B. Meddeb

Predictive factors of all-trans-retinoic acid related complications during induction therapy for acute promyelocytic leukemia

Abstract Background: The combination of all-trans-retinoic acid (ATRA) and chemotherapy has made acute promyelocytic leukemia (APL) a highly curable leukemia. However, several complications are reported with this treatment the most serious and life threatening being Retinoic Acid Syndrome (RAS). We aimed at identifying factors that could predict complications caused by ATRA during induction treatment of APL. Patients: Forty-two patients with confirmed APL (by t(15;17) and/or PML/RARA) treated at our institution (University hospital of Tunis) between January 1998 and June 2006 using two consecutive protocols: European APL93 trial (24 patients) until February 2004 and Spanish PETHEMA LPA99 trial (18 patients) more recently. Induction regimen consisted of ATRA 45 mg/m2/d until CR combined to DNR 60 mg/m2/d×3+Cytarabine 200 mg/m2/d×7 (APL93) and Idarubicin 12 mg/m2 d2, 4, 6, 8 (LPA99). Prednisone (0·5 mg/kg d1–d15) was added if WBC >10×109/L to prevent RAS in LPA 99. Results: Median age was 36 yr (7–64 yr), M/F=16/26 (0·61), median WBC was 2·4×109/L (range 0·6–100×109/L). WBC >10×109/L was noted in 14 patients (33%). Additional cytogenetic abnormalities were seen in 12/42 (28%). Median body mass index (BMI=weight/height2:N 20–25) was 24 kg/m2 (range 16–40 kg/m2), BMI >30 was noted in nine patients (8F and 1M). Thirty-three patients achieved CR (78·57%):18/24 (75%) in APL93 versus 15/18 (83%) in LPA99. Nine patients (21·42%) had early death. Causes of early death were: RAS (6) and CNS hemorrhage (3). Complications due to ATRA were: RAS (10), Scrotal ulcerations (3), Sweet syndrome (2), Perineal ulcerations (1), and Pseudotumor cerebri (1). Prognostic factors for complications of ATRA (Fisher exact test) were: BMI >35 (p=0·055), induction treatment without cytarabine (LPA99 trial) (p=0·047), whereas age (p=0·74), gender (p=0·51), initial WBC (p=0·47), and additional cytogenetic abnormalities (p=0·83) were not predictive. Retinoic Acid Syndrome was more reported in patients with initial WBC >10×109/L (p=0·08). Conclusion: We found high BMI (>35) in female and treatment without Cytarabine to increase the risk of developing complications with ATRA.

A first case of congenital TTP on the African continent due to a new homozygous mutation in the catalytic domain of ADAMTS13

Hereditary thrombotic thrombocytopenic purpura (TTP) is a rare disorder characterized by occlusive microvascular thrombosis, consumptive thrombocytopenia, and microangiopathic hemolytic anemia. Homozygous or compound heterozygous mutations in the ADAMTS13 gene result in a congenital severe ADAMTS13 deficiency and subsequent accumulation of ultra-large von Willebrand factor multimers, which tend to form platelet thrombi in the microcirculation. We report a first case of congenital TTP on the African continent with a new, homozygous mutation in the metalloprotease domain of ADAMTS13. An initially oligo-symptomatic presentation was followed by acute exacerbation with ischemic stroke and acute renal failure highlighting the severity of this syndrome.

Transfusion-related acute lung injury (TRALI) during remission induction course of acute myeloid leukemia: A possible role for all-transretinoic-acid (ATRA)?

Transfusion-related acute lung injury (TRALI) is a clinical syndrome characterized by sudden onset of respiratory distress due to pulmonary edema during or following transfusion. Two proposed pathophysiologic mechanisms for TRALI were proposed: the antibody hypothesis and the two-event hypothesis. The two-event hypothesis postulates that a pathway to neutrophil activation and aggregation can occur without leukocyte antibodies. We report a case of TRALI occurring during remission induction course of acute myeloid leukemia in a 27-year-old woman who received All-transretinoic-acid (ATRA). We postulate that ATRA may have played a role in this life-threatening complication by priming neutrophil and enhancing their adherence and their activation in the pulmonary endothelium. TRALI improved with non-invasive ventilation support and use of high dose corticosteroids.

Quantitative detection of bcr-abl transcripts in chronic myeloid leukemia.

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 patients 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.

