we determined a FANCA mutational spectrum with 130 pathogenic alleles. Some of these mutations were further characterized for their distribution in populations, mode of emergence, or functional consequences at cellular and clinical level. The world most frequent FANCA mutation is not the result of a mutational “hot-spot” but results from worldwide dissemination of an ancestral Indo-European mutation. We provide molecular evidence that total absence of FANCA in humans does not reduce embryonic viability, as the observed frequency of mutation carriers in the Gypsy population equals the expected by Hardy-Weinberg equilibrium. We also prove that long distance Alu-Alu recombination can cause Fanconi anemia by originating large interstitial deletions involving FANCA and 2 adjacent genes. Finally, we show that all missense mutations studied lead to an altered FANCA protein that is unable to relocate to the nucleus and activate the FA/BRCA pathway. This may explain the observed lack of correlation between type of FANCA mutation and cellular phenotype or clinical severity in terms of age of onset of hematologic disease or number of malformations.

No relevant conflicts of interest to declare.

The familial platelet disorder with associated myeloid malignancy (FPDMM) is an autosomal dominant platelet disorder, caused by germline RUNX1 mutations, with predisposition to develop hematologic malignancies, especially acute myeloid leukemia. In many of the FPDMM families reported, the platelet defect was a delta-storage-pool disease (d-SPD) which can also be found without leukemia propensity. However, it has not been studied whether the two types of d-SPD have a common nature. Platelet ultrastructure, previously very little studied, may be one of the aspects to be analyzed to solve this question.

MYH9-related disorders are a group of disorders (May-Hegglin anomaly, Fechtner syndrome, Sebastian syndrome) characterized by macrothrombocytopenia and ribosome inclusions in granulocytes. They are caused by mutations in the MYH9gene and, depending on the location of the mutation, are associated with extrahematological pathologies (sensorineural hearing loss, progressive nephropathy, presenile cataracts) of variable intensity.

Introduction.Myelodysplastic syndromes (MDS) are clonal heterogeneous disorders in which molecular studies and cytogenetics are essential for diagnosis, classification and prognosis. Cell-free DNA (cfDNA) analysis has been reported as a reliable non-invasive approach for detecting molecular abnormalities in MDS. However, there is limited information about cytogenetic alterations and monitoring in cfDNA from MDS patients.

Besses: Gilead: Research Funding. Salar: Janssen: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Gilead: Research Funding; Celgene: Consultancy, Speakers Bureau. Bellosillo: Thermofisher Scientific: Consultancy, Speakers Bureau; Roche: Research Funding, Speakers Bureau; Qiagen: Consultancy, Speakers Bureau.

No relevant conflicts of interest to declare.

Sierra: Jazz Pharmaceuticals: Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Daiichi Sankyo: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Astellas: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead-Kite: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background

Hydrops fetalis is rarely caused by congenital dyserythropoietic anemia (CDA). We report a patient with hydrops fetalis as a result of severe anemia. This patient needed intrauterine transfusions from 21 weeks of gestation until birth. The hematologic study showed an atypical CDA (hydrops fetalis–associated CDA) characterized by features resembling CDA type II, but negative acidified serum lysis test (HEMPAS negative). The patient was regularly transfused for a year, after which an allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling was successfully carried out. His actual hemoglobin is 127 g/L, and he has not received transfusions for more than a year. In conclusion, intrauterine transfusions and BMT could cure an otherwise lethal atypical CDA.

Hydrops fetalis is rarely caused by congenital dyserythropoietic anemia (CDA). We report a patient with hydrops fetalis as a result of severe anemia. This patient needed intrauterine transfusions from 21 weeks of gestation until birth. The hematologic study showed an atypical CDA (hydrops fetalis–associated CDA) characterized by features resembling CDA type II, but negative acidified serum lysis test (HEMPAS negative). The patient was regularly transfused for a year, after which an allogeneic bone marrow transplantation (BMT) from an HLA-identical sibling was successfully carried out. His actual hemoglobin is 127 g/L, and he has not received transfusions for more than a year. In conclusion, intrauterine transfusions and BMT could cure an otherwise lethal atypical CDA.

