HU Treatment Research Articles

Reducing DNA Damage Associated with Endogenous Genotoxic Stress in Hematopoietic Stem Cells

Fanconi anemia (FA) is a bone marrow failure (BMF) syndrome that arises from mutations in a network of FA genes essential for DNA interstrand crosslink (ICL) repair and replication stress tolerance. Endogenous formaldehyde is ubiquitous within cells as it is produced as a byproduct during normal cellular metabolism such as histone, RNA and DNA demethylation. Previous studies show that endogenous formaldehyde is a hematopoietic stem cell (HSC) genotoxin and metabolic carcinogen. We previously showed that endogenous formaldehyde is removed by the enzyme alcohol dehydrogenase 5 (ADH5), and Adh5-deficient mice therefore accumulate formaldehyde adducts in DNA that require the FA pathway to repair. Mice lacking Adh5 together with the FA pathway central component Fancd2 exhibit bone marrow failure and early lethality. Adh5 haploinsufficiency combined with Fancd2 deficiency, or Fancd2 -/- Adh5 +/- mice, demonstrates severely compromised HSC activity. However, the contributing factors that cause DNA damage and the protective mechanisms by which cells reduce DNA damage during endogenous genotoxic stress are not fully understood. Here, we show that the genetic inactivation of Lnk/Sh2b3 in Fancd2 -/- Adh5 +/- mice restores HSC function. Adh5 haploinsufficiency exacerbated Fancd2 deficiency, while Lnk deficiency rescued HSC homeostasis and transplantability of Fancd2 -/- Adh5 +/- mice. Moreover, Lnk deficiency increased HSC quiescence and reduced endogenous DNA damage in Fancd2 -/- Adh5 +/- mice. Importantly, Lnk deficiency does not participate in repairing formaldehyde-induced DNA damage, nor does it metabolize formaldehyde or reduce genomic formaldehyde-deoxyguanine adduct in vivo. Rather, Lnk deficiency reduced single-strand DNA (ssDNA) breaks and ssDNA binding protein RPA in HSCs during the cell cycle, thereby suppressing the ATR pathway activation upon endogenous replication stress. Moreover, loss of Lnk increased S-phase progression and fork recovery in Fancd2 -/- Adh5 +/- HSPCs at the stressed replication fork upon HU treatment, thereby preventing replication fork collapse and DNA damage. These findings highlight a new mechanism by which Lnk deficiency mitigates replication stress-induced DNA damage in response to endogenous genotoxic insult from cellular metabolism during HSC regeneration.

Preclinical and Clinical Use of AB1, a DNMT1 Protein Depleter, to Upregulate Fetal Hemoglobin in Townes Sickle Cell Disease (SCD) Mice and Patients with SCD

Background: Sickle Cell Disease (SCD) is an inherited hematologic disorder characterized by lifelong hemolysis, vaso-occlusion and severe complications including pain, acute chest syndrome and stroke among others. Its complex pathophysiology is driven by polymerization of mutated sickle hemoglobin (HbS) in red blood cells. Fetal hemoglobin (HbF) has been well documented to interfere with HbS polymerization, however, γ-globin gene transcription is silenced after birth through the action of a repressor complex including DNA methyltransferase 1 (DNMT1) during hemoglobin switching. We previously demonstrated the ability of AB1, a potent non-cytotoxic agent which depletes DNMT1 protein, to induce HbF in sickle erythroid progenitors. To expand these findings, we aimed to 1) perform preclinical studies in the Townes SCD mouse model to determine the ability of AB1 to induce HbF and deplete DNMT1 protein; and 2) evaluate the safety of oral AB1 in patients with SCD through an open-label, dose escalating phase 1/2 study (NCT05261711). Methods: Preclinical. Townes SCD mice, 4 months old (6 mice per group) were treated by intraperitoneal injections of escalating AB1 doses of 0.5, 1.5 and 3.0 mg/dose, hydroxyurea (HU; 100 mg/kg/dose) alone or HU combined with AB1 (0.5mg/dose) and water control, 5 days/week for 6 weeks. Every 2 weeks, complete blood counts with differentials were performed along with flow cytometry for HbF positive cells (F-cells) and reticulocytes; RT-qPCR for γ- and β-globin mRNA levels was completed and the spleen was harvested for weights and DNMT1 protein levels by Western blot. Clinical Trial. After informed consent, patients with confirmed HbSS or HbSb 0 thalassemia, on stable HU doses (if on treatment), and no recent/chronic transfusions or pain crisis were approached for the study. Patients were enrolled at Duke and Augusta University, starting on 2 mg of AB1 with 4 additional cohorts (4mg, 8mg, 16mg, and 32mg) to be recruited. Doses were taken daily by mouth for 8 weeks. Safety and tolerability (primary outcomes), as well as pharmacokinetics, pharmacodynamics and HbF response (secondary outcomes) were assessed. The F-cells and mean fluorescence intensity were measured by flow cytometry and HbF% by HPLC. Results: Preclinical. In general, SCD mice treated with AB1 showed good weight gain without toxicity. Three deaths occurred in the HU and combination treatment groups along with a significant decrease in platelets, granulocytes, and monocytes; by contrast, one death occurred in the 3mg/kg AB1 group, but the blood or reticulocyte counts were not affected. By week 6, HU increased F-cells by 2.1-fold (p=0.02) compared to a 2.2-fold (p=0.006) and 1.5-fold (p=0.03) increase by AB1 1.5mg/kg and 3.0 mg/kg respectively. Likewise, γ-globin mRNA increased, and β-globin mRNA decreased at the highest dose. Spleen analysis showed decreased weights and an 80% decrease in reactive oxygen species by AB1. Finally, DNMT1 protein levels were depleted by AB1 in spleen tissue. Clinical Trial. The clinical trial opened for enrollment in the winter of 2022. Two patients completed cohort 1 and two patients have enrolled and started cohort 2. Both cohort 1 patients had HbSS with a median age of 29.5 years (range 28-31). No change in total hemoglobin or reticulocyte counts was observed in either subject treated with 2mg AB1 daily. During the treatment period two adverse events occurred (rash-drug related and elevated liver enzymes-unrelated), however there were no serious adverse events. AB1 increased F-cell levels in subject 1 by 5.7-fold (2.9% to 16.4%). Expanded analysis of subject 2 showed an increase in F-cells 3-fold (4.8% to 14.4%) and 3-fold increase in F-reticulocytes. At the lowest 2mg dose, by HPLC analysis both patients had an increase in HbF% from a mean of 6.3% to 7% supporting protein induction by AB1. Discussion: The goal of this project is to develop an oral-active HbF inducer for the treatment of individuals with SCD. Data generated in the SCD mouse showed a significant increase in F-cells and γ-globin gene transcription, along with DNMT1 protein depletion. Subsequent oral AB1 treatment of patients with HbSS confirmed increased F-cells and HbF protein levels without clinical toxicities. These findings support continuing the ongoing phase 1/2 study, which will provide further insights into the ability of AB1 to increase HbF without hematologic side effects in patients with SCD.

