ALBERT

Description: Introduction/ Background : Chronic lymphocytic leukemia (CLL) is a heterogeneous disease which can present as an aggressive and life threatening leukemia or as an indolent form that will not require treatment for decades. This heterogeneity has important consequences which will impact on clinical approaches, treatment strategies, and survival times from diagnosis. Prognostic markers such as expression of specific proteins in or on CLL cells (ie, CD38, 70-kD ζ-associated protein or CD49d), cytogenetic abnormalities (del 13q, del 11q, del 17p and trisomy 12) quantified by FISH and immunoglobulin heavy chain (IgVH) gene mutation have all been very useful. Futhermore, patients with early-stage disease, with biologically aggressive disease and shorter survival times can be distiguished. However, these prognostic tests are expensive and require considerable technical expertise and equipment and thus are not available to many patients with CLL living in developing countries. Therefore less expensive prognostic markers are needed. In this study, we evaluated the prognostic significance of smudge cells percentage on a blood smear in CLL patients. Patients and Methods : In this prospective study, 42 untreated patients with CLL have participated after signing a consent form. Patients were seen at our center between July 2011 and May 2015. Patients were diagnosed on the basis of an absolute lymphocyte count greater than 5.109/L and a demonstration of monoclonality using flow cytometry (panel comprising CD19, CD5, CD22, CD23, FMC7, and surface Ig). Staging was done according to the Binet staging system. CD38 surface expression was determined by flow cytometry in all patients. The cytogenetic abnormalities : del 13q, del 11q, del 17p and trisomy 12, were performed by FISH and available for 25 patients.Smudge cells were defined as broken cells with no intact cytoplasm and a disrupted nuclear membrane (Figure 1). The smudge cell percentage is estimated by counting 200 lymphocytes and/or smudge cells; the smudge cell number is then divided by total number of cells counted (smudge cells + intact lymphocytes) and multiplied by 100. Each slide was evaluated by 2 hematologists and the blood smears were prepared using a manual wedge method. Categorical variables were analyzed using the χ2 test or Fisher exact test (p

Description: Key Points Severe baseline anemia is associated with leg ulcer, microalbuminuria, and echographic pulmonary hypertension in African SCD patients. These vascular complications of SCD are not independently associated with indirect markers of increased hemolysis.

Description: Abstract 1633 Malaria is a real public health problem in Africa, more than 300 million new cases and approximately two million deaths arise every year. Sickle-cell disease (SCD) patients are at high risk of developing malaria which is a major contributor to morbidity and mortality in this disease. In Senegal, malaria transmission is high during rainy season, between July and October, and it was noted that sickle-cell crisis are frequent during this period. Then we carried out a double-blind randomized controlled trial to compare the impact of monthly sulfadoxine-pyrimethamine (SP) during the high-transmission season versus placebo on malaria incidence and morbidity of sickle-cell anemia. Sixty (60) SCD patients were randomized either to receive three intermittent preventive treatment (ITP) with SP or placebo using the random permutation table with nine elements. The drug was administrated as follows: sulfadoxine 25 mg/kg and pyrimethamine 1.25 mg/kg and this treatment was given once during the following months: September, October, and November. Overall four episodes of malaria disease were diagnosed, all these cases in the placebo arm. Thus, overall prevalence was 6.6% and there was no other case of malaria in the SP arm during the study period. Parasitological diagnosis confirmed the presence of Plasmodium falciparum in all four cases. No patient death was encountered during the study. SP treatment was well tolerated as only one patient (1.6%) in the SP arm reported pruritis. A significant reduction of patients’ complaints (p= 0.002) and blood requirements (p = 0.001) was noted in the SP group; whereas, no impact was observed on vaso-occlusive crisis and hospitalization occurrence. Malaria prophylaxis by monthly intake of SP during the transmission period of the parasite reduced the prevalence of malaria and was safe in SCD patients leaving in malaria endemic area. Disclosures: No relevant conflicts of interest to declare.

