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Background Deletions in the β-globin cluster are uncommon and cause thalassemia (thal) with hereditary persistence of fetal hemoglobin. They constitute a heterogenous group of disorders characterized by absent or reduced synthesis of adult hemoglobin (Hb A) and increased synthesis of fetal hemoglobin (Hb F). Although the clinical severity of these disorders are asymptomatic owing to the increased Hb F levels, the molecular basis is very heterogenous due to the large deletions in the β-globin cluster spanning both HBD and HBB genes. Here, we describe a Tunisian family carrying a novel deletion mutation causing (δβ)°-thalassemia. Methods The amounts of hemoglobin fractions were measured by capillary electrophoresis of hemoglobin. Amplification and sequencing of different regions on the β-gene cluster were performed by Sanger method. Results Family study and genetic analysis revealed a large deletion mutation in the β-globin cluster of 14.5 kb (NG_000,007.3:g. 58,253 to g.72837del14584) at the homozygous state in the patient and at heterozygous state at the other members of the family. This deletion removes the HBD and HBB genes. Conclusions In our knowledge, this new large deletion is described for the first time in the Tunisian population and in the world, designed Tunisian(δβ)0 in Ithanet database (IthaID: 3971). Therefore, it is important to identify the deletion leading to δβ-thalassemia carriers at the molecular level, to highlight the importance of recognizing the clinical features and implementing appropriate testing to clarify the diagnosis and manage the condition.

Chronic myeloid leukemia (CML) in childhood is rare accounting for less than 10 % of all cases of CML and less than 3% of all pediatric leukemia. We conducted a retrospective study of children and young adults (CYA) with CML diagnosed in Tunisia from June 2003 through December 2022. Diagnosis of CML was confirmed by RT-PCR for all patients. Data were analyzed regarding epidemiological characteristics, clinical presentations, molecular findings, treatment response and side effects of Tyrosine kinase inhibitors (TKI). Of the 1876 patients diagnosed with CML during this period 61 (3.25%) were under 18 years. 37 were male and 24 were female. The chronic phase (CP) was the most common phase of CML seen in 43 patients. While 9 patients were in accelerated phase (AP) and 9 had inaugural blast crisis. Mean white blood cell count were 232 x10 /L. All patients were positive for bcr-abl transcript : 32 patients had b2a2, 25 had b3a2. They were given oral TKI. Only 5 patients underwent hematopoietic stem cell transplantation. 51 achieved complete hematologic remission on imatinib. 27 patients achieved major molecular remission and 13 are currently maintaining a bcr-abl ratio under 0.01% which corresponds to MR ,6 patients progressed under TKI within 7 months. This retrospective study represents the first series of CYA with CML in Africa. Our data are comparable with the literature findings regarding epidemiological characteristic, clinical presentations, and response to TKI.

Several causes are known to be at the origin of neonatal cyanosis among them methemoglobinemia is by inheritance of an hemoglobin (Hb) M variant. This is a rare condition never been reported in Tunisia so far. Here, we report a Tunisian newborn with refractory cyanosis since birth. As cardiac and respiratory diseases were ruled out, methemoglobinemia was suspected. Hematological parameters, concentration of methemoglobin, capillary electrophoresis, and amplification sequencing of the HBB gene were performed. Computational analysis was achieved by different in silico tools to investigate the mutation effect. The diagnosis was established by a raised MetHb, confirmed by the presence HbM-Saskatoon [Beta63 (E7) His>Tyr] by capillary electrophoresis and molecular analysis. The identified mutation occurred as a de novo mutation. In silico analysis confirmed the pathogenicity of the mutation. To our knowledge, this is the first time that this mutation has been reported in the Tunisian population. In view of its low incidence rate, clinicians might misdiagnose cyanosis caused by HbM, which can lead to inappropriate treatment and clinical complications. An up-to-date literature review of HbM disease is presented in this study.

The genetic investigation of essential thrombocythemia(ET) has highlighted the presence of driver mutations in ET. Janus kinase JAK2V617F and calreticulin(CALR) mutations are the most frequent driver mutations and have significantly improved the molecular diagnosis of ET. The impact of genetic heterogeneity on clinical features has not been fully elucidated. This is the first study which aimed to determine the frequency of JAK2V617F and CALR exon9 mutations in Tunisian ET patients and to establish the correlation between hematological characteristics and mutational status.This study included Tunisian patients suspected with ET and was conducted between September 2017 and March 2021. Genomic DNA of patients was isolated from peripheral blood samples. JAK2V617F was detected by AS-PCR and CALR mutations were detected by PCR/direct sequencing. Clinical and hematological characteristics were also analyzed.Two hundred and fifty ET patients were enrolled in this study. JAK2V617F mutation was found in 166/250 (66.4%) of patients, whereas CALR mutations were detected in 27/84 (32.1%) patients without JAK2V617F. Compared with JAK2V617F-positive patients, those with CALR mutations showed lower hemoglobin level and lower leucocytes count (p = 0.007 and p = 0.004,respectively). CALR type 2 was the most frequent mutation of CALR detected in 55.55% of CALR mutated. Six of seven patients with thrombotic events harbored JAK2V617F mutation.The prevalence of driver mutations JAK2V617F or CALR mutations was 77.2% in Tunisian ET patients. Moreover, patients with JAK2 V617F mutation had a higher risk of thrombosis. The mutational status is necessary to improve the diagnosis and contribute to the therapeutic decisions.