Do Hematologic Parameters Predict Thrombosis or Disease Transformation in Essential Thrombocythemia?

Background: Patients with essential thrombocythemia (ET) are burdened with an increased risk of thrombosis and disease transformation, both significant contributors to morbidity and mortality. However, based on the available literature, there are limited tools available to predict the development of these complications. Particularly, the association between hematologic parameters and thrombosis is unclear, making risk stratification and disease management challenging. Utilizing a large retrospective database, we assessed the impact of hematocrit (Hct), red blood cell count (RBC), white blood cell count (WBC), and platelet count (Plt) on the subsequent development of thrombosis and disease transformation to myelofibrosis (MF), polycythemia vera (PV), and acute leukemia. Methods: We included ET patients treated at our institution between 2007 and 2021. The index time was date of diagnosis or date of first contact at Mount Sinai Hospital for patients with a prior history of ET. Hematologic parameters (Hct, RBC, WBC, Plt) were documented at three-month intervals after the index time. Patients were included in the study if they had at least three sets of laboratory values prior to the outcome of interest. For each hematologic parameter, we performed univariate cox analysis of time to thrombosis or progression to either MF, PV, or acute leukemia. Each lab parameter was analyzed as a time dependent covariate, taking into account the length of time the patient was above or below the threshold of interest. Hct and plt were specifically evaluated as dichotomous variables, with a cut off of less than or greater than 45% for Hct based on CYTO-PV results. For plt, two cut-off values were used: less than or greater than 400 (based on ELN response criteria) and less than or greater than 600 (based on Cortelazzo et al HU treatment goal) in plt/microliter. Results: We identified 430 patients who met criteria and were included in the analysis. Our cohort of patients was mostly female (61%), non-Hispanic (66%), white (64%), JAK2 mutated (60%), with an average age at diagnosis of 56 years and a median follow-up time of 33 months. 41 (7%) patients had a history of prior thrombosis. There was a total of 23 major thromboses that occurred after the index time with 12 venous and 11 arterial thrombotic events. 38 patients progressed to post ET-MF, 20 patients developed post ET-PV, and 10 patients transformed to acute leukemia. For the assessment of thrombotic risk, there were no significant associations between any of the hematologic parameters and this outcome. We also evaluated the risk of thrombosis in the JAK2 positive subset of patients (n of 15), and did not find any significant association with any of the lab parameters (in particular, for hct < 45%, p > 0.99). For transformation to MF, plt < 400 x10 9/L was associated with an increased risk of the development of post-ET MF (HR 2.2, p 0.016), and the same was seen with plt < 600 x10 9/L (HR 2.2, p 0.044). The development of PV was less likely with a hct lower than 45% (HR 0.03, p < 0.001), with similar results seen for plt < 400 x10 9/L (HR 0.2, p 0.03) and plt < 600 x10 9/L (HR 0.2, p 0.002). For transformation to acute leukemia, plt < 400 x10 9/L (HR 8.3, p 0.008) was associated with an increased risk of this complication. Conclusions: In this retrospective cohort of 430 patients with ET, we found no associations between four different hematologic parameters over time with thromboses. In particular, this data once again argues against aiming to achieve a particular platelet goal to minimize thrombosis in ET patients. The lack of association between hematologic parameters and thrombotic risk was similarly exemplified in our subset of JAK2 mutated ET patients. Pertaining to disease transformation, plt count appeared to be the most predictive out of all of the parameters, with lower plt values corresponding to increased risk of both post-ET MF and acute leukemia, which both warrant further exploration. A hct level of less than 45% appeared to be protective against the development of post-ET PV. Significant limitations of our study include the retrospective nature as well as the small number of events of interest that our patients experienced. Future studies must continue to focus on surrogate endpoint validation in order to more reliably predict thrombotic events as well as disease progression in patients with ET given the significant morbidity and mortality that such events incur.

Double-Blind Randomized Study to Evaluate the Effect of Hydroxycarbamide on Albuminuria in Adults with Sickle Cell Disease: Sikamic (SIklos on Kidney Function and AlbuMInuria Clinical Trial)

Background Sickle cell disease (SCD) is associated with a high frequency of chronic kidney disease, which is an independent risk factor for mortality in this population. SCD-associated nephropathy (SCAN) is an emerging concern, characterized by significant albuminuria and progressive deterioration of renal function with a prevalence of up to 26% to 68% in SCD adult patients (Falk 1992, Powars 2005, Yeruva 2016, Ataga 202). Because of rapid decline of estimated glomerular filtration rate (eGFR) is a frequent finding in this population (Derebail 2019, Ataga KI 2021), early diagnosis of SCAN and identification of associated clinical and biologic risk factors are crucial for initiating kidney-protective therapy at early stages of renal impairment. Albuminuria serves as a relevant biomarker for detecting early glomerular damage in SCD patients (Audard 2017). To date, Hydroxycarbamide (HU) remains the cornerstone treatment for managing SCD patients, and its efficacy is primarily attributed to its ability to increase fetal hemoglobin (HbF) levels (Segal 2008). Several observational studies conducted in adult SCD patients have shown that HU may have renal protective effects, making it a promising therapeutic option, but until now, randomized clinical trials confirming this hypothesis are missing (Bartolucci 2015, Laurin 2014). Method SIKAMIC (SIklos on Kidney Function and AlbuMInuria Clinical Trial) is an international (France and Africa), phase IIb, double-blind, randomized, placebo-controlled study. The main objective is to assess the effect of HU on albuminuria levels in SCD adult patients after 6 months of treatment. Eligible patients are of the SS or Sβ0 genotype with a mean albuminuria value (assessed by albumin-to-creatinine ratio: ACR) above 3 and less than 100 mg/mmol, on three urine samples taken one day apart. Patients should not have received HU treatment within the previous 6 months and should not be taking conventional kidney-protecting measures (ACE inhibitors or ARA2) and having blood transfusion in the last 3 months. Patient with an eGFR rate < 60 and ≥ 140 ml/min/1,73m² are excluded from the study. Patients receive either HU at a dosage of 15mg/kg/day or placebo for 6 months, and responders (patient achieving at least a 30% decrease of the ACR baseline at 6 months) have the option to continue the study for an additional six months. Recruitment started in August 2019 in France and in October 2021 in Africa and was extended until June 2023 due to challenge in finding HU-Naïve patients. The end of the study is planned in June 2024 with safety monitoring conducted by an Independent Data Monitoring Committee throughout. Results During the recruitment period, 179 patients were screened, 99 were randomized, 34 in France, 65 in Africa (Senegal: 31, Mali: 23, Ivory Coast: 11) ( Figure 1). The main reason for screen failure was mean ACR < 3mg/mmol. Two patients were excluded from the analysis due to non-compliance with exclusion criteria. Among the 97 patients analyzed, 32 completed the study by June 30, 2023, while 46 were still ongoing treatment, and 19 withdrew before the end of the 6 months follow-up period. The study includes a higher proportion of women (68%), which can be attributed to challenges faced in conducting semen analysis in African countries and the hesitancy to initiate HU treatment without prior semen analysis. The mean age at enrolment was 29.7 ± 9.2 years. Recruitment significantly improved after opening African sites. The study encountered several difficulties, primarily stemming from the challenge of finding HU-naïve patients in France and the high rate of screen failure attributed to non-expected ACR measurement variability and eGFR outside the accepted limits. No safety issues were reported, with 15 well-known adverse events related to HU occurring in 11 patients. Results of the study are expected by the end of 2024. Conclusion This study is the first randomized placebo-controlled study investigating the effect of HU on albuminuria in SCD patients. By employing a complex and robust study design, with ACR measurements at multiple time points, the study will provide valuable insights into the impact of HU on albuminuria levels. Moreover, the collaboration with African countries has highlighted the significance of considering country-specific factors and fostering partnerships to address the unique challenges faced by patients in these regions.