Description: The incidence of CLL in Africa is not known for several reasons, among them the difficulty to access the diagnosis since there is only clinical features and cytology which are available in several countries in francophone Africa, especially in Senegal. The WHO criteria which have been published since 2001 from the first revised WHO classification of tumours of haematopoitic and lymphoid tissues cannot be applied since there is no facility for immunophenotyping and cytogenetic. Moreover, no research in place can be set up without any precise diagnosis. In this study, we have developed the flow cytometry technique in the laboratory of Immunology in CHU Le Dantec in Dakar to obtain the immunophenotype looking at the expression of Kappa/lambda light chains, CD19, CD22, CD20, FMC7, CD5, CD10, CD23, CD38. Peripheral blood smears were fixed according to the technique for FISH to detect the following cytogenetic abnormalities: trisomy12, deletion 13q14, deletion 11q22-23, deletion 17p. RNA to test by qRT-PCR the expression of microRNA mir15a, mir16-1, mir181a, mir181b, and mir 34a and 34b was obtained from peripheral blood lymphocytes. Twelve cases of CCL aged ranging from 50 to 80 year old were characterized at clinical level according to the Binet’s system: 1 stage A, 5 stage B, 6 stages C. In most of cases (9 cases) hyperlymphocytosis of small lymphocytes with clumped chromatin and scanty cytoplasm was very high (122.000 to 336 000/mm3). The “Matutes” score obtained by immunophenotyping was 5 or 4 for 11 patients in favor of typical CLL. The expression of CD38 which is a marker of unfavorable prognosis is positive in 9 patients. Nine patients had cytogenetic abnormalities: 3 trisomy 12, 6 del 13q14, 2 del 11q22-23, 1 del17p. Four patients had two simultaneous cytogenetic abnormalities (tri 12, tri 13 ; del13q14, del11q22-23 ; del13q24, del17p ; del13q24, tri12). The analysis of mi-RNA showed high expression of mir15a and mir 16-1 and low value of mir181a and mir 181b which were described in aggressive CLL ; however the expression of mir 34a and mir34b was also high. This study shows the aggressiveness of CLL in Senegal, probably due to the delay of the diagnosis. We have also demonstrated the possibility to set up immunophenotype and the possibility of a precise diagnosis of lymphoproliferative disorder in place, in western Africa and the series will be extended. Moreover we have also demonstrated the possibility to construct translational research project from samples characterized in the University Hospital in Dakar gathering the cases from different sanitary structures in the country. Such experiment would be a model for epidemiologic, clinico-biologic and translational research studies in order to set up in the country the capacity building for diagnosis and research. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction About 80 % of sickle cell disease (SCD) patients live in sub-Saharan Africa whereas most studies on SCD are performed in Europe and America. Hyperhemolysis is thought to play a major role in the pathological process of SCD vasculopathy. Nevertheless, the paradigm of hyperhemolysis is controversial and has never been studied in the African context. We aim to analyze the association between hemolysis and clinical vascular complications among SCD African patients. Methods CADRE is a multinational cohort of SCD African patients aged 3 years and older, included prospectively and explored in a steady state. All patients from the CADRE cohort in Ivory Coast, Cameroun and Mali were included in the present study. Patients were classified in SS-Sβ0 phenotype or SC-Sβ+ phenotype. SCD vascular complications (pulmonary arterial hypertension (PAH), microalbuminuria, leg ulcers, priapism, stroke and osteonecrosis) were assessed using clinical examination, laboratory exams and echocardiography. Hemolysis was measured by a composite score, created with a principal component analysis, that included LDH, hemoglobin and bilirubin rates and clinical icterus. The association between hyperhemolysis (upper quartile of the hemolysis score) and the vascular complications was assessed using multivariate regression analysis in the whole population with further stratification by hemoglobin phenotype. Results We included 2409 patients among which 1751 SS-Sβ0 patients and 658 SC-Sβ+ patients. Compared to SC-Sβ+ patients, SS-Sβ0 patients