Objective In this study, we report a Tunisian newborn boy referred for neonatal hemolytic anemia with yellowish skin and enlarged spleen due to coinheritance of hemoglobin O (HbO) Arab and β-thalassemia. Study Design Hematological parameters were collected using an automated blood cell counter. The amounts of Hb fractions were measured by capillary electrophoresis of Hb. Amplification and sequencing of the HBB gene were performed by Sanger's method. Results Family study and genetic analysis revealed that the proband was a carrier of two hemoglobinopathies: HbO Arab and β0-thalassemia. Conclusion The coexistence of these two pathologies complicated the general state of the newborn boy and led to a severe anemia at birth. Key Points

Acute myeloid leukemia (AML) is a heterogeneous disease. The discovery of novel discriminative biomarkers remains of utmost value for improving outcome predictions. Enhancer of zeste homolog 2 (EZH2) is a histone methyltransferase of H3K27me3. It is frequently up-regulated in human cancers and associated with silencing of differentiation genes. We aimed herein to investigate the prevalence and prognosis impact of somatic EZH2 mutations and their potential associations with other prognostic markers FLT3, NPM1, DNMT3A and IDH2.Our study population was composed of 211 Tunisian patients with de novo AML and 14 healthy donors. The 11 last exons coding the set domain of EZH2 were investigated by PCR and Sanger sequencing.EZH2 mutations were identified in 66/211 (31%) patients with a sex ratio of 1.06. The presence of EZH2 mutations was statistically significantly associated with failure consolidation therapy (p = 0.004). There were no differences in the incidence of EZH2 mutations and FLT3-ITD, NPM1, DNMT3A and IDH2 mutations. When EZH2 mutations were associated with those of FLT3 or IDH2, a short duration of progression free survival was observed (p 0.05). Moreover, CD7 aberrant markers conferred a poor prognosis in EZH2 mutated patients (p 0.05).Given these data we conclude that EZH2 mutations are frequent in our patients, and can be used as a prognosis marker in combination with FLT3, IDH2 mutations and CD7 marker, to stratify AML patients and to guide therapeutic decisions.

Background/Aims: Hereditary Spherocytosis (HS) is the most common erythrocyte membrane disorder causing hemolytic anemia. The wide heterogeneity of both clinical and laboratory manifestations of HS contributes to difficulties associated with the diagnosis of this disorder. Although massive data previously reported worldwide, there is yet no data on HS among the Tunisian population. Here we aim to characterize HS in Tunisian patients at biochemical and cellular levels, identify the membrane protein deficiency, and compare the accuracy of the diagnostic tests to identify the most appropriate assay for HS diagnosis. Methods: We investigated 81 patients with hemolytic anemia and 167 normal controls. The exploration of HS based on clinical and family history, physical examination, and the results of laboratory tests: blood smear, osmotic fragility test (OFT), cryohemolysis test (CT), pink test (PT), eosine-5’-maleimide (EMA) test, and erythrocyte membrane protein electrophoresis. Results: We identified 21 patients with HS, classified as severe (6/21;28.5%), moderate (10/21;47.6%), and mild (5/21;23.8%). The most prevalent protein deficiency was the band 3 protein detected in ten Tunisian HS patients. The EMA test showed a high specificity (97.5%) and sensitivity (94.7%) for HS diagnosis compared to the other screening tests. Interestingly, fourteen among sixteen patients presenting with homozygous sickle cells HbSS showed an increase of EMA fluorescence intensity compared to other anemic patients. Conclusion: Our study highlights the efficiency of the EMA dye for the detection of HS whatever the nature of the involved protein deficiency. We report for the first time, the most prevalent protein deficiency among Tunisians with HS. Moreover, we found that the combination of the EMA-binding test with PT or incubated OFT improves the diagnosis sensitivity while maintaining a good specificity.

This study was aimed to establish local reference values for hematological indices and hemoglobin (Hb) fractions in umbilical cord blood (UCB) for the northern population of Tunisia.Our study included full-term newborns by vaginal deliveries. Hematological parameters were collected using an automated blood cell counter. The amounts of Hb fractions were measured by capillary electrophoresis of Hb. Statistical analysis was performed using R software.A total of 328 cord blood samples were analyzed. Among them, 154 (male: 44.8%, female: 55.2%) were used to establish reference values. The normal reference values of complete blood count (CBC) and Hb fractions were calculated. Mean neonatal Hb was 14.75 ± 2.26 g/dL. Gestational age affects the expression of CBC values as red blood cell (RBC), Hb, hematocrit (Hct), mean corpuscular volume (MCV), white blood cell (WBC), and the Hb profile. Umbilical blood hemogram parameters and Hb profile are affected by the environment; higher in newborns from urban regions but not affected by gender ratio.Reference ranges of normal CBC indices and Hb fractions have been successfully established in Tunisian neonates' UCB. Our data suggest reference values that could be useful for neonatal patients' laboratory results and clinical interpretation.· Reference values for CBC and hemoglobin fractions have been established.. · Hematological reference for UCB is useful to identify hemolytic anemia cases early.. · UCB hematological values are influenced by gestational age and probably by environmental factors..