Differences in Clinical Outcomes between Hydroxyurea-Resistant and -Intolerant Polycythemia Vera Patients

Previous retrospective studies have revealed that a substantial portion of PV patients experience resistance to hydroxyurea (HU-R) and intolerance to HU (HU-I). Currently, the therapeutic options for both groups of patients do not differ, and most clinical trials investigating second-line therapies have enrolled patients with HU-R or HU-I without considering potential clinical distinctions between the two groups. Direct comparisons between patients with HU resistance and HU intolerance have not been conducted, and prognostic factors related to clinical outcomes, such as thrombosis, bleeding, disease progression, and survival, remain unclear for patients experiencing HU resistance or intolerance. A total of 90 patients with HU-R/I were retrospectively anlyzed in this study. Among them, 44 and 46 patients fulfilled the ELN criteria defining resistance or intolerance to HU, respectively. Patients with HU-R were older at the time of diagnosis of PV (66.7 ± 13.2 years, vs. 59.8 ±11.2 years in the HU-I group, P = 0.016), had longer times to developing HU-R/I, and thus, were older at the time of HU-R/I development compared with those with HU-I. The majority of the study patients continued HU treatment with dose modification or intermittent discontinuation with anagrelide added to control platelet counts until disease progression or death because of limited second-line treatment options during the observed period. The cumulative incidence of thrombosis after 7 years of HU-R/I was 21.4%, and the incidence of disease progression was 22.5%. Comparing the HU-R and HU-I groups, the HU-R group had a significantly higher rate of disease progression (36.7% vs. 0.56%, P = 0.009), while there was no significant difference in thrombosis incidence (19.0% vs. 22.9%, P = 0.463). The type of thrombosis did not differ between the two groups (P = 0.628). Multivariate analysis revealed that HU-R was an independent prognostic factor for progression-free survival (hazard ratio = 5.57, 95% CI: 1.22 - 25.54, P = 0.027). The patients in the HU-R group had significantly lower PFS (40.2% vs. 66.1% in the HU- I group, P < 0.01). They also showed a trend toward lower TFS (51.0% vs. 62.1% in the HU-I group; P = 0.055) and lower OS (57.5% vs. 67.7% in the HU-I group; P = 0.060) compared to patients with HU-I. The mortality at seven years was 42.5% in the HU-R group and 32.3% in the HU-I group (P = 0.06). Higher LDH levels, multiple cardiovascular risk factors, and prior thrombosis were identified as unfavorable predictors of overall survival. These findings suggest that HU-resistant patients have a higher risk of hematological transformation but a comparable risk of thrombosis to HU-intolerant patients. These distinctions should guide decisions on second-line treatment options and clinical trials involving these patients.

Clinical and biological profile of Sickle Cell Anemia children in a rural area in Central Africa

ABSTRACTBackground:Sickle Cell Anemia (SCA) is the most common genetic disease worldwide caused by a single mutation in the gene HBB. The disease severity is very variable and depends on many factors. We evaluated the clinical and biological profile of sickle cell anemia children in rural Central Africa.Methods:This cross-sectional study was conducted in the Hôpital Saint Luc de Kisantu, located 120 km away from Kinshasa-DR Congo in an area of 35 km around Kisantu with a population of roughly 80 000 individuals. We included SCA patients aged 6 months to 18 years. We collected clinical and hematological data. The SCA scoring system proposed by Adegoke et al. in 2013 was applied to determine the disease severity. We searched for factors associated to the disease severity.Results:This study included 136 patients, 66 males and 70 females (sex-ratio M/F 0.94). The mean severity score was 8.21 ± 5.30 (ranges 0–23). Fifty-nine (43.4%) children had mild disease, 62 (45.6%) moderate and 15 (11%) severe disease. Girls had higher levels of HbF than boys (p = 0.003). An inverse correlation was observed between fetal hemoglobin and the disease severity (p = 0.005, r −0.239, IC95% −6.139; −1.469). Some factors such age influence the occurrence of certain chronic complications such as avascular bone necrosis.Conclusion:In conclusion, the disease severity of SCA depends on multiple factors. In this study, fetal hemoglobin was the main modulator of the disease severity. These data may also serve as a baseline to initiate HU treatment in this setting.

Changes in soil CO2 and N2O emissions in response to urea and biochar-based urea in a subtropical Moso bamboo forest

The application of biochar-based fertilizers is an effective strategy for enhancing plant growth and soil organic carbon stocks; however, the impacts of such practices on soil CO2 and N2O emissions and associated mechanisms in subtropical forests are poorly understood. A two-year field trial was conducted to determine effects of urea and biochar-based urea on variations in soil CO2 and N2O emissions, as well as various soil environmental factors in a subtropical Moso bamboo forest. Five treatments were established: a control (without fertilization), urea with low and high application rates (100 and 300 kg N ha−1, LU and HU), and biochar-based urea with low and high application rates (100 and 300 kg N ha−1, LBU and HBU). The soil CO2 emissions were increased by LU, HU, and HBU treatments during the first year compared to controls (P < 0.05). However, this stimulatory response was observed only under the HU treatment during the second year. The soil N2O emissions increased under the LU and HU treatments during the first year but were decreased under HBU (P < 0.05); during the second year, only HU had a stimulatory effect. Importantly, the soil CO2 emissions and their Q10 values under the biochar-based urea treatment were lower than those with urea (P < 0.05). The application of urea increased contents of NH4+-N, NO3−-N, water soluble organic C/N (WSOC/WSON), as well as activities of invertase, β-glucosidase, urease, and protease (P < 0.05). In comparison, application of biochar-based urea enhanced contents of WSOC, NH4+-N, and NO3−-N but decreased WSON and activities of β-glucosidase, urease, and protease (P < 0.05). Regardless of treatment, the CO2 emissions were correlated with WSOC concentration and invertase/β-glucosidase activities; the N2O emissions were associated with (P < 0.05) NH4+-N, NO3−-N, WSON and urease/protease activities. Structural equation models revealed application of urea/biochar-based urea influenced the soil CO2/N2O emissions primarily through altering the pool size of labile C/N fractions and the activities of enzymes regarding C/N cycling. In conclusion, compared with urea, application of biochar-based urea provided more advantages for mitigating soil CO2/N2O emissions in subtropical forest ecosystems.