Rio: Rocket Pharmaceuticals, Inc.: Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Navarro:Rocket Pharmaceuticals, Inc.: Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents. Segovia:Rocket Pharmaceuticals, Inc.: Equity Ownership, Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Wang:GeneWerk: Employment. Casado:Rocket Pharmaceuticals, Inc.: Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents. Galy:Genethon: Employment. Cavazzana:SmartImmune: Other: Founder. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH, Heidelberg, Gemrany: Equity Ownership; German Cancer Research Center, Heidelberg, Germany: Employment. Díaz de Heredia:Novartis: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Sevilla:Rocket Pharmaceuticals, Inc.: Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

Nine Fanconi anemia patients complementation group A (FA-A), age 2-6 years, have been infused with autologous hematopoietic cells after genetic correction with the therapeutic PGK-FANCA.Wpre* lentiviral vector. In all instances patients underwent CD34+cell mobilization with G-CSF and plerixafor and were subsequently infused in the absence of any pre-conditioning regimen, in order to avoid genotoxic side effects in a population characterized by DNA repair defects and cancer predisposition. The first four patients were treated between January 2016 and March 2017 and were infused with an estimated number of 170,000 and 410,000 transduced CD34+cells/Kg. The other five patients were treated more recently with cell numbers that ranged between 50,000 to 1.6x106corrected CD34+cells/kg. The analyses of the first four patients showed the presence of corrected cells both in BM and PB after six months post-infusion and progressive increases of gene marking were observed thereafter in all these patients until the most recent follow-up (2 to >3 years post-infusion). Gene marking in BM CD34+cells correlated with the survival of the CFCs to mitomycin-C, with levels up to 70% at 3 years post-infusion. Additionally, progressive decreases in the percentage of PB T cells with diepoxybutane-induced chromosomal breaks were observed in the patients with higher levels of gene marking. Similarly, stabilized PB cell counts have been observed in patients with higher percentages of gene corrected cells. Insertion site analyses revealed the absence of genotoxic events, and demonstrated the engraftment of pluripotent HSCs and a pattern of oligoclonal reconstitution, consistent with the number of infused corrected CD34+cells and the absence of conditioning. In the five additional patients treated more recently, the presence of gene corrected PB cells has been confirmed; levels of gene marking have been consistent with data observed in the first four treated patients and with the number of infused CD34+cells. Our results confirm the engraftment of gene corrected HSCs in non-conditioned FA-A patients, in some cases through more than 3 years of follow-up, suggesting the relevance of this therapeutic approach in FA.

Bueren: Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Navarro:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Segovia:Rocket Pharmaceuticals Inc: Consultancy, Equity Ownership, Patents & Royalties, Research Funding. Casado:Rocket Pharmaceuticals Inc: Patents & Royalties. Schwartz:Rocket Pharmaceuticals: Employment, Equity Ownership. Schmidt:GeneWerk GmbH: Employment; German Cancer Research Center: Employment; bluebird bio: Consultancy. Rio:Rocket Pharmaceuticals Inc: Equity Ownership, Patents & Royalties, Research Funding. Sevilla:Rocket Pharmaceuticals Inc: Honoraria, Patents & Royalties.

TITLE:A retrospective multicenter analysis of the safety and efficacy of Brentuximab Vedotin (BV) as monotherapy or in combination with Bendamustine (BV-B) in relapsed refractory Classic Hodgkin Lymphoma (cHL) in Argentina.

ITP is a heterogeneous diagnosis of exclusion. Diagnostic methods capable of distinguishing the effects of impaired platelet production and rapid platelet consumption would enable classification of ITP patients on the basis of these basic pathophysiologic mechanisms. Data from in vivo platelet consumption studies may allow this, but it has been difficult up to now to interpret such studies in terms of the concurrent and interactive effects of random consumption, lifespan-dependent platelet consumption, and altered platelet production rates. Here we used a recently described numerical model to re-interpret previously published in vivo 111In-labeled platelet consumption studies in a series of 39 adult ITP patients who had failed to respond adequately to prednisone. All underwent splenectomy after the study, to which four of the patients did not respond adequately (“non-responders”).