were younger (15 years old versus 21 years old) and exhibited more leg ulcers (166 (9.5%) versus 24 (3.7%)) and microalbuminuria (573 (42.4%) versus 119 (20.0%)). The hemolysis score was higher in SS-Sβ0 patients as compared to SC-Sβ+ patients (median 1.18 versus 0.43, Figure). After adjustment for age, sex and country, hyperhemolysis was associated with microalbuminuria (OR = 1.59 [1.17-2.16]) and priapism (OR=1.58 [1.01-2.49]) (Table). In SS-Sβ0 patients, hyperhemolysis was associated with microalbuminuria (OR=1.54 [1.10-2.15]) and there was a trend to an association with PAH (OR=1.65 [0.91-2.98]) and priapism (OR=1.50 [0.95-2.39]). In SC-Sβ+ patients, only the association between hyperhemolysis and microalbuminuria remained significant. Sensibility analysis in the adult population showed that hyperhemolysis was significantly associated with all the SCD vascular complications, except for osteonecrosis. Conclusion In African SCD patients, associations between hyperhemolysis and SCD vascular complications were statistically significant but of modest magnitude, and depended on the hemoglobin phenotype. These results suggest that in the African context, hemolysis is not a major determinant of the development of SCD vasculopathy. Table. Associations between SCD complications and hemolysis score (quartiles 1 to 3 versus quartile 4) in the whole population: multivariate analysis with adjustment for age, sex, country and hemoglobin phenotype % of patients with the complication Multivariate analysis N event/N total Quartiles 1 to 3 Quartile 4 OR IC 95% P value PAH* 115/431 6.14 7.08 1.58 0.89-2.83 0.123 Urine albumin/creatinine 〉 30mg/g 724/1991 24.88 45.36 1.59 1.17-2.16 0.004 Leg ulcer, lifetime 190/2409 6.80 11.01 1.20 0.81-1.78 0.355 Priapism, lifetime ** 160/1095 13.27 17.90 1.58 1.01-2.49 0.046 Stroke, lifetime 25/2409 0.79 1.77 1.32 0.49-3.54 0.578 Osteonecrosis, lifetime 277/2409 11.22 12.28 1.00 0.70-1.43 1.000 PAH: Pulmonary Arterial Hypertension. * Patients from Mali and Cameroun only ** Males only Figure 1. Distribution of the hemolysis score: global population, SS-Sβ0 patients and SC-Sβ+ patients Figure 1. Distribution of the hemolysis score: global population, SS-Sβ0 patients and SC-Sβ+ patients Disclosures No relevant conflicts of interest to declare.

Description: Introduction Although most individuals with sickle cell disease (SCD) live in sub-Saharan Africa, the history of the disease on this continent remains largely unknown. SCD is characterized by the association of chronic hemolytic anemia with episodes of acute vaso-occlusive events and progressive vascular organ damage. Several pathophysiological pathways in SCD result in the activation of circulating blood cells and the release of microparticles (MPs). In the present study, we investigated cell-derived MPs in patients with SCD living in Africa and analyzed their relationship with clinical complications. Patients and Methods This cross-sectional case-control study is nested in the CADRE cohort (clinical trials.gov identifier NCTO3114137). We included 232 SS adults in two African centers: Bamako (Mali) and Dakar (Senegal). Patients were chosen depending on the absence or the presence of at least one of the following complications: tricuspid regurgitant jet velocity (TRJV) 〉3 m/s (which may indicate pulmonary hypertension), macroalbuminuria, leg ulcer, priapism, aseptic osteonecrosis, and retinopathy. Overall, 7 groups of 40 SS patients were constituted (20 in each center). Patients were investigated at steady state (i.e.,at least 15 days after a vaso-occlusive crisis, 8 days after fever or infectious disease, and 3 months after a transfusion). MPs were isolated in the African centers immediately after blood sampling by successive centrifugations at increasing speed: 2,500g x2 and 21,000g x2. MPs pellets were stored at -80 °C. The cellular origin of the MPs, erythrocyte, reticulocyte, endothelial, platelet, and leucocyte, was determined using antibodies directed against CD235a, CD71, CD106, CD41, and CD45, respectively, at the National Institute of Blood TransfusioninParis. To maintain the background at an acceptable level, events of 0.16 µm size were excluded (Fig 1A). Only MPs positive for Annexin V and the cell-type-specific labelling were retained. Potential associations between cell-derived MPs, hematological parameters, and vascular complications were assessed using logistic