Preliminary Study on 53BP1-Mediated DNA Double-Strand Break Response in Spermatogonial Stem Cells.

53BP1 mediates DNA repair process in somatic cells; however, the function of 53BP1 in germline stem cells still remains unclear. In the present study, animals and cells DNA damage repair (DDR) model was established by irradiation and HU treatment; immunofluorescence staining and laser confocal microscopy were used to detect the expression of 53BP1, p-CHK2, and p-P53 in the DDR process of mSSCs. 53BP1 knockdown expression mSSCs cell line conducted by Trp53bp1-shRNA was established and EdU staining was adopted to analyze cell cycle and cell proliferation. Moreover, NHEJ reporter vector was applied to detect the repair efficacy after Trp53bp1 knocked-down (KD) expression. Results showed that 53BP1 could form foci signals in mSSCs during DDR process both in vivo and in vitro, which was independent of γH2AX. 53BP1 downstream protein, p-P53, and p-CHK2 were involved and dynamically expressed in DDR response. Knocking down of Trp53bp1 expression in mSSCs could not dramatically inhibit cell proliferation, but may increase cell sensitivity to HU. The NHEJ repair efficacy was sharply decreased in Trp53bp-KD SSCs via flow cytometry analysis. We revealed the specific mechanism of 53BP1 in SSCs DDR process, which is expected to provide a new theoretical basis and insights for the diagnosis and treatment of male infertility.

Determinants of hydroxyurea use among doctors, nurses and sickle cell disease patients in Nigeria.

Hydroxyurea (HU) is an evidence-based therapy that is currently the most effective drug for sickle cell disease (SCD). HU is widely used in high-income countries with consequent reduction of morbidity and mortality. In Nigeria, HU is prescribed by physicians while nurses are mainly involved in counseling the patients to ensure adherence. The extent of utilization and the determinant factors have not been sufficiently evaluated in Nigeria. To assess the frequency of use of HU and factors affecting utilization among healthcare providers, patients, and caregivers for SCD. A questionnaire was administered online and in- person to assess the frequency of HU use and the factors that promote and limit its use. The data were analyzed by descriptive statistics using IBM SPSS software version 23 and the result was presented in frequency tables and percentages. A total of 137 physicians, 137 nurses, and 237 patients/caregivers responded to the survey. The rate of prescription of HU by doctors in the past 6 months was 64 (46.7%), 43 (31.4%) nurses provided counseling and 36 (15.6%) patients were on HU. Among doctors, adequate knowledge (91.3%), clinical benefits and safety (94.8%), and inclusion of HU in management guidelines (86.9%) were motivators for prescribing it while inadequate knowledge (60.9%) and unawareness of treatment guidelines (68.6%) constituted barriers. Among nurses, reduction of crisis (91.6%) and safety (64.8%) were the major motivators while barriers were high cost (79.1%) and intensive monitoring (63.1%) of HU treatment. Among the patients, the major motivator was the reduction of crises (80.3%) while poor knowledge (93.2%), high cost of the drug (92.2%) while monitoring (91.2%), non-availability (87.7%) and side effects (83.9%) were the major barriers for the utilization of HU. HU prescription and utilization are still poor among healthcare providers and patients. Inadequate knowledge, non-availability and high cost of HU as well as unawareness of treatment guidelines constitute major barriers to prescription and utilization.

P1478: INTEGRATIVE DIAGNOSIS OF SICKLE CELL DISEASE PATIENTS FOR PERSONALIZED MEDICINE

Background: Sickle cell disease (SCD) is a chronic life threatening autosomal recessive disorder, caused by the presence of structurally abnormal adult hemoglobin S (HbS). Under low oxygen saturation, HbS forms hemoglobin polymers that deform the red blood cell structure, referred to as ‘sickling’. Sickled erythrocytes result in hemolytic anemia and recurrent vaso-occlusive crisis, which lead to long-term morbidity and ultimate early death. The patient specific pO2 at which sickling starts (PoS) along with RBC deformability at normoxia (EImax) and upon deoxygenation (EImin) can be measured by oxygen gradient ektacytometry (Laser Optical Rotational Red Cell Analyzer (LoRRca)). In the GenoMed4ALL project, oxygen gradient ektacytometry data will be integrated with genomics, metabolomics and clinical data of 1000 SCD patients. This will allow better characterization of SCD and development of Artificial Intelligence (AI) algorithms for personalized medicine. Aims: To analyze the correlation of Lorrca parameters (PoS, EImax and EImin) with SCD genotypes (SS, Sβo, SC and Sβ+), hydroxyurea treatment (HU-, HU+) and HbF. Methods: SCD patients with SS, Sβo, SC and Sβ+ genotypes, older than 1yo at steady state with no transfusion in the last 3 months were analyzed by oxygen gradient ektacytometry. All samples were processed in duplicates. Derived parameters were correlated with genotypes, and for severe SCD (SS, Sβo) with hydroxyurea options (HU-, HU+) and HbF. An Anova analysis has been carried out to test the relation of the parameters among groups. The post-hoc test Scheffe has been used to test which groups are different two by two. Results:: 49 samples were analyzed: 32 SS, 2 Sβo, 12 SC and 3 Sβ+. Mean and standard deviation values for PoS, EImax and EImin are shown in Table 1. PoS and EImin allowed patients’ clustering in 2 groups according to genotype: SS PoS: 34.34±5.0 Elmin: 0.23±0.1 vs Sβo, SC and Sβ+ SS PoS: 24.62±3.7 and Elmin: 0.34±0.1. EImin showed statistically significant Scheffe test value in discriminating SS vs Sßo and SS vs SC, while PoS was statistically significant only in discriminating SS vs SC. 31 samples from severe SCD patients (SS, Sβo) were further analyzed for correlation with HU (7 HU-, 24 HU+) and HbF. Mean and SD values are shown in Table 1. The differences in mean values for PoS, EImax and EImin between HU- and HU+ were of -27%, 54% and 135% respectively. The best correlation between Lorrca parameters and HbF was found in group of patients without treatment (HU-). EImin showed the highest correlation being 93% in HU- and 81% in HU+ group. Image:Summary/Conclusion: Our results demonstrate the value of oxygen gradient ektacytometry to the characterization of SCD patients. PoS and Elmin were able to distinguish between SS and other genotypes, regardless of HU treatment. In addition, PoS, EImax and EImin all showed differences between HU+ and HU- groups, being Elmin the most valuable one. Elmin also showed the highest correlation with HbF. It is highly likely that PoS could be impacted by other variables: 2,3 DPG, pH, RBC hydration, genetic variants or metabolomics. Pooling data of 1000 SCD patients from at least 9 EU centers in the GenoMed4LL project is needed to improve results robustness. Standardization of Oxygenscan data generation should be ensured for data comparison and development AI algorithms for personalized medicine.