ITP is a heterogeneous diagnosis of exclusion. Diagnostic methods capable of distinguishing the effects of impaired platelet production and rapid platelet consumption would enable classification of ITP patients on the basis of these basic pathophysiologic mechanisms. Data from in vivo platelet consumption studies may allow this, but it has been difficult up to now to interpret such studies in terms of the concurrent and interactive effects of random consumption, lifespan-dependent platelet consumption, and altered platelet production rates. Here we used a recently described numerical model to re-interpret previously published in vivo 111In-labeled platelet consumption studies in a series of 39 adult ITP patients who had failed to respond adequately to prednisone. All underwent splenectomy after the study, to which four of the patients did not respond adequately (“non-responders”).The model (Strom, TS; PLoS One. 2013;8(1):e55087) quantifies random and lifespan-dependent processes for each patient as a random destruction rate constant (RD, %/hr), a lognormally distributed lifespan (LS, hr), and a standard deviation for ln LS (SD). Modeled consumption curves are optimized for each patient via mapping of least squared residuals for all possible parameter value combinations. The model also generates the net turnover rate (equivalent to the platelet production rate (PR)), the fraction of platelets lost to lifespan dependent consumption (LSDC), and the platelet age distribution. Because the individual data sets demonstrate consumption plateaus, a fourth parameter (% long lived species, or LL) was also optimized for each data set. Figure 1 shows examples of optimal consumption curves.As a guide to interpreting the optimal parameter value sets, predicted values were generated for three alternative mechanisms for inducing thrombocytopenia:1) Reduced PR: Assuming that the absolute RD rate from individuals with normal platelet counts represents a hemostatic minimum requirement which is maintained as platelet count is reduced, we modeled the effects of impaired PR on RD and platelet count.2) Increased RD: Assuming that PR is constant, we modeled the effects of augmented (immunologic) RD on platelet count.3) Increased RD and PR: Assuming that PR increases in proportion to reduced platelet count, and again assuming a minimum hemostatic RD requirement, we modeled the simultaneous effects of increased RD and a homeostatic increase in PR on platelet count.The observed and modeled findings (figure 2) indicate that 90% of the patients in the study show no substantial (> 50%) compensatory increase in PR. Approximately 1/3 of the patients show parameter set values consistent with a thrombocytopenia due only to impaired platelet production. And a small group of patients (∼4 of 39) show consumption of an unexpectedly high fraction of their platelets by LSDC. Modeled values also show that the immature platelet fraction (IPF) is not consistently predictive of altered rates of platelet production and consumption (figure 3). Thus the IPF cannot substitute for PR and RD values inferred from in vivo consumption studies.Our analysis demonstrates a pathophysiologic basis for predicting therapeutic responses in ITP patients. Patients showing only impaired PR should be expected to respond optimally to thrombopoietin receptor agonists, and not to splenectomy. Conversely, patients for whom enhanced platelet production cannot keep up with rapid RD would be expected to respond best to interventions targeting platelet consumption, such as splenectomy.These findings indicate that impaired platelet production, or the absence of a homeostatic increase in platelet production, is a more common factor in corticosteroid non-responsive ITP patients than is increased immunologic platelet clearance.Strom: University of Tennessee Health Science Center: Employment, The numerical analysis model utilized in this study is disclosed in a patent application filed in the USPTO December 2012, naming me as the inventor., The numerical analysis model utilized in this study is disclosed in a patent application filed in the USPTO December 2012, naming me as the inventor. Patents & Royalties.

Zieger: CSL Behring Hattersheim: Research Funding.