regression with adjustment for age, sex and country. Results The MP pellets of 106 SS patients from Bamako and 126 from Dakar were analyzed in Paris. In these patients, at a mean age of 29 years (+/- 11), high TRJV was present in 64, microalbuminuria in 84, leg ulcers in 33, priapism in 43, aseptic osteonecrosis in 45, and retinopathy in 31 patients whereas 28 patients had no complication at the time of sampling. As a typical result, Fig 1B shows erythrocyte-derived MPs labelled by Annexin V and CD235a (quarter Q2). The MPs distribution was as follows: erythroid 52% [reticulocytes (CD235a+CD71+) 14%, erythrocytes (CD235a+ CD71-) 38%], leukocyte 18%, platelet 21%, and endothelial 9%. Neither erythrocyte- nor reticulocyte-derived MPs significantly correlated with hemolysis markers (LDH, unconjugated bilirubin or reticulocytes) or hemoglobin levels. Erythrocyte- and reticulocyte-derived MPs were significantly lower in patients with retinopathy (OR=0.48, p=0.003 and OR=0.68, p=0.005, respectively). Reticulocyte-derived MPs were negatively associated with a history of priapism (OR=0.76, p=0.020) and positively associated with a history of leg ulcers (OR=1.23, p=0.040). No correlation was found between MPs of other cellular origin and chronic complications, except for a negative association between endothelial-derived MPs and priapism (OR=0.76, p=0.036). Conclusion In our African patients with SCD, erythroid-derived MPs, although recognized cellular products of hemolysis, were not associated with other markers of hemolysis. We hypothesize that erythroid MPs are not only derived from hemolysis but also probably from the sickling process in this population. They were negatively associated with retinopathy and priapism and positively associated with leg ulcers, but not with the other complications classically associated with the hyperhemolytic sub-phenotype. The search for pertinent biomarkers of SCD complications in Africa is an essential challenge. To our best knowledge, this report is the first illustrating the feasibility of high-technology experiments in an African context. Disclosures No relevant conflicts of interest to declare.

Description: Introduction Sickle cell disease (SCD) is not only responsible for acute vaso-occlusive events but also for chronic vasculopathy that affects many organs including kidneys. Animal studies have suggested that SCD vasculopathy mainly results from chronic hemolysis. However hemolysis markers have not been consistently associated with clinical vascular complications. Moreover, SCD vasculopathy events have been almost exclusively studied in the USA or in Europe, although more than 80% of SCD patients are born and living in sub-Saharan Africa, a very different environment. We have settled the first multinational African SCD cohort to measure the incidence of SCD vascular complications and looked for their predictive factors in sub-Saharan Africa. We present here our first results, focusing on SCD nephropathy with glomerular involvement and urinary loss of albumin. Methods CADRE is an ongoing cohort of SCD patients in five African countries: Cameroon, Gabon, Ivory Coast, Mali, and Senegal. Included subjects are enrolled at steady state and undergo clinical exam, blood sampling for hemoglobin electrophoresis, blood count, creatinine, lactate dehydrogenase (LDH) and bilirubin levels, and urine sample for albumin and creatinine levels, as well as echocardiography. Main outcome for the present study was micro or macroalbuminuria defined as urine albumin/creatinine ratio 〉30 mg/g, that has been shown to be predictive of chronic renal insufficiency in SCD. We differentiated two main SCD groups: 1) SS and Sβ0 and 2) SC and Sβ+. Frequency of albuminuria at inclusion was calculated in both groups, with further stratification according to age. We looked for clinical and biological correlates to albuminuria in both groups using multivariate logistic regression. Results In June 2013, 3850 SCD patients have been recruited, among them 3032 (2290 SS, 445 SC, 202 Sβ+, 95 Sβ0) had available complete inclusion data. Median age was 15 and 20 years in SS/Sβ0 and SC/Sβ+ group, respectively, with similar female/male ratio of 1.2. Frequency of albuminuria was 36.9% [34.4-38.7] in SS/Sβ0 and 16.1% [13.2-19.9] in SC/Sβ+ patients, and increased significantly with age in SS/Sβ0 patients (Fig 1). Albuminuria remained significantly higher in SS/Sβ0 group than in SC/Sβ+ group, after adjustment for age, sex, country, blood pressure and hemoglobin level (p