Real-world data on voxelotor to treat patients with sickle cell disease.

The aim of this single-center chart review was to quantify the hematologic response and validated reported outcomes with voxelotor treatment. Real-world data were collected retrospectively in patients with sickle cell disease (12-70 years old) who were treated with standard-of-care procedures. Data were collected before and during voxelotor treatment. A total of 77 patients with a mean age of 30.4 years were included in the analysis; 30% of patients were children <21 years old. Most patients were female (62%), had a homozygous hemoglobin S (HbSS) genotype (86%), and were treated with concomitant hydroxyurea (HU; 82%). The mean baseline Hb level was 8.3g/dl, reticulocyte percentage was 11.5%, and total bilirubin was 3.5mg/dl. The mean duration of voxelotor treatment was 9.7months (range: 1.9-17 months). Favorable responses to voxelotor treatment and signs of hematologic response after voxelotor treatment included increased Hb levels, decreased reticulocyte percentage, and decreased total bilirubin. In patients treated with concomitant HU, a more robust improvement was noted versus voxelotor alone, suggesting a complementary effect. Recorded adverse events were rare, mild, and self-limited and resolved with dose modification. Hematologic improvements were observed after voxelotor treatment, with a potential additive benefit with concomitant HU treatment.

Hydroxyurea ameliorates atherosclerosis in ApoE-/- mice by potentially modulating Niemann-Pick C1-like 1 protein through the gut microbiota.

Rationale: The efficacy and mechanism of hydroxyurea in the treatment of atherosclerosis have rarely been reported. The goal of this study was to investigate the efficacy of hydroxyurea in high-fat diet-fed ApoE-/- mice against atherosclerosis and examine the possible mechanism underlying treatment outcomes. Methods: ApoE-/- mice were fed a high-fat diet for 1 month and then administered hydroxyurea by gavage continuously for 2 months. Aortic root hematoxylin-eosin (H&E) staining and oil red O staining were used to verify the efficacy of hydroxyurea; biochemical methods and ELISA were used to detect changes in relevant metabolites in serum. 16S rRNA was used to detect composition changes in the intestinal bacterial community of animals after treatment with hydroxyurea. Metabolomics methods were used to identify fecal metabolites and their changes. Immunohistochemical staining and ELISA were used for the localization and quantification of intestinal NPC1L1. Results: We showed that aortic root HE staining and oil red O staining determined the therapeutic efficacy of hydroxyurea in the treatment of atherosclerosis in high-fat diet-fed ApoE-/- mice. Serological tests verified the ability of hydroxyurea to lower total serum cholesterol and LDL cholesterol. The gut microbiota was significantly altered after HU treatment and was significantly different from that after antiplatelet and statin therapy. Meanwhile, a metabolomic study revealed that metabolites, including stearic acid, palmitic acid and cholesterol, were significantly enriched in mouse feces. Further histological and ELISAs verified that the protein responsible for intestinal absorption of cholesterol in mice, NPC1L1, was significantly reduced after hydroxyurea treatment. Conclusions: In high-fat diet-fed ApoE-/- mice, hydroxyurea effectively treated atherosclerosis, lowered serum cholesterol, modulated the gut microbiota at multiple levels and affected cholesterol absorption by reducing NPC1L1 in small intestinal epithelial cells.

Disease Burden and Pre-Transplant Health-Related Quality of Life in Pediatric Sickle Cell Disease Patients Receiving Nonmyeloablative HLA-Identical Sibling Donor Transplantation

Title: Disease Burden and Pre-transplant Health-related Quality of Life in Pediatric Sickle Cell Disease Patients Receiving Nonmyeloablative HLA-identical Sibling Donor TransplantationBackground: Sickle cell disease (SCD) is a hemoglobinopathy characterized by recurrent pain crises, severe organ damage, and reduced health-related quality of life (HRQL). Bone marrow transplantation (BMT) is a proven curative treatment for SCD that can improve both disease burden and HRQL. Despite the benefits, relatively few patients pursue a cure through BMT. The decision to receive a BMT is nuanced and likely to be influenced by a number of factors. One study by Roth et al. showed SCD patients interested in BMT had a higher HRQL than those uninterested in BMT, suggesting some variability between SCD patients pursuing transplant and the general SCD patient population. To date the factors that may affect pre-transplant HRQL of pediatric SCD patients remain unclear, particularly in the nonmyeloablative setting.Objective: To evaluate the impact of disease-burden, patient demographics, and hydroxyurea treatment on pre-transplant HRQL.Methods: Children and young adults with SCD set to receive a nonmyeloablative HLA-identical sibling donor BMT were administered HRQL surveys (PedsQL) in the six months leading up to the start of the conditioning regimen. Patients' disease history and demographic information were captured and analyzed for their relationship to HRQL. PedsQL parent surveys were used when patient surveys were unavailable. The mean difference in HRQL between groups were compared using an unpaired t test or linear regression.Results: 32 patients were enrolled onto this study. Six of the 32 patients HRQL were not included due to withdrawal, delay of transplant, or completion of the survey outside of window. The median age at the time of survey administration was 13.5 years (range 2-21). 57.7% of patients were male and 88% of patients had genotypes hemoglobin SS or Sβ 0thalassemia. 12% of patients had a history of overt stroke. 52% of patients were hospitalized while receiving HU treatment. 32% of patients received chronic blood transfusions leading up to transplant. The median number of hospitalizations in the two years leading up to transplant was 2 (range 0-10). 16.7% of patients met the AAPT diagnostic criteria for chronic pain. 75% of patients received HU treatment leading up to transplant. The median hemoglobin F percentage was 10.4. The median HRQL for all patients was 77.17 (range 36.9-98.91, SD +/- 15.99).Sickle cell genotype (P=0.88), history of overt stroke (P=0.79), hospitalization while receiving HU treatment (P=0.78), and the number of hospitalizations in the two years before transplant (P=0.39) were not associated with lower HRQL. History of chronic transfusion leading up to transplant approached statistical significance for lower HRQL (P=0.06). Older age (P=0.17) and female gender (P=0.89) were not associated with worse HRQL. Patients receiving HU treatment had significantly higher HRQL than patients not receiving HU (P=0.0034), with median PedsQL scores of 80.06 and 58.64, respectively. The date of survey administration was not associated with HRQL (P=0.40). The number of eligibility criteria met (P=0.63), chronic pain (P=0.17), total number of RBC transfusions (P=0.45), and pre-transplant hemoglobin (P=0.25) were not associated with HRQL.Discussion: Patient demographics and several markers of disease burden appear to have minimal impact on HRQL. Interestingly, the use of HU therapy was associated with significantly higher pre-transplant HRQL. The etiology of this finding is unclear given that disease burden was not associated with HRQL and warrants further investigation. The absence of association between disease burden and HRQL through current disease severity eligibility paradigms suggests that other factors may impact HRQL in SCD patients choosing a low toxicity nonmyeloablative BMT. DisclosuresNo relevant conflicts of interest to declare.