Abstract 707FN2This icon denotes a clinically relevant abstract

In 2008 Spaepen et al. (The Oncologist 2008;13:596–607) published a cost analysis on the treatment of chemotherapy-induced anemia with the different available erythropoiesis stimulating agents (ESA), based on data derived from a Belgian Hospital Disease Database. The objective of this study was to assess the applicability of that analysis in the Austrian setting and to evaluate if there were differences in cost between ESAs.The original study was performed based on a hospital disease database including 34% of the Belgian hospital beds and day clinic visits. As ESAs can only be provided by the hospital pharmacy, all ESA use in cancer patients was captured. To adapt this database for use in the Austrian setting, it was necessary to adjust for discrepancies regarding the epidemiology and treatment patterns of cancer patients in both countries, as well as the costs of medical care. This was achieved using a mixed-effects model stratifying for propensity score quintiles, as described in Spaepen et al (2008). Data on the following variables were compared between countries and adaptations were performed if necessary: (1) gender and age distribution of the general population; (2) distribution of cancer types; (3) use of chemotherapy; and (4) use of ESAs and blood transfusions. Austrian unit costs (Euro, 2010) where applied to the original resource use data.The general demographic country profile revealed similarities between Austria and Belgium in terms of average age and gender (41.01 vs. 40.72 average age; 39.48% vs. 39.39% proportion male), and distribution per age. The overall cancer incidence was somewhat lower in the Austrian population (0.45% vs. 0.52%). However, important differences were found in the prevalence of 4 specific cancer types (hematologic, female breast, lung, female genital cancers), which were less present in Austria. Therefore, the relative weight of those cancer types was reduced in the original dataset, based on the prevalence ratios of Austria compared to Belgium [hematologic cancers (0.6377); female breast cancer (0.5102); lung cancer (0.5618); female genital cancer (0.7787)]. In terms of chemotherapy use, IMS Health sales data was used to derive the total use (in mg) of the different molecules for both countries. The ratio of total annual Austrian over total annual Belgian consumption (38%) was calculated per molecule to adjust the analysis for Austria. No major differences were found regarding the use of ESAs or the administration of blood transfusions for chemotherapy patients. Table 1 and Table 2 present the results of the propensity-score adjusted cost analysis for Austria (Euro, Health Care payer perspective, mean ± se).By using published epidemiologic and treatment pattern data, it was possible to adapt the Belgian Hospital database to the Austrian population. After adjusting for country differences, the results of the Austrian analysis reflected that patients treated with darbepoetin experienced lower overall costs than patients treated with epoetin-α or epoetin-β. Patients in the darbepoetin group incurred the lowest cost in terms of ESAs used. In terms of anemia-related costs, the overall costs of treatment were lower in patients receiving darbepoetin compared to patients receiving epoetin-α or epoetin-β. These findings are in line with those from the Belgian analysis.Duran: Amgen: Consultancy. Spaepen:IMS Health: Consultancy. Lamotte:Amgen: Consultancy. Walter:Amgen: Consultancy. Kutikova:Amgen: Employment. Pujol:Amgen: Employment. Annemans:IMS Health: Consultancy.

Abstract 3811

Background: Therapeutic plasma exchange (PE) is the mainstay of treatment of thrombotic thrombocytopenic purpura (TTP). In many patients, prolonged PE is required to achieve remission and between 30% and 60% of patients relapse after a variable period of months to years, with a further episode of TTP or a chronic picture of recurrent relapses. Various immunosuppressive agents have been used in acute refractory and chronic relapsing cases. Despite advances in treatment, mortality remains at 15–20%. Limited clinical data from small groups of patients suggest that the administration of anti-CD20 antibody (Rituximab) may also be useful in acute refractory and chronic relapsing TTP. However, whether rituximab could be and effective strategy for adults with relapsed or refractory TTP remains unresolved. Patients and Methods: We have studied the clinical response to rituximab in 22 adult patients (16 F/6 M; median age, 43 years, range 24–72) from 11 Spanish centers who presented with acute refractory (13 cases) or an acute relapse (9 cases) episode of idiopathic TTP. Second-line therapies previously administered included vincristine (11 cases) and splenectomy (2 cases). At the time of the study, five (28%) patients presented neurological symptoms, three (17%) renal failure, and two (17%) fever. On admission, every patient received daily PE and corticosteroids during the acute episode. Results: Rituximab was administered a median of 16 days (range, 7–53) after starting PE. It was administered immediately after PE at a dose of 375 mg/m2 each week for a median (range) of 4 (1–8) doses. No severe acute or delayed toxicity associated with rituximab was observed. Two (9%) patients died due to TTP-related causes 24 and 48 hours after relapse and were not evaluable for response. The remaining 20 (91%) patients achieved complete remission in a median of 21 days after initiating rituximab (range, 2–35). After a median follow-up of 13 months (range, 1.5–33), 17 patients are in remission and 3 patients have relapsed at 7, 29, and 29 months after rituximab administration. Conclusion: Based on these results, rituximab appears a safe, effective, targeted therapy associated with a high response rate in adult patients with acute refractory/relapsing idiopathic TTP.