Sickle cell disease(SCD)is a monogenic disease, in which the severity and symptoms vary widely.The sickle cell mutation is the origin of all functional and structural alterations of red blood cell(RBC)which induces the acceleration of eryptosis and formation of Circulating blood cells microparticules(MPs)which has the consequence of acute anemia and increasing the thrombotic risks in sickle cell patients. Eryptosis is the suicidal erythrocyte death characterized by erythrocyte membrane scrambling by phosphatidylserine(PS)externalization. MPs are an extracellular vesicules released following cellular activation, stress or apoptosis. These parameters are able to modulate many biological functions, and considered as biomarkers of preventive diagnostic of SCD. This present study aims to determine the cellular biomarkers to implementation new and innovative methods of the preventive diagnosis of SCDacute complications. We propose to study the mechanisms involved in the triggering of eryptosis and to quantify microparticles derived from platelets and erythrocytes of homozygous sickle cell patients. Following clinical diagnosis, homozygous SCDpatients and healthy donors were sampled for hematological and cellular assays. The exploration of eryptosis and MPs was performed by a flow cytometry by determining the viability parameters of red blood cells. The outcome of our study indicated that Eryptosis in sickle cell patients is triggered essentially by high Ca2 + entry and narrowing of red blood cells. However, the pathway of ceramides and ERO can also considered a stimulating factor of eryptosis. Eryptosis ensures healthy erythrocyte quantity in circulation whereas excessive eryptosis is the cause of acute anemia and may contribute to vaso-occlusive crisis in SCD patients.For MPs study,microparticles derived from platelets and erythrocytes clearly increased in SCD patients. This suggests that erythrocytes MPs may contribute to thrombotic risk andchronic hemolytic anemia. Key messages Potentiel biomarkers in the preventive diagnosis of sickle cell disease. Eryptosis and plasmatic microparticules as potentiel biomarkers.

Background Recurrent pregnancy loss (RPL) is a significant adverse pregnancy complication, with an incompletely understood pathology. While many entities were proposed to elucidate the pathogenic basis of RPL, only few were significant enough to warrant investigation in all affected couples.. The aim of this study was to provide novel insights into the biological characteristics and related pathways of differentially expressed miRNA (DEMs) and genes (DEGs), in RPL, and construct a molecular miRNAs–mRNAs network. Methods miRNAs and gene expression data were collected, and a number of DEMs and (DEGs) were obtained, and regulatory co-expression network were constructed. Function and enrichment analyses of DEMs were conducted using DIANA-miRPath. DEGs were screened, and were used in generation of protein-protein interaction (PPI) network, using STRING online database. Modularity analysis, and pathway identification operations were used in identifying graph clusters and associated pathways. DEGs were also used for further gene ontology (GO) analysis, followed by analysis of KEGG pathway. Results A total of 34 DEMs were identified, and were found to be highly enriched in TGF-β signaling pathway, Fatty acid metabolism and TNF signaling pathway. Hub miRNAs were selected and were found to be involved in several functional pathways including progesterone-mediated oocyte maturation and Thyroid hormone signaling pathway. Five dysregulated feedback loops involving miRNA and TFs were identified and characterized. Most notably, PPI network analysis identified hub-bottleneck protein panel. These appear to offer potential candidate biomarker pattern for RPL diagnosis and treatment. Conclusions The present study provides novel insights into the molecular mechanisms underlying RPL.

Imatinib mesylate (IM) is considered as a highly effective therapy for chronic myeloid leukemia (CML) patients. However, a minority of patients fail to achieve optimal response due to impaired bioavailability of IM. The human organic cation transporter 1 (OCT1; SLC22A1) has been reported to be the main influx transporter involved in IM uptake into CML cells. Genetic variants and/or hOCT1 expression changes may influence IM response. In this study, we aimed to investigate the impact of both hOCT1 polymorphisms located in exon 7 and hOCT1 mRNA levels on the clinical outcome in CML patients. hOCT1 expression profile was determined using the quantitative real-time polymerase chain reaction in 69 CML patients treated with IM (35 responders to IM patients and 34 IM-resistant patients), while genotyping of 69 cases and 51 controls for hOCT1 polymorphisms was performed by direct sequencing after amplification of exon7. Our results showed that the hOCT1 gene was significantly downregulated in the samples of the IM-resistant group when compared with the IM-responder group (p = 0.0211). Moreover, sequencing data show an association in all cases between the SNP 408V>M (g.1222G>A) and an intronic 8 bp (base pairs) insertion of GTAAGTTG (rs36056065) at the 3′ end of exon 7. The genotype and allele distribution of the different SNPs did not differ significantly between the two groups of patients. hOCT1 mRNA expression may serve as a clinical biomarker of response to imatinib and could be useful to predict IM therapy outcome of CML patients.

Targeted therapy in the form of selective breakpoint cluster region-abelson (BCR/ABL) tyrosine kinase inhibitor (imatinib mesylate) has successfully been introduced in the treatment of the chronic myeloid leukemia (CML). However, acquired resistance against imatinib mesylate (IM) has been reported in nearly half of patients and has been recognized as major issue in clinical practice. Multiple resistance genes and microRNAs (miRNAs) are thought to be involved in the IM resistance process. These resistance genes and miRNAs tend to interact with each other through a regulatory network. Therefore, it is crucial to study the impact of these interactions in the IM resistance process. The present study focused on miRNA and gene network analysis in order to elucidate the role of interacting elements and to understand their functional contribution in therapeutic failure. Unlike previous studies which were centered only on genes or miRNAs, the prime focus of the present study was on relationships. To this end, three regulatory networks including differentially expressed, related, and global networks were constructed and analyzed in search of similarities and differences. Regulatory associations between miRNAs and their target genes, transcription factors and miRNAs, as well as miRNAs and their host genes were also macroscopically investigated. Certain key pathways in the three networks, especially in the differentially expressed network, were featured. The differentially expressed network emerged as a fault map of IM-resistant CML. Theoretically, the IM resistance process could be prevented by correcting the included errors. The present network-based approach to study resistance miRNAs and genes might help in understanding the molecular mechanisms of IM resistance in CML as well as in the improvement of CML therapy.