Management of Male Fertility in Sickle Cell Disease before and after Hydroxycarbamide

BackgroundHydroxycarbamide (hydroxyurea or HU) is an orphan medicinal product for the prevention of recurrent vaso-occlusive crises and acute chest syndrome in adults (US and Europe) and children older than 2 years (Europe) with sickle-cell disease (SCD).HU is generally well tolerated; however, it could have adverse effects on male fertility. Despite the fact that male fertility issues such as delayed puberty, sperm abnormalities and low testosterone level could be directly linked to the underlying SCD, thorough patient information and if it is possible, cryopreservation are recommended before starting HU. It has been demonstrated in a prospective comparison of sperm parameters in SCD adult patients before and after a 6 months treatment with hydroxyurea, impairment in sperm parameters (Bachir et al, 2012).ESCORT-HU (European Sickle Cell Disease Cohort-Hydroxyurea) is a European multicenter, prospective, non-interventional study initiated in Europe to collect more information about the safety profile and morbidity-mortality in SCD patients treated with Siklos® (HU). Our purpose in the present study was to verify whether recommendations regarding male fertility are really implemented and if cryopreservation is a common process in Europe through the preliminary results of ESCORT-HU study.Methods and patientsThe data used were from ESCORT-HU study. The cut-off date of data extraction was set as June 19th, 2017. Between January 2009 to June 2017, 1841 patients were enrolled from 63 centers in France, Germany, Greece and Italy. For this analysis only data (age, cryopreservation, sperm analysis results) from male patient were analyzed, no male patient has been excluded. 824 male-patients were aged 0.7 to 78 years at the time of study initiation among which 422 had reached puberty (>15 years of age), with a mean age of 31 years. The main hemoglobin genotype was SS (336 patients), followed by Sß+ (28 patients) and Sß0 (31 patients).ResultsOn the 422 patients aged 15 years and older, 170 cryopreservations (169 patients) have been reported, among them 121 semen analyses have been recorded. The mean age for performing these examinations was 30 years. Cryopreservations realized systematically with a sperm analysis were mainly performed in France, in contrary to Germany where only children were enrolled and moreover cryopreservations were not supported (Table 1).Before initiation of a treatment with HU, 67 sperms analysis (66 patients) were performed and 50 results (49 patients) were reported. Among them, 33 results (49%) were considered normal and 17 (25%) with abnormalities (at least 1 abnormal criterion: sperm mobility, sperm count, appearance).During HU treatment, 24 sperms analyses (24 patients) were performed and a result was reported for 18 of them. The rate of abnormalities during HU treatment increased to 50% (12 abnormal sperms analysis). Reported abnormalities were asthenospermia, hypospermia, oligospermia, azoospermia and atypical forms (Table 2).ConclusionThere were no patients with sperms analysis performed before and during the treatment in order to compare the results in our database.These results confirm information reported in a recent review (Table 3) about abnormalities include reduced sperm density and motility, reduced number of spermatozoa with normal morphology, and reduced semen volume.Intensification with HU during childhood might change these related-disease events however HU is itself deleterious for the sperm cells. Therefore, male fertility assessment before any HU therapy but also during the treatment should be carried out. Cryopreservation may be very useful if the resolution of HU-induced lesions is prolonged and if the patient wishes to become father. [Display omitted] DisclosuresRibeil:Novartis: Other: Non-financial support; Addmedica: Membership on an entity's Board of Directors or advisory committees; Vitalaire: Other: Non-financial support. Bartolucci:Fondation Fabre: Research Funding; Addmedica: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees; GBT: Membership on an entity's Board of Directors or advisory committees. Galacteros:Addmedica: Membership on an entity's Board of Directors or advisory committees.

Outcome of Severe Vaso Occlusive Crisis in Sickle Cell Disease Adults Admitted to Referral Centers in Africa and Europe. Preliminary Results from the Presev 2 Study

▪Introduction : Vaso-Occlusive Crisis (VOC), the most common manifestation of sickle cell disease (SCD), is the first cause of death, particularly when complicated by an acute chest syndrome (ACS).In the PRESEV1 study, we developed a predictive score of secondary ACS in SCD patient admitted in the hospital for VOC (AUC = 0.840 [95% CI: 0.780 - 0.900]), with a negative predictive value of 98.8% for the low level risk and a positive predictive value of 39.5%. The aim of the PRESEV-2 study is to validate the predictive score of ACS on adults and children with SCD in Europe and Africa. Herein we present preliminary data on 235 adults with a severe VOC included in 11 referral centers from 4 countries.Patients and Methods: We conduct an international multicenter prospective observational study. The primary outcome measure is the occurrence of an ACS. The ACS is defined as a new auscultatory abnormality (crepitation or bronchial breathing) OR the association of a new radiologic infiltrate AND chest pain or decreased breath sounds. Secondary outcome measures are length of hospitalization, transfusion, morphine consumption, mortality and hospitalization in intensive care unit. Inclusion criteria are: SS or S-b0 thalassemia patients, VOC admitted at the emergency unit (VOC is defined as pain or tenderness affecting at least one part of the body not attributable to other causes) that required hospitalization and opioids (level 3). Exclusion criteria are: no inaugural ACS, homeless patients, pregnancy, deprived of their liberty or under guardianship, unable to understand the purpose and conditions of carrying out the study, unable to give consent.Results : From march 2016 to june 2017, 112 adult patients were included in Africa and 122 adults in Europe. Patients in Africa were younger than in Europe with a mean age, respectively, of 26 ± 7 yrs and 29 ± 8 yrs (p=0.008). Sex ratio (M/F) in Africa was 0.84 and 0.95 in Europe. Use of hydroxyurea was significantly higher in Europe (65%) than in Africa (6%) (p<0.0001). The ACS incidence was significantly higher in Africa than in Europe, respectively 22% and 11% (OR=2.24; p=0.03), in both areas the time for ACS appearance was the second day. Transfusion was performed in 27% in Africa and 6% in Europe (p<0.001), ACS was the main cause of transfusion (53.3% in Africa and 62.5% in Europe). Patients with ACS were more transfused in Africa than in Europe, respectively 64% and 35.7% but the difference was not significant. Mortality was 3.6% in Africa and was not observed in Europe; circumstances of death were stroke (1), profound anemia (1), kidney failure (1) and VOC (1).Discussion and conclusion: Our preliminary results provide epidemiologic insights about VOC and ACS incidence in Europe and Africa. A better availability of HU treatment in Africa is a health care priority. DisclosuresGalactéros:Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Addmedica: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