Description: Introduction : Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations, being rarer in Asian and African people. It has been suggested that patients with CLL from Africa might have a more aggressive disease compared with Causasien patients. In this study, we aimed to identify genetic factors that may account for this difference Methods: We collected peripheral blood mononuclear cells (PBMCs) from a total of 75 patients with CLL, 25 from Senegal (Africa), and 50 from Siena. Since it is well known that there are differences in germline IGH repertoires between different populations, we also collected PBMCs from five healthy Senegalese individuals as control. We analyzed immunoglobulin heavy chain (IGH) genes mutational status by performing next-generation sequencing in these 2 groups of patients. Results: We found that Senegalese patients more frequently had adverse prognostic factors and an unmutated profile. Furthermore, we documented that IGHV1 (IGHV1-69), IGHD3, and IGHJ6 were significantly more frequent in Senegalese patients, whereas IGHV3-30 was common and limited to the Italian cohort. Stereotyped receptors commonly detected in the white population were not recorded in our Senegalese series. Conclusion: The different IGH repertoire we observed in the Senegalese cohort may reflect the diverse genetic and microenvironmental (ie, polymicrobial stimulation) background. Disclosures Gozzetti: Takeda: Honoraria; Amgen: Honoraria; Janssen: Honoraria, Research Funding.

Description: Introduction Growth failure has been a well-known complication of sickle cell disease (SCD) since the 70s. More recent studies show that the proportion of underweight children with SCD has decreased significantly, thanks to modern treatments (in particular hydroxyurea and iterative transfusions), with a tendency towards overweight and even obesity. However, most studies have been carried out in high-income countries, while 80% of the affected children are born in sub-Saharan Africa, where the environment and the medical care are entirely different. We carried out a case-control study nested in a multinational African cohort to study the growth of sickle cell children and the possible factors influencing growth failure. Methods We performed a case-control transversal study nested in the CADRE cohort that includes SCD patients from five African countries: Cameroon, Gabon, Côte d'Ivoire, Mali and Senegal. All children aged 5 to 21 years-old from this cohort were included in our study. Healthy controls were recruited among the patients' siblings or the children of health workers from each center. The main parameters studied were: medical history, height, weight, blood pressure; hemoglobin phenotype (SS, Sβ0, SC or Sβ +); complete blood count; hemolysis markers (LDH and bilirubin); microalbuminuria; echocardiographic parameters. The primary endpoint was growth failure, defined as a weight, height or BMI below the 5th percentile of the WHO growth charts. We described the frequency of growth failure according to hemoglobin phenotype, age and sex. Then we assessed by multivariate logistic regression in two SCD phenotypic groups (SS or Sβ0 and SC or Sβ +) the association between growth failure and the biological characteristics or the history of SCD-related complications. Results 2296 patients (1799 SS, 114 Sβ0, 287 SC, 96 Sβ+ patients and 287 controls were enrolled in Cameroon (n=735), Ivory Coast (n=380), Gabon (n=298), Mali (n=589) and Senegal (n=581). Overall, 48% of the patients were male and their median [interquartile range] age was 12 [8-16] years-old. Growth failure was diagnosed in 51% of SS, 58% of Sβ0, 44% of SC, 38% of Sβ+patients and 32% of controls, with deeper underweight than linear growth retardation. Beyond the age of 18, the mean BMI of SCD patients was again similar to that of controls in girls, but remained lower in boys, whereas the mean height was similar to that of controls regardless of sex (Figures a to d). Growth failure was more frequent in boys than in girls and maximal between 13 and 16 years-old (Figures e and f). In univariate analysis, the prevalence of growth retardation was associated with the country (highest in Senegal, lowest in Cameroon), age, male sex, hemoglobin phenotype, levels of anemia and hemolysis (p