Recent genome-wide association studies (GWAS) focusing on pediatric acute lymphoblastic leukemia (ALL), the most common malignancy in children younger than 15 years old, have found evidence that single-nucleotide polymorphisms (SNPs) in IKZF1 (7p12.2), ARID5B (10q21.2), CDKN2A (9p21.3), and CEBPE (14q11.2) are strongly associated to the risk of developing pediatric ALL. These studies have been conducted in European and Thai populations, and it is unclear whether these observations generalize to other populations with a lower incidence of pediatric ALL. In order to explore the impact of these variants on pediatric ALL risk in the Tunisian population, we genotyped 58 cases of pediatric ALL and 150 controls for SNPs rs4132601 (7p12.2), rs7089424 (10q21.2), rs3731217 (9p21.3), and rs2239633 (14q11.2). Our results, which are consistent with findings in European populations, show that 3 SNPs, i.e., rs4132601 (P = .00116, odds ratio [OR] = 2.78, 95% confidence interval [CI] = [1.42, 5.87]), rs7089424 (P = .0022, OR = 0.49, 95% CI = [0.31, 0.79]), and rs2239633 (P = .0010, OR = 0.47, 95% CI = [0.29, 0.75]) are significantly associated with a higher risk of developing pediatric ALL (P.05). Furthermore, we show differences in allele frequencies in SNPs between Tunisian and Caucasian and/or Thai populations (e.g., CEBPE, rs2239633; population attributable risk [PAR] ∼15-fold the PAR of Thai population). These differences, combined with differences in linkage disequilibrium structure between populations and differences in size between populations, may contribute to racial differences in pediatric ALL incidence.

Introduction Unconjugated hyperbilirubinemia (UCB) is a feature of Gilbert's syndrome (GS) and Crigler-Najjar's syndrome (CNS), which are two hereditary defects in bilirubin metabolism. Both syndromes are linked to mutations in the UGT1A1 gene, which cause either the decrease or the absence of the UGT1A1 enzymatic activity. Here, we investigated the molecular basis of the UGT1A1 gene in Tunisian patients presenting with unconjugated hyperbilirubinemia. Methods Twenty-four patients with UCB were investigated. The screening protocol for hemoglobinopathies, enzymopathies, and membrane defects was executed in all patients. Afterward, the molecular analysis of the entire UGT1A1 gene was performed by DNA Sanger sequencing. Several bioinformatic tools were used to explore the effects of novel mutations. Results Fifteen different UGT1A1 variations were identified, among which four are described here for the first time. In exon 5, the c.1412C > G; p.(Ala471Gly) and c.1589C > T; p.(Ser530Phe) mutations were detected in patients presenting with CNS type I and GS, respectively. In the 3′UTR region of UGT1A1, the c.*90C > T mutation was detected in 3 patients with CNS type I. In the same region, the c.*388C > T defect was found in a GS patient. A deleterious and damaging effect on the UGT1A1 protein were predicted for both exonic mutations. Furthermore, novel microRNAs were identified as targetting the mutated sequences for the 3′UTR mutations. Conclusion Our study provides novel data on UCB among Tunisians. Furthermore, we report four novel mutations associated with both GS and CNS. The identification of these mutations increases the spectrum of the UGT1A1 mutations and contributes to an understanding of the molecular abnormalities associated with unconjugated hyperbilirubinemia.

bcr-abl fusion gene is the hallmark of chronic myeloid leukemia (CML). RQ-PCR provides an accurate measure of the total leukemia cell mass and the degree to which bcr-abl transcripts are reduced by therapy correlates with progression free survival.We report molecular assessment of residual disease in CML Tunisian patients.Between June 2003 and December 2014 we measured bcr-abl mRNA levels in peripheral blood from all Tunisian patients by quantitative real time polymerase chain reaction (RQ-PCR).A total of 708 patients with a mean age of 42 years were included in this study. Based on European Leukemia Net 2013, 80% of the patients achieved an optimal response 20% were in treatment failure. 38% of the patients achieved RM4 which corresponds to a bcr-abl/abl ratio0.01%, 13% of the patients achieved RM4.5corresponding to bcr-abl/abl ratio of 0.0032%.CML patients had a good response to tyrosine kinase inhibitors treatment. RQ-PCR is helpful in detecting any residual disease and determining the depth of the treatment response.

International audience; Data are limited in developing countries regarding the clinicopathologic features and response to therapy of chronic myeloid leukemia (CML) in the era of imatinib (IM). The objective of this study is to report on the clinicoepidemiologic features of CML in Tunisia, to evaluate the long-term outcome of patients in chronic (CP) or accelerated phase (AP) treated with IM 400 mg daily as frontline therapy, and to determine imatinib's efficacy and safety. From October 2002 to December 2014, 410 CML patients were treated with IM in six Tunisian departments of hematology. Response (hematologic, cytogenetic, and molecular responses) and outcome-overall survival (OS), event-free survival (EFS), and progression-free survival (PFS)-were evaluated. The following prognostic factors were analyzed for their impact on the European leukemia net (ELN) response, OS, EFS, and PFS at 5 years: age, sex, leukocyte count, Sokal score, European Treatment and Outcome Study (EUTOS) score, CML phase, time to starting IM, and impact of adverse events. The median age was 45 years (3-85 years). Two hundred ten (51.2%) patients were male. Splenomegaly was present in 322 of the 410 (79%). Additional cytogenetic abnormalities were encountered in 25 (6.3%) patients. At diagnosis, 379 (92.4%) patients were in CP, 31 (7.6%) were in AP. The Sokal risk was low in 87 (22.5%), intermediate in 138 (35.7%), and high in 164 patients (41.9%). The EUTOS risk was low in 217 (74%), and high in 77 (26%) patients. The rates of cumulative complete cytogenetic response (CCyR), major molecular response (MMR), and molecular response 4/5 log (MR4.5) in CP/AP-CML patients were 72, 68.4, and 46.4%, respectively. The median time to reach CCyR, MMR, and MR4.5 was 6 months (3-51), 18 months (3-72), and 24 months (3-100), respectively. According to the ELN criteria, optimal, suboptimal response, and failure were noted in 206 (51.8%), 61 (15.3%), and 125 (31.4%) patients, respectively. Five-year event-free survival (EFS), progression-free survival (PFS), and overall survival (OS) were 81, 90, and 90%, respectively. By multivariate analysis, AP, high EUTOS risk, and baseline WBC ≥ 150G/l remained independent predictive factors of non-optimal response to IM. The adverse events (AE) of IM were moderate and tolerable. With the caveats that the monitoring of the disease was not optimal, response rates were similar to those reported in previous studies. It is clear to us that improvements should be made in treatment of AP-CML and high Sokal risk group of CP-CML. The frontline use of second-generation tyrosine kinase inhibitor (TKI) is expected to improve the results of the first-line treatment of these high-risk Tunisian patients, but cost and accessibility of this therapy remain the problems in developing countries.