Introduction : Primary cutaneous T cell lymphomas (CTCL) represent 70% of all cutaneous lymphomas. The most frequent is mycosis fungoides/Sézary syndrome (MF/ SS). In this entity, few high resolution cytogenetic studies have been performed. Our aim was to analyze chromosomal abnormalities in MF tumoral stage by array comparative genomic hybridization (ArrayCGH) and to describe potential candidate genes related to this disease. Patients and methods : Forty-one patients (22 males/19 females) with MF tumor stage were included from centres collaborating in the EORTC Cutaneous Lymphoma Group. DNA was extracted from frozen tissues containing more than 70% of tumor cells. ArrayCGH tecnhology was performed to detect genomic imbalances (gains/losses) using the Humane Genome CGH Microarray Kit 44B (Agilent Techologies). This array consists on 44.000 oligo probes of 60 bp covering all the human genome with a mean resolution of 50–100 Kb. CGH-Analytics 3.2.25 and InSilicoArray CGH (http://isacgh.bioinfo.cipf.es) was used for array analysis and to define SORIs (Smallest Overlapping Region of Imbalance). Results: Genomic abnormalities were observed in thirty-two cases (76%). Losses (62.36%) were more frequently detected than gains (37.64%). The mean chromosomal imbalances per case were 3.5 gains (0–14) and 5.6 losses (0–30). The minimal common regions altered were gains of 7q33.3 (55%), 17q21.1q21.3 (42.5%) and 8q24.21. Regarding to the losses, 9p21.3 (42,5%), 17p13.1 (42,5%) and 10p11.22 (17,5%) were the most frequent detected. SORIs and potential candidates genes from the most frequently altered regions are summarized in the following table. Type of change Cytoband First gene start (Kbp) size (Mb) Frequency Candidate genes Loss 9p21.3 MTAP 21795 0,2 42,50% MTAP, CDKN2A, CDKN2B Loss 17p13.1 DULLARD 7094 1,01 27,5% TP53 Loss 10p11.22 chr10:031132968 31132 1,5 17,5% TCF8 Gain 7q33.3 BG495318 135521 14,1 55% BRAF Gain 17q21.1q21.3 SMARCE1 36050 4,7 42,5% STAT5A/STATB Gain 8q24.21 M13930 128816 0,75 32,5% C-MYC Conclusions : Our results have shown high recurrent chromosomal abnormalities. Moreover, arrayCGH technology has allowed us to define in detail new common regions and to describe potential candidate genes in MF tumor stage as STAT5A/STA5B (17q21.2), BRAF (7q33) and TCF8 (10p11.22). Our findings are similar to those recently published by Vermeer et al in Sézary Syndrome, which lead to confirm the relationship between these two entities. Deletion of 9p21.3 (CDKN2A, CDKN2B, MTAP genes) and 17p13.1 (TP53) are concordance to previous studies.

Introduction: Primary cutaneous T cell lymphomas (CTCL) represent 70% of all cutaneous lymphomas. The most frequent is mycosis fungoides/Sézary syndrome (MF/ SS). In this entity, few high resolution cytogenetic studies have been performed. Our aim was to analyze chromosomal abnormalities in MF tumoral stage by array comparative genomic hybridization (ArrayCGH) and to describe potential candidate genes related to this disease.