A New Tool for HbF Quantification Demonstrates That Hydroxyurea Decreases Mostly RBCs Containing Very Low Amounts of HbF

Introduction :Fetal hemoglobin (HbF) expression is a major modulator of sickle cell disease (SCD) severity. Hydroxyurea improves SCD clinical manifestations in part through its ability to induce HbF synthesis which decreases the HbS polymer formation. However, the distribution of HbF is heterogeneous in red blood cells (RBCs) from SCD patients. In the hypothesis of an HbF “threshold” for inhibiting the HbS polymerization, accurate measurement of the HbF content in RBC is an important issue. Several techniques have been developed to quantify HbF. Nevertheless, these technics are qualitative, or semi quantitative, allowing only to detect the RBCs containing a mild/high HbF level, called “F-cells”.Methods : we developed a simple and accurate method to quantify the HbF in each single RBCs and to evaluate the HbF distribution in SS RBCs during a 6 month Hydroxyurea therapy, as a proof of concept. RBCs adults carrying hereditary persistence of HbF (HPFH), β-Thalassemia or db-Thalassemia, were collected. Pancellular homogenous HbF distribution was controlled by flow cytometry. The mean amount of HbF (MCHbF) was calculated from the %HbF determined by HPLC and the mean corpuscular haemoglobin (MCH). The mean fluorescence intensity (MFI) after HbF staining by a monoclonal antibody was quantified by flow cytometry and normalized using quantitation beads.RBCs from homozygous SCD patients were collected before treatment (D0), between 15 days and 3 months (D15-M3) and after 6 months of HU treatment (M6). RBCs were frozen at -80°C. Non-inclusion criteria were vaso-occlusive crisis, blood transfusion within 3 months preceding the enrolment, and on-going pregnancy. After thawing, RBCs were analysed by flow cytometry after HbF staining.Results : Homogenous HbF distribution was proven by flow cytometry in 12 adults with HPFH, δβ thalassemia or β thalassemia. A linear regression between MCHbF and MFI, on 11 different experiments, was performed and provided an equation allowing the quantification of HbF (pg) by RBC (R²=0.94; p<0.0001). A mathematical model calculated the 95% confidence interval. Sixteen homozygous SCD adults were included for the study on HbF distribution under HU. RBCs containing 0 to 2 pg of HbF (table 1) decrease by 25% after 6 months of HU (p<0.0001) whereas a non-significant increase in the other categories of HbF by RBC was found.Conclusion : This simple technique, based on flow cytometry, could represent a new important tool in sickle cell and thalassemia diseases to better quantify the HbF expression and then better evaluate patient's pronostic and therapeutic effects. [Display omitted] DisclosuresGalactéros:Addmedica: Membership on an entity's Board of Directors or advisory committees. Bartolucci:Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding; Novartis US: Membership on an entity's Board of Directors or advisory committees.

Hydroxyurea: Clinical and Hematological Effects in Omani Patients with Sickle Cell Disease

Background and Aim:•It is well known that hydroxyurea impacts on clinical and hematologic indices in sickle cell disease (SCD), we aimed to evaluate the effect of hydoxyurea on clinical and hematological improvement of Omani sickle cell disease pateints.Methods and Materials•In this retrospective single-center study, we selected 18 Omani sickle cell disease patients aged above 18 years, who were started on Hydroxyurea 5-10mg/kg/day as per their standard of care and in whom the inclusion criteria fulfilled.•Inclusion criteria were history of more than three admissions with vaso-occlusive crises /year, history of acute chest syndrome, history of priapism, history of splenic sequestration crises.•Patients were excluded if they had follow-up of Less than six months, blood transfusion during the study, pregnant ladies.•In our study, we looked at the SCD patients who were followed in Haematology clinic in AFH every three months. As per the routine follow up of these patients in this hospital, patients were assessed in each visit for the number of crises, HPEL, CBC, LFT, UEG, and any reported side effects.•Our aim was specifically to observe the number of ER crises, number of hospital admissions, HBF, HB, MCV, WBC over six months post HU treatment .•The data collected based on the doctors notes & lab results from the Hospital information system. Side effects of the drug if any were observed from the prescription history.•The study was approved by the ethical committee in Armed Force Hospital in Muscat, Oman.•Data has been stored and analyzed in the SPSS program.Results•In this study, we looked at 18 patients with SCD who were treated with HU 5-10mg/kg/day .•These patients consisted of 6 females and 12 males.•We found that in patients with sickle cell disease Hu significantly increased the HBF (P=0.008) and MCV (p=0.011) after six months.•There was no statistically significant difference between the baseline and after six months in HB (p=0 .131) and WBC (P=0.078 ).•There was statistically decrease in the number of ER between baseline and six months (p = 0.011) .•There was statistically significant decrease in the number of Hospitalizations between the baseline and the six months (p= 0.026).•Our study showed HU therapy was well tolerated by our patients and remarkable adverse effects were not reported in patients after six months treatment with HU.Conclusion:•The results showed that the use of hydroxyurea 5-10 mg/kg/day for 6 months duration on patients with SCD significantly improve the HBF and MCV and decreased the number of ER crises and hospital admissions without any remarkable adverse effects. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Management of Renal Dysfunctions in Escort-HU Study