The introduction and success of imatinib mesylate have become a paradigm shift in chronic myeloid leukemia (CML) treatment. However, despite its high efficiency, resistance to imatinib has emerged as a significant problem, which may in part be caused by pharmacogenetic variability. Three single-nucleotide polymorphisms (C1236T, G2677T/A, C3435T) and/or mRNA expression changes of ABCB1 gene were demonstrated to be associated with inter-individual variability of imatinib response in CML patients. In this study, we aimed to examine whether genetic variations and/or altered expression of ABCB1 gene may influence response to imatinib. Sixty nine CML Tunisian patients, undergoing imatinib therapy, were enrolled in this study. These were divided into two groups: responders and non-responders to imatinib. The relative transcript expression levels of ABCB1 gene and the distribution of allele and genotype frequency of ABCB1 SNPs were compared between these two categories of patients. Linkage disequilibrium tests and haplotypes analysis were also studied. Our results showed that the mRNA expression level of ABCB1 gene did not differ significantly between the two categories of patients. In addition, results obtained from ABCB1 polymorphisms study and their correlation with imatinib response showed that the optimal response rate to imatinib did not differ significantly between C1236T, G2677T/A or C3435T genotypes. However, haplotype analysis showed that the 1236C–2677A–3435C haplotype was observed only in imatinib non-responders’ patients suggesting that CAC haplotype was linked to higher risk of imatinib resistance. Furthermore, analyses of ABCB1 haplotypes should be taken into account to study the relationship between ABCB1 genotypes and imatinib efficacy.

Resistance to imatinib has been recognized as a major challenge for the treatment of chronic myeloid leukemia (CML). Aberrant expression of miR-451 has been reported to participate in anticancer drug resistance. However, the role of miR-451 in imatinib resistance has not been investigated. The present study was undertaken to determine the expression of miR-451 in order to find a possible association between the expression of this miRNA and imatinib resistance in Tunisian CML patients.First, real-time RT-PCR was performed to identify the expression of miR-451 in peripheral leukocytes of 59 CML patients treated with imatinib. Then, bioinformatics analysis was carried out to understand the regulatory roles of miR-451 in imatinib-resistant process.Downregulated miR-451 was observed in imatinib-resistant CML cases. In silico analysis identified MYC as a potential target of miR-451. We further revealed the existence of an MYC-binding site in MiR-451 promoter region. On the other hand, increased level of MYC was detected in imatinib-resistant CML cases which may explain the causative role of MYC in CML cases and the downregulation of miR-451.Taken together, our findings suggest that miR-451 and MYC form together a regulatory loop which may act as a potential therapeutic target, and disruption of suggested regulatory loop could help to improve CML therapy.

The KIT gene encodes a class III tyrosine kinase receptor in which specific somatic mutations have been found to be associated with many diseases. In this work, we investigated the prevalence of KIT mutations in patients with chronic and acute myelogenous leukemia (CML and AML) and their prognostic significance. A total of 157 subjects were included in the present study (84 patients with CML, 33 with AML, and 40 healthy controls). Patients were analyzed at the first diagnosis, and the KIT mutations were screened by polymerase chain reaction (PCR) and direct sequencing technologies. The results demonstrated the presence of a G/A transition at codon 796, which is associated with the R796K protein variation. This mutation was detected at 21.42% in the CML subgroup and was absent in both AML patients and healthy controls; however, no correlation was found between this mutation and clinical parameters such as the molecular response to Gleevec. In conclusion, we retain that the KIT gene is highly mutated in the CML subgroup, but its role as a prognostic factor needs to be further elucidated.

Acute promyelocytic leukemia (APL) has now become the most curable of all subtypes of acute myeloid leukemia. A cure rate of 75-80% can be anticipated with a combination of all-trans retinoic acid (ATRA) and anthracyclines. In Tunisia, the ATRA era began in 1998 with the use, consecutively, of two regimens of a combination of ATRA with anthracycline and cytarabine (APL93), and without cytarabine (LPA99). From 2004, 39 patients with confirmed APL either by t(15;17) or PML/RARA were treated by the PETHEMA LPA 99 trial. The rationale of this protocol by avoiding cytarabine is to reduce death in complete remission (CR) without increasing the incidence of relapse. Thirty-three patients achieved CR (84.6%). The remaining six patients were considered as failure due to early death: three caused by differentiation syndrome (DS) and three died from central nervous system hemorrhage. Baseline blood cell count (WBC) >10 x 10(9)/l (P=0.26) and creatinine >1.4 mg/dl (P=0.42) were not predictive of mortality. DS was observed in 11 patients (30.5%) with a median onset time of 12 days (range: 3-23 days) and median WBC of 29 x 10(9)/L (range: 1.2 x 10(9)-82.7 x 10(9)/l). DS was severe in seven cases, moderate in four, and fatal in three cases. Body mass index > or =30 (P=0.044) and baseline WBC > or =20 x 10(9)/l (P=0.025) are independent predictors of DS. The median follow-up of this study is 36 months. Thirty patients are alive in continuous complete remission; two patients died in CR from septic shock and secondary myelodysplastic syndrome respectively; one patient died 47 months after achieving two relapses. Event free survival from diagnosis was 80% and overall survival was 82%. Our results are quite acceptable and can be improved by reducing mortality rate.