Background: CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) therapy is standard treatment for aggressive Non-Hodgkin’s Lymphoma (NHL), and frequently results in myelosuppression. The resulting febrile neutropenia (FN) and chemotherapy-induced anaemia (CIA) may be treated with Granulocyte Colony Stimulating Factor (G-CSF) and Erythropoiesis-Stimulating Agents (ESAs), respectively. IMPACT is an observational study, ongoing since February 2007, to examine supportive care management practices in NHL patients undergoing CHOP chemotherapy. Since the beginning of this study, European treatment guidelines for use of ESAs in CIA changed: from initiation at 11 g/dL with the goal to achieve hemoglobin (Hb) within 11–13 g/dL, to, as of February 2008, initiation at 10 g/dL and target range of 10–12 g/dL. This planned interim analysis describes anaemia and transfusion outcomes from this ongoing study as of April 2008. Methods: European patients with diffuse large-B-cell lymphoma (DLBCL), planning at least 3 cycles of CHOP-14 or -21 +/− rituximab were included in the study. This subset analysis evaluated only those patients not participating in a clinical trial. Patients were retrospectively or prospectively followed up to the end of chemotherapy treatment to a maximum of 8 chemotherapy cycles. CIA treatment was according to local standard of care, either with or without ESAs. Data were collected on Hb at ESA initiation, reasons for ESA treatment, Hb achieved during the ESA treatment phase, Hb level at first transfusion, and transfusion incidence. Results: For this interim analysis, data were available for 454 patients (mean age 60.5 [SD 14.8] years and 54% male), of whom 64% received CHOP-21; 35% of patients received treatment for CIA comprising ESA and/or transfusion. Among patients who received ESAs (N=112), the most frequent reasons for initiating treatment included Hb level (79%) or perceived risk of CIA (24%). Forty-one percent of ESA-treated patients had Hb levels <10 g/dL at ESA initiation; overall, the mean (SD) Hb at initiation was 10.16 (1.22) g/dL. The median (Q1, Q3) chemotherapy cycle at which patients received their initial dose of ESA was 3 (1, 4). Among patients treated with ESAs, 61% achieved a Hb level between 10–12 g/dL during the ESA treatment phase; and 27% experienced a Hb rate of rise 31g/dL in 14 days. Twenty percent (n=22) of ESA-treated patients received transfusions. Among these, the mean (SD) Hb level at first transfusion was 9.15 (0.94) g/dL; 36% (n=8) of ESA-treated patients received their first transfusion in cycle 1. Conclusions: While the number of the patients available for this interim analysis is small, the results do provide some perspective on anaemia treatment in this population. European patients with DLBCL receiving CHOP-14 or -21 may experience treatment-related anaemia that can be managed with ESAs. Any ESA1 (N=112) Darbepoetin alfa (N=59) Epoetin alfa (N=21) Epoetin beta (N=30) 1Two additional patients labeled as receiving ‘Other’ ESA treatment included. 2Assessment of Achieved Hb excludes Hb values within 28 days after a transfusion. Note: week 1 indicates first week of ESA initiation. Age, mean (SD), years 64.3 (12.8) 63.5 (13.8) 65.3 (10.1) 65.7 (12.4) Men, n (%) 56 (50) 31 (53) 11 (52) 13 (43) ECOG 0–1, n (%) 88 (79) 46 (78) 13 (62) 27 (90) CHOP-21, n (%) 81 (72) 43 (73) 13 (62) 23 (77) Hb (g/dL) at initiation of ESA treatment, n (%) Achieved Hb (g/dL) week 1 to end of ESA treatment, n (%)2 Hb at initiation <10 46 (41) 22 (37) 11 (52) 13 (43) Achieved Hb ≥10 g/dL 32 (70) 15 (68) 8 (73) 9 (69) Achieved Hb 10–12 g/dL 28 (61) 14 (64) 6 (55) 8 (62) Hb at initiation <11 87 (78) 47 (79) 17 (81) 21 (70) Achieved Hb ≥11 g/dL 44 (51) 27 (57) 7 (41) 10 (48) Hb at initiation <12 100 (89) 54 (92) 18 (86) 26 (87) Achieved Hb ≥12 g/dL 35 (35) 19 (35) 5 (28) 11 (42)

The remission rate with plasma exchange (PE) in thrombotic thrombocytopenic purpura (TTP) exceeds the 80%, but the disease relapses in up to 20–30% of the cases. Clinical characteristics and response to treatment of relapsed TTP is not well defined. The objective of the present study was to analyze the presenting clinical and biological features of relapsed TTP as compared with de novo TTP in a series of 102 TTP episodes (70 de novo and 32 relapses) followed prospectively and treated by daily PE and corticosteroids according to a homogeneous protocol. In comparison with de novo TTP, relapsed TTP episodes showed a higher Hb level (122 g/L versus 91 g/L, p<0.001) and lower serum LDH (2.2 versus 4.5 fold above the upper limit of normality, p<0.001). Neurological symptoms and fever were less frequent in patients with relapsed TTP than in patients with de novo TTP. There were no statistically significant differences in the percentage of patients with a severe deficit of ADAMTS13 activity (72% in de novo TTP versus 81% in relapsed TTP, p not significant) or the presence of inhibitory autoantibodies (87% versus 81%, p not significant). Patients with relapsed TTP needed fewer PE sessions (5 versus 10, p= 0.02) and a smaller volume of plasma (221 ml/kg versus 468 ml/kg, p=0.004) to achieve remission than those with de novo TTP. There were no significant differences in the rate of recrudescence under treatment, the need for complementary treatments (e.g. rituximab or splenectomy) or the frequency of refractoriness to PE therapy. In conclusion, relapsed TTP presents with a milder clinical and biological profile and responds easier to PE than de novo TTP.