▪BACKGROUNDRenal impairment is a progressive chronic complication of sickle cell disease (SCD) that begins in childhood and may progress until renal failure. It is responsible for mortality in 12% of SS SCD adults (Tehseen S, 2017).Causes of renal damages in SCD patients are multiple: hemolysis, vaso-occlusive episodes which lead to renal injuries, papillary necrosis, medullary fibrosis and focal segmental glomerulosclerosis (Rees D, 2010).Hydroxycarbamide (hydroxyurea or HU) is a myelosuppressive drug marketed since 1968 for the treatment of hematological malignancies, and authorized since 2007 in Europe as an orphan medicinal product for the prevention of recurrent vaso-occlusive crises and acute chest syndrome in adults and children older than 2 years with SCD. HU also seems to protect kidney function in SCD by decreasing proteinuria (Bartolucci P, 2015).As renal excretion is a pathway of HU elimination (60% of dose excreted in urine), consideration should be given to decrease the dose of HU in patients with renal impairment.We propose to study SCD patients with renal diseases treated with hydroxycarbamide (Siklos) and enrolled in ESCORT-HU (European Sickle Cell Disease COhoRT - HydroxyUrea), European multicenter prospective non interventional study.METHODSFrom January 2009 to June 2017, 1841 patients were enrolled in the ESCORT-HU study. 1016 patients were adults and 824 were children (1 date of birth missing). Among them, renal lesions have been reported in the study database in 220 patients (194 adults, 25 children, 1 date of birth missing).At inclusion the renal status of patients are reported in the electronic Case Report Form (e-CRF) using different clinical approaches. It allows to distinguish patients with a medical history of renal impairment (i.e. patient with renal insufficiency of etiology not reported, with an irreversible and progressive reduction in renal function) from patients with a history of sickle cell (SC) nephropathy (patients with any renal alteration related to SCD and not necessarily irreversible and progressive).RESULTSCompared to the global cohort without any kidney involvement, patients with renal lesions were older (35.8 years vs 21.0 years) as expected, with less children (11.4% vs 50.2%) and mainly with SS genotype (94.5% vs 82.2%) (Table 1).64.1% of patients with renal lesions were already treated with HU other than Siklos before their enrollment in the study, in line with the fact that kidney lesions are correlated with the ageing of the SCD population.HU was initially prescribed at lower doses in SCD patients with renal disease and naïve of HU treatment as recommended. For patients already treated with HU other than Siklos (the dose at enrolment ~ maintenance dose), even if we observed an increase of the dose until maintenance dose, this subpopulation received HU at lower daily doses (Table 2).SCD Patients with kidney diseases reported more frequent and more severe adverse events not related to SCD (AENOTSCD) than patients without renal lesions (2.30 AE per patient vs 1.49 AE per patients and 15.4% of severe AE vs 11.2%) (Figure 1). However these events are less serious (15% of serious AE vs 25% in patients without renal lesions) (Figure 2). Similarly, when focusing on AENOTSCD causally related to HU (as judged by the investigators), patients with renal lesions reported more AE especially in patients with history of renal impairment.As recommended by some guidelines (Habibi 2015, Sharpe 2014), renin-angiotensin system (RAS) inhibitors may be used is SCD patients with renal damage in order to limit the kidney disease progression. In ESCORT-HU study, RAS inhibitors were used in 47.3% of patients with renal lesions whereas it was used in 3.0% of patients without renal lesions. Consistently RAS inhibitors are mainly used in patients with a history of SCD nephropathy (54.54% vs 21.15% in the population with renal impairment) probably because damages in this subpopulation were irreversible (Table 3).CONCLUSION12% of SCD patients, mainly adults, enrolled in the ESCORT-HU study had renal dysfunctions at enrolment. As expected this subpopulation is older than the global population of the cohort. Although HU is prescribed at lower doses, these patients go on to have more adverse events and this justifies a close monitoring of them. Controlled trials are required to evaluate the role of HU on the renal failure. [Display omitted] DisclosuresBartolucci:Novartis US: Membership on an entity's Board of Directors or advisory committees; Addmedica: Research Funding; GBT: Membership on an entity's Board of Directors or advisory committees; Fondation Fabre: Research Funding. Ribeil:Addmedica: Membership on an entity's Board of Directors or advisory committees, Research Funding. Lobitz:Addmedica: Membership on an entity's Board of Directors or advisory committees. Galacteros:Addmedica: Membership on an entity's Board of Directors or advisory committees.

Treatment of Essential Thrombocythemia with Anagrelide Is Associated with an Increased Risk of Worsened Kidney Function

Background and Purpose: When choosing a cytoreduction method for patients suffering from essential thrombocythemia (ET), it is important to know the safety profile of the medicine used. Few articles have been published about the effects of hydroxycarbamide (hydroxyurea, HU) and anagrelide (ANA) on renal function in ET patients. This study is the largest analysis of nephrotoxicity of cytoreductive drugs used in ET therapy so far, which additionally includes risk factors for the progression of kidney disease and coexisting genetic mutation. Experimental Approach: The retrospective study included 310 patients diagnosed with ET. Demographic data, comorbidities, Cr, and estimated glomerular filtration rate (eGFR) were all taken into account prior to diagnosis and after 6 months of HU and ANA treatment. Key Results: A statistically significant difference was found between Cr and eGFR levels at baseline and after 6 months of treatment (p < 0.001). The applied treatment (HU and ANA) had the greatest impact on kidney function. ANA significantly increased the risk of worsening renal function in contrary to hydroxycarbamide after 6 months of treatment (eGFR change: median +1 mL/min/1.73 m² [interquartile range (IQR) (−4)–(+7)] in the HU group vss. median −13 mL/min/1.73 m² [IQR (−18)–(−6)] in the ANA group, odds ratio [OR] 7.92 95% confidence interval [95% CI] [4.17–15.08], p < 0.001). Lowering of eGFR <60 mL/min/1.73 m² occurred in 31 patients (31.0%) from the ANA group and 10 people (4.8%) treated with HU (p = 0.000). In 1 patient from the ANA group, >50% decrease in eGFR was observed. The chance for an increase in Cr levels was higher in people with pre-existing arterial hypertension (OR 1.92 CI = 95% [1.21–3.05], p = 0.006). Sex, type of mutation found (JAK2 V617F or CALR), and previous renal impairment did not affect renal function after 6 months of treatment. In addition, there was no difference in the efficacy of ET treatment between HU and ANA (p = 0.998). Conclusions and Implications: The observations indicate that ANA should be used in patients with ET with great caution and taking into account the risk of worsened kidney function.

PSII-4 Changes in abundance of ruminal bacteria associated with urea level and tannin supplementation in grazing beef cattle

Abstract The study was carried out to evaluate the effect of tannins extract (TN) associated to two levels of urea in the supplement of grazing beef cattle on ruminal bacterial abundance. Were used eight rumen cannulated Nellore steers (293 kg ± 5.6 BW) in a double Latin square 4 x 4 (four periods of 28 each and four supplements: LU = low urea without TN; LUT = low urea plus TN; HU = high urea without TN and HUT = high urea plus TN). Steers grazing Urochloa brizantha cv. Marandu and were supplemented at 3 g/kg BW cwith soybean meal, corn, mineral mix and two levels of urea (3% or 5%) associated or not to TN (SilvaFeed-ByPro at 0.7 g / kg). Samples of ruminal content were collected after supplementation (+ 3h), V3/V4 regions of 16SrRNA gene was sequencing using the Illumina MiSeq. Quantitative Insights into Microbial Ecology (QIIME v.1.9.1) were used to filter reads and determine Operational Taxonomic Units (OTUs). DESeq2 differential abundance analysis were performed using each steer on LU supplement as own control on others supplements and evaluated by Kruskal-Wallis rank sum test considering statistical significance at P &amp;lt; 0.10. Only 5 OTUs differed significantly between treatments. Enterorhabdus.spp population were less abundant in HU, but TN was able to reduce the abundance in both LUT and HUT(P = 0.049). The ruminal abundance of Streptococcus.spp (P = 0.026), Fusobacterium.spp (P = 0.053) and Synergistes.spp (P = 0.048) was reduced by TN, but had no effect of urea level. Enterorhabdus.spp, Streptococcus.spp and Fusobacterium.spp are associated with inflammatory bowel diseases, ruminal acidosis and to liver abscesses in cattle, respectively. Betaproteobacteria class were less abundant in HU treatment (P = 0.048), this heterogeneous class bacterial content some nitrogen fixing bacteria and some pathogens important in plants and animals. In conclusion, TN reduced the ruminal population of important ruminal pathogens bacterial.