International audience; Hypereosinophilic syndromes (HES) are a heterogenous group of rare disorders characterized by sustained and otherwise unexplained overproduction of eosinophils with organ involvement and consecutive dysfunction. Detection of the FIP1L1-PDGFRA fusion gene or the corresponding cryptic 4q12 deletion in HES supports the diagnosis of chronic eosinophilic leukemia (CEL) and provides a molecular explanation for the pathogenesis of this disorder. We screened seven Tunisian patients fulfilling the WHO criteria of HES for the presence of the FIP1L1-PDGFRA fusion gene using nested reverse transcription polymerase chain reaction on peripheral blood samples. Four of the seven patients were positive for this fusion gene. Sequence analysis revealed a substantial heterogeneity of the fusion transcripts due to the involvement of several FIP1L1 exons. All patients were male. The median age at diagnosis was 24 years (range, 18-50); one patient had a history of hypereosinophilia of more than 10 years. Two patients had clinically important and symptomatic eosinophilic endomyocardial disease with thrombotic events. Splenomegaly was constant in FIP1L1-PDGFRA positive CEL but not in the other HES patients (only 1/3).; Les hyperéosinophilies essentielles sont caractérisées par une hyperéosinophilie supérieure à 1500 éléments/mm 3 persistante au-delà de sixmois, après exclusion d ’ une éosinophilie réactionnelle. Différentes altérations viscérales peuvent s’y associer leur pronostic est souvent redou-table et jusqu’à une période récente, il n’existait pas de traitement spécifique ou réellement efficace. En 2003 un gène de fusion :FIP1L1-PDGFR alpha a été retrouvée de façon récurrente dans ces syndromes, ce gène de fusion est généré à la suite d’une délétion interstitielle en 4q12 qui est cryptique à l’ analyse cytogénétique standard, la protéine chimérique résultante a une activité tyrosine kinase constitutive qui répond au traitement par l’ imatinib utilisé par ailleurs pour le traitement de la leucémie myéloïde chronique. Nous rapportons quatre cas d’hyperéosinophilie associéesà ce remaniement génétique

International audience; Human DNA repair mechanisms protect the genome from DNA damage caused by endogenous and environmental agents. Polymorphisms in DNA repair genes and differences in repair capacity between individuals have been widely reported in different cancers. In this study we aimed to evaluate the associations between XPCLys939Gln (rs2228001), XPDLys751Gln (rs13181) and XPG Asp1104His (rs17655) polymorphisms and leukemia risk in a Tunisian population. Genotypes were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) in 206 patients with leukemia and 206 healthy controls. We found increased risk of leukemia among subjects carrying the XPC 939Gln/Gln genotype (odds ratio [OR] = 2.48, 95% confidence interval [CI] = 1.353-4.560, p = 0.0042). Moreover, in subgroup analysis according to clinical types, patients with chronic myeloid leukemia (CML) showed a higher risk than patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) (OR = 3.87, 95% CI = 1.820-8.237, p = 0.0003). However, the XPD 751Gln allele may be protective against CML and AML development, and no significant differences in genotype frequencies were observed for the XPG gene between patients and controls. Further studies with larger samples and risk factor information are needed.

Differentiation syndrome (DS) is a life-threatening complication observed in patients with acute promyelocytic leukemia (APL) receiving induction therapy with all-trans-retinoic acid (ATRA). A bimodal incidence of DS has been observed, with a majority of cases occurring during the first week of ATRA treatment ("early" DS), but a substantial number of cases occurring during the third or even fourth week of ATRA treatment ("late" DS). However, to our knowledge occurrence of both early and late DS in the same patient has not been reported. We report an APL patient treated with the AIDA regimen, who experienced both early and late DS, a situation where differential diagnosis was difficult.

Hypereosinophilic syndromes (HES) are a heterogeneous group of disorders characterized by marked peripheral blood and tissue eosinophilia resulting in organ damage. Recent advances in molecular biology have led to the identification of a FIP1L1-PDGFRA fusion gene as a recurrent abnormality in some patients with HES. This fusion gene results from a cryptic 4q12 interstitial deletion involving an 800 kb region. Recent reports indicate that this subtype of HES is imatinib responsive with rapid and complete haematological remissions. Here we report two patients successfully treated with imatinib.

International audience; Mutations in the KRAS gene have been shown to play a key role in the pathogenesis of a variety of human tumours. However the mutational spectrum of KRAS gene differs by organ site. In this study, we have analysed the mutational spectrum of KRAS exon 1 in bladder tumours, colorectal cancer (CRC) and chronic myeloid leukemia (CML). A total of 366 patients were included in the present study (234 bladder tumours, 48 CRC and 84 CML). The KRAS mutations are absent in BCR/ABL1 positive CML. This result suggests that BCR/ABL1 fusion gene and KRAS mutations were mutually exclusive. The frequency of KRAS mutations in bladder cancer was estimated at 4.27 %. All of mutations were found in codon 12 and 90 % of them were detected in advanced bladder tumours. However the correlation between KRAS mutations and tumour stage and grade does not report a statistical significant association. The KRAS mutations occur in 35.41 % of patients with CRC. The most frequent mutations were G12C, G12D and G13D. These mutations were significantly correlated with histological differentiation of CRC (p = 0.024). Although the high frequency of KRAS in CRC in comparison to bladder cancer, these two cancers appear to have the same mutational spectrum (p > 0.05).