Background: Therapeutic plasma exchange (PE) is the mainstay of treatment of thrombotic thrombocytopenic purpura (TTP). In many patients, prolonged PE is required to achieve remission and between 30% and 60% of patients relapse after a variable period of months to years, with a further episode of TTP or a chronic picture of recurrent relapses. Various immunosuppressive agents have been used in acute refractory and chronic relapsing cases. Despite advances in treatment, mortality remains at 15–20%. Limited clinical data from small groups of patients suggest that the administration of anti-CD20 antibody (Rituximab) may also be useful in acute refractory and chronic relapsing TTP. However, whether rituximab could be and effective strategy for adults with relapsed or refractory TTP remains unresolved.

The remission rate with plasma exchange (PE) in thrombotic thrombocytopenic purpura (TTP) exceeds the 80%, but the disease relapses in up to 20–30% of the cases. Clinical characteristics and response to treatment of relapsed TTP is not well defined. The objective of the present study was to analyze the presenting clinical and biological features of relapsed TTP as compared with de novoTTP in a series of 102 TTP episodes (70 de novoand 32 relapses) followed prospectively and treated by daily PE and corticosteroids according to a homogeneous protocol. In comparison with de novoTTP, relapsed TTP episodes showed a higher Hb level (122 g/L versus 91 g/L, p<0.001) and lower serum LDH (2.2 versus 4.5 fold above the upper limit of normality, p<0.001). Neurological symptoms and fever were less frequent in patients with relapsed TTP than in patients with de novoTTP. There were no statistically significant differences in the percentage of patients with a severe deficit of ADAMTS13 activity (72% in de novoTTP versus 81% in relapsed TTP, p not significant) or the presence of inhibitory autoantibodies (87% versus 81%, p not significant). Patients with relapsed TTP needed fewer PE sessions (5 versus 10, p= 0.02) and a smaller volume of plasma (221 ml/kg versus 468 ml/kg, p=0.004) to achieve remission than those with de novoTTP. There were no significant differences in the rate of recrudescence under treatment, the need for complementary treatments (e.g. rituximab or splenectomy) or the frequency of refractoriness to PE therapy. In conclusion, relapsed TTP presents with a milder clinical and biological profile and responds easier to PE than de novoTTP.

Background:CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone) therapy is standard treatment for aggressive Non-Hodgkin's Lymphoma (NHL), and frequently results in myelosuppression. The resulting febrile neutropenia (FN) and chemotherapy-induced anaemia (CIA) may be treated with Granulocyte Colony Stimulating Factor (G-CSF) and Erythropoiesis-Stimulating Agents (ESAs), respectively. IMPACT is an observational study, ongoing since February 2007, to examine supportive care management practices in NHL patients undergoing CHOP chemotherapy. Since the beginning of this study, European treatment guidelines for use of ESAs in CIA changed: from initiation at 11 g/dL with the goal to achieve hemoglobin (Hb) within 11–13 g/dL, to, as of February 2008, initiation at 10 g/dL and target range of 10–12 g/dL. This planned interim analysis describes anaemia and transfusion outcomes from this ongoing study as of April 2008.

The Coulter LH750® is a last generation automated hematology analyzer. In 1999 we evaluated the optimization of immature granulocytes/bands (IG/B) detection in the Coulter GEN*S (Blood Vol.94 n° 10 sup.1, 1999 page 45 b), in which we observed that the Mean Volume of the Neutrophil population (M NE V) and the standard deviation of the neutrophil volume (SD NE V) were the most useful parameters to improve the detection of the presence of IG/B. Recently the utility of SD NE V has been proposed in the diagnosis of septic patients.(ISLH 05). The purpose of this study is to confirm that the NE morphometric parameters in the LH750 have the same values and cut-off as the GEN*S to discriminate the cases with lefts shift and if it is possible to create rules/flags that use these parameters to allow the automated detection of IG/B with cut-off levels specific to the origin (emergency room, outpatients, intensive care etc.) of the patient sample. In addition we investigated if the parameters are useful when taking into account not only the % of IG/B, but also their absolute number as recommended by the NCCLS. The overall intent being to optimize the efficiency of our laboratory in the detection of IG/B by decreasing the number of smears, but without increasing the number of false negatives. We have selected from the routine samples of a 2000 beds hospital, 340 consecutive cases on the LH750® that triggered the bands /Immature granulocytes flags, Immature NE 1 (NE1) or Immature NE 2 (NE2). We considered the samples with more than 0,9 IG/B (103/mL) as positive. Blood smears were performed on all the positive samples (340) and a WBC differential was performed according the NCCLS protocol (H 20A). The percentage and absolute number of IG/B were collected. The statistical analysis was performed using Medcalc® version 8.1.0.0. A ROC Curve and the cut-off for the detection of positive samples with immature IG/B using the SD NE V and M NE V was calculated. RESULTS: For the flag NE1 the best parameter to select the postive cases with left shift was the SD NE V with a cut-off of >24,2. (although this flag is actually defined for a moderate left shift). For the flag NE2 the best parameter to select the postive cases with left shift was the M NE V with a cut-off of > 161. CONCLUSION: Before the study performed in 1999 we validated the Imm NE1 and Imm NE2 flags according to the NE% and NE# or the WBC values, that statistically demonstrated limited utility. Parameters which permitted to optimize the detection of IG/B, were the Neutrophile morphological parameters; M NE V and SD NE V. The values of the LH 750 are slightly different to those of the GEN*S. We will subsequently study their utility and advantages compared to the IG/B counting in the detection of bacterial infections and sepsis due to better reproducibility.

The recent introduction of novel proteomic techniques to evaluate normal human platelet proteins and their modifications following activation may also help to elucidate the causes and improve diagnoses of platelet-related bleeding disorders. Previous studies from this and other laboratories have demonstrated the sensitivity and reliability of this new technique to detect minor changes (+/−20%) in the protein content of platelets. More than 600 proteins shared by normal platelets were identified, and >200 in platelet-derived microparticles (MPs) generated by ionophore or Thrombin Receptor Activating Peptide (TRAP) activation. As a prelude to our ultimate goal of establishing this new technology, we applied the iTRAQ method (multiplexed relative protein quantitation by mass spectrometry) in the study of two unrelated patients with the Gray Platelet Syndrome (GPS). Megakaryocytes of patients with GPS can synthesize alpha-granule proteins and enclose them within membranes. However, the membranes lack structure linked latency, and enclosed proteins leak out of the organelles before platelets are delivered to circulating blood. Thus, GPS platelets contain empty alpha-granule vacuoles instead of alpha-granules. Due to large changes in platelet properties, it was surprising to find that cytoskeletal proteins of GPS were present in similar abundance to normal platelets (i.e. in the range of 0.80–1.20). Receptor proteins showed a similar distribution. As expected, the content of most alpha-granule proteins in the platelets from the 2 GPS patients was markedly reduced, with individual protein abundance ratios of 0.10 to 0.52 compared to normal platelets. However, certain trans-membrane proteins of alpha-granules (such as vesicle-associated proteins and P-selectin) were better preserved in GPS platelets, with ratios in the range of 0.73 to 1.16 compared to normal platelets. These results showed that many structural components (such as cytoskeleton and receptor proteins) are unchanged in GPS platelets. The results support our previous data indicating that detection of a change of as little as 20% may indicate a pathological change in the platelet proteome that may impact normal function. Our results therefore demonstrate that highly sensitive methods, capable of detecting small protein changes, may be needed to fully appreciate pathological disorders. The iTRAQ technique is able to detect low level changes and may be a leading tool that can generate new insights into the molecular pathophysiology and genetic variations of platelet disorders.