Interleukine 6 (IL-6) is the most important cytokine involved in malignant plasma cells growth and survival.To analyse bone marrow plasma cells IL6 receptor gene expression in both multiple myeloma patients at diagnosis and healthy bone marrow donors.Clinical and biological patients' features and responses to Dexamethasone-Thalidomide induction therapy were gathered. 47 patients and 16 case controls were analyzed: Bone marrow plasma cells were isolated; and IL6 receptor gene expression was quantified using Taqman quantitative PCr technology and 2-ΔCT formula.Quantitative and qualitative IL6 receptor gene expression were negatively correlated with the degree of response to therapy (p= 0.02). In this study, plasma cells IL6 receptor gene expression seems to be decisive in predicting the response to treatment.Understanding the mechanisms involved in plasma cells IL6 receptor gene expression may offer a better appreciation of the physiopathologic and anti-oncogenic ways of drug resistance in multiple myeloma and consequently the discovery of new specific drugs.

Introduction: Chronic myeloid leukemia (CML) accounts for 7%-15% of all leukemias affecting adults. The incidence in Algeria is 0.4/100,000 inhabitants in 2009. The aim of this study is to establish an Algerian-Tunisian epidemiological approach of CML and to know the characteristics of the disease in both countries. Materials and methods: This is a retrospective, longitudinal and multicenter study, including Algerian and Tunisian patients with CML diagnosed between January 2010 and December 2014. Through a data form distributed to various hematology departments, we collected and analyzed the following information: Patient's general characteristics, profession, circumstances of discovery of the disease, clinical and para-clinical examinations outcomes at the time of diagnosis including blood count, blood smear, bone marrow aspiration, cytogenetics, molecular biology, stages of the disease and the Sokal and Eutos prognostic classification scores. Bio-statistical tests: incidence, prevalence and rate of prevalence or relative prevalence (reported to 100,000 inhabitants / year). The descriptive analysis of quantitative and qualitative variables as percentages and 95% confidence interval. The Chi2 test is used to compare two variables. Results: We collected 1349 cases, including 325 from 06 Tunisian hematology units and 1024 from 18 Algerian units. The incidence in the Algerian-Tunisian population was 0.67/100,000 inhabitants with a prevalence rate of 2.72/100,000 inhabitants. The incidence in Tunisia was 0.50 / 100,000 inhabitants with a prevalence of 227 cases in 2014. In Algeria the incidence was 0.53/100,000 inhabitants with a prevalence of 1030 in 2014. The median age is 48 years (03-90) with a peak incidence in the age group (45-49 ans) and slight male predominance (sex ratio: 1,2). There was any notion of risk exposure. The average time between the start of the unrest and the date of diagnosis is 127 days (1-667). The circumstances of discovery: fortuitous in 30.5% (n = 355), splenomegaly in 39.7% (n = 463), asthenia in 24.6% (n = 287), a complication in 8.4% (n =95). Clinical examination includes general signs in 424 cases (36.4%): Weight loss 22.6% (n = 263), profuse sweating 13.8% (n = 13, 8%), bone pains found in 7.8%, splenomegaly in 81.7% (n = 952) with an overhang splenic average of 11.5 ± 5.3 cm (1-28), cutaneous and subcutaneous bleeding: 13.5% (n = 97), thrombosis 0.9% (n = 09). Biological characteristics: the Complete blood count (n = 1185) shows a white blood cells average rate of 171,223 G/L (34,700-984,800), hemoglobin average rate of 10.2 g/dl (4-17), platelets at 394,070 g/l (85-1340). Blood smear 96.3% (n = 1121): the average myelemia was 43.2% (10-98%). The Myelogram is practiced in 55% (n = 641), the average rate of the granular 76,5% (40-99%), erythroblasts 10.5% (0-82%), average blasts 3.6%. The karyotype 38.1% (n = 444), the Philadelphia chromosome was found in 423 cases (95, 3%); additional abnormalities were found in 17 cases (3.8%). The Fish was practiced in 281 cases (24.1%) and transcribed bcr/abl was found in 257 cases (91.4%). Molecular biology is practiced in 672 cases (57.7%) the transcript bcr/abl is found in 100%, the transcript of the type is specified in 373 cases, it is kind of b2a2 in 159 cases (42.6%), a b3a2 type in 180 cases (48.3%) and other transcribed in 34 cases (9.1%). CML chronic phase is diagnosed in 88.8% (n = 1051), acceleration phase in 9% (n = 107) acutisation phase in 3.1% (= 37). The distribution of pts according to Sokal prognostic classification (n = 948) describes a predominance of intermediate risk in 54% (n = 511), high risk in 30.3% (n = 287) and low risk in 16% (n = 152). The Eutos score is specified in 769 cases (66%), it is less than 87 in 661 cases (86%) and more than 87 in 108 cases (14%). Conclusion: The incidence of CML in the Algerian-Tunisian population is 0.67/100,000 population with a prevalence rate of 2.72/100,000 inhabitants. The young adult is more affected with a peak incidence between 45 and 49. The average time between the onset of the disease and the diagnosis remains long and the delay probably explains the frequency of tumor forms encountered in Algeria and the prevalence of high and intermediate risk, according to Sokal prognostic classification. Disclosures No relevant conflicts of interest to declare.

Acute lymphoblastic leukemia (ALL) is the major pediatric cancer in developed countries. The etiology of ALL remains poorly understood, with few established environmental risk factors. These risks were influenced by co-inheritance of multiple low-risk genetic polymorphisms such as variants within cytochrome P450A1 (CYP1A1), NADPH: quinone oxidoreductase (NQO1) and Thiopurine methyltransferase (TPMT) genes. In this work, we conduct a case–control study to assess the impact of CYP1A1*2A (CYP1A1 T6235C); NQO1*2 (NQO1 C609T); TPMT*2 (TPMT G238C) and TPMT A719G polymorphisms on the risk of developing ALL. The frequencies of TPMT*2, TPMT A719G, NQO1*2 and CYP1A1*2 variants were examined in 100 patients with ALL and 106 healthy controls by allele specific PCR and/or PCR–RFLP methods using blood samples. We have found that NQO1 609CT genotype was overrepresented in patients and was associated with an aggravating effect compared to the reference group with NQO1 609CC genotype (p = 0.028, OR = 1.41; CI 95 %: 1.04–1.93). However, TPMT*2, TPMT 719*G and CYP1A1*2 variants did not appear to influence ALL susceptibility (p > 0.05). Moreover we have not found a significant correlation between the studied variants and Bcr-Abl transcript. In conclusion we retain that leukemogenesis of ALL is associated with carcinogens metabolism and consequently related to environmental exposures.

Leukemia is a type of cancer of the blood or bone marrow that is characterized by an abnormal increase of white blood cells. Leukemia is clinically and pathologically subdivided into a variety of large groups. The risk of developing leukemia may be influenced by polymorphisms of xenobiotic metabolizing enzymes. In this work, we conduct a case-control study to assess the impact of polymorphisms in GSTM1 , GSTT1 and NAT2 genes on the risk of developing leukemia. Our data have shown that GSTM1*0 and GSTT1*0 were respectively associated with 2.05 and 4.36 increased risk for acute lymphoblastic leukemia (ALL). We have also shown that GSTM1*0 and GSTT1*0 act additively to increase the risk for ALL. Indeed, patients harbouring the “ GSTM1*0/GSTT1*0 ” genotype were at 11.81-fold increased risk for developing ALL ( P = 2 10 -5 ). The risk for developing acute myeloid leukemia (AML) increases on patients with “rapid or intermediate NAT2 genotypes”. Finally, the comparison of leukemia subgroups according to GSTM1 , GSTT1 and NAT2 genotypes, suggests that leukemogenesis of different leukemia subgroups is very distinct. In conclusion, our findings suggest that leukemogenesis is associated with carcinogen metabolism and consequently related to environmental exposures.

In Tunisia, the ATRA era began in 1998 with the use, consecutively, of two regimens combining ATRA and an anthracycline with cytarabine (APL93), and without cytarabine (LPA99). From 2004, 51 patients with confirmed APL either by t(15;17) or PML/RARA were treated according to the PETHEMA LPA 99 trial. Forty three patients achieved CR (86%). The remaining seven patients had early death (one died before treatment onset): four caused by differentiation syndrome (DS) and three died from central nervous system hemorrhage. Multivariate analysis revealed that female gender (P=0.045), baseline WBC> 10 G/L ( P=0.041) and serum creatinine > 1.4mg/dl ( P=0.021) were predictive of mortality during induction. DS was observed in 16 patients (32%) after a median onset time of 15 days from treatment onset (range, 2–29). Body mass index ≥ 30 (P=0.01) was the only independent predictor of DS. Occurrence of hypertensive peaks significantly predicted occurrence of DS (P=0.011) and was significantly associated with high BMI (p=0.003). With a median follow-up of 50 months, 5 year cumulative incidence of relapse, event free and overall survival were 4.7%, 74% and 78%, respectively.

Reports on childhood APL from developing countries are scarce. We treated 65 APL with two consecutive trials combining ATRA and chemotherapy. Twenty (30.7%) were aged less than 20 years including 11 girls and 9 boys, with a median age of 12 years. Fever at presentation (P = 0.002) and variant APL (P = 0.044) were more frequent in children, while there were no significant difference between children and adults for WBC count, Sanz’s score distribution and additional cytogenetic abnormalities. The CR rate was 95% (19/20) in children and 80% (36/45) in adults (P = 0.13). Differentiation syndrome (DS) was less often observed in children (1/20) than in adults (13/45) (P = 0.031). Two children relapsed and died during salvage therapy, and 2 died in CR from infection and from cardiac failure attributed to anthracyclines, while other children remained alive in CR. With a median follow-up of 4 years, 4-year EFS was 75% in children and 71.1% in adults (P = 0.57), while 4-year OS was 75% in children vs. 73.3% in adults (P = 0.72). Our results suggest that, even in the absence of optimal socio-economic condition, ATRA combined with anthracycline-based chemotherapy gives adequate results in childhood APL, as in adults.

The optimal management of malignant haematological disorders depend on the degree of tumor load reduction after therapy. Chronic myeloid leukemia constitutes a clinical model for molecular detection and therapy surveillance of malignant disease since this entity was the first leukemia shown to be associated with a specific bcr-abl fusion gene in the patient's leukemia cells. Molecular monitoring of bcr-abl transcript levels by real-time quantitative PCR is increasingly used to assess treatment response in patients with chronic myeloid leukemia (CML). This has become particularly relevant in the era of imatinib therapy when residual levels of leukaemia usually fall below the level of detection by bone marrow cytogenetic analysis. We monitored bcr-abl transcript levels by quantitative real time PCR in 50 tunisian patients treated with imatinib for chronic myeloid leukemia in chronic phase for a median of 29 months (3-60) after they started imatinib.