Molecular analysis in two Tunisian families with combined factor V and factor VIII deficiency

Summary.  Combined factor V (FV) and factor VIII (FVIII) deficiency (F5F8D) is a rare autosomal recessive disorder caused by mutations in LMAN1 or MCFD2 genes which encode proteins that form a complex involved in the transport of FV and FVIII from the endoplasmic reticulum to Golgi apparatus. We report two novel mutations in MCFD2 gene and one recurrent mutation in LMAN1 gene that caused combined FV and FVIII deficiency in two unrelated Tunisian Muslim families. For the first family two patients were homozygous for a new missense mutation Asp81His in exon 3 of MCFD2 and heterozygous for a second new missense mutation Val100Asp in the same exon. Replacement respectively of the hydrophilic Asp residue with hydrophobic positively charged His and of the hydrophobic neutral Val residue with the Asp residue most likely disrupts the MCFD2–LMAN1 interaction, thus leading to the disease phenotype. For the second family a reported Arg202X mutation in exon 5 in the LMAN1 gene was identified in the homozygous state.

Secondary chronic myelomonocytic leukemia with monosomy 7 after successful treatment of acute promyelocytic leukemia

Current APL chemotherapy protocols usually include high-dose anthracyclines, mitoxantrone, and epipodophillotoxins, which are topoisomerase II inhibitors of high leukemogenic potential. In the last years, several case reports of myelodysplastic syndrome (MDS) or AML (different from APL), occurring during the course of APL have been made. We report herein a first case of CMML with monosomy 7 occurring after treatment of APL.

Low dose prophylaxis in Tunisian children with haemophilia.

Low dose prophylaxis could be recommended in countries with limited resources.

Structural analysis of two novel mutations in MCFD2 gene causing combined coagulation factors V and VIII deficiency

Combined factor V and factor VIII deficiency (F5F8D) is a rare autosomal recessive bleeding disorder reported usually in the context of consanguinous marriage. F5F8D is characterized by mild-tomoderate bleeding and coordinate reduction in plasma FV and FVIII levels, as well as platelet FV level (OMIM 227300) [1]. The disease is caused by mutations in genes encoding lectin mannose binding protein (LMAN1) and multiple coagulation factor deficiency 2 (MCFD2), which are the components of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC-53) involved in the FV and FVIII intracellular transport [1, 2]. LMAN1 is a type-I integral membrane protein that was first described as a 53-kDa marker of the ERGIC [3], whereas MCFD2 is a soluble luminal protein

Identification of novel and recurrent mutations in Tunisian haemophilia B patients

Haemophilia B disease is a recessively inherited X-linked bleeding disorder which results from deficiency of factor IX (F9). Haemophilia B has a frequency of approximately 1 in 25 000 men worldwide [1]. Haemophilia B results from heterogeneous mutations spread throughout the F9 gene [2]. According to the World Federation of Hemophilia Report on the annual global survey 2007, 51 haemophilia B in Tunisia have been reported [3]. In this first study on Tunisian haemophilia B, we report the molecular analysis of 16 unrelated haemophilia B families. Patients involved in this study were from the Hemophilia Treatment Center, Aziza Othmana hospital, Tunisia. Informed consent was obtained from all patients. Molecular analysis was performed using the following strategy: polymerase chain reactions for the entire coding sequence of the F9 gene were prepared as described previously [4]. The mutation detection protocol was performed by dHPLC on a WAVE DNA Fragment Analysis System (Transgenomics, San Jose, USA). Altered profiles detected by dHPLC were sequenced using ABI Dye Terminator Cycle Sequencing (Perkin-Elmer Applied Biosystems, Foster City, CA, USA) and analysed using a capillary sequencer Genetic Analyser ABI PRISM310 (Perkin-Elmer Applied Biosystems, Foster City, CA, USA [4]. Results were analysed using BLAST (http://www.ncbi.nlm. nih.gov/blast) program against the normal F9 gene sequence (GenBank Accession No. K02402) and the mutations were compared with the haemophilia B mutation database (http://www.umds.ac.uk/molgen). To evaluate the nature of missense mutations, we used PolyPhen (Polymorphism Phenotyping) (http://genetics.bwh.harvard.edu/pph). Our cohort is composed of 30 patients belonging to 16 unrelated families who represent 60% of total haemophilia B Tunisian population. A total of 15 different mutations were detected (Table 1), except for one family that did not show any mutations. In addition, the polymorphism g.20421A>G in exon 6 was also identified in five families. Five novel mutations were identified in five patients including 2 missense mutations, 1 nonsense mutation, 1 splice site mutation and one small deletion. For patient Hb 2, a deletion of CAG sequence from the 17795 to 17797 position inducing the loss of the last acid Ala173 in exon 5 (The numbering of the amino acids is according to the Swiss-Prot PZES (P00740)). For patient Hb10, a T to A substitution at nucleotide position 113 which changes a Cys acid in a codon stop (Cys27X) in exon 1, which will result in nonsense-mediated RNA decay and produce a severe phenotype as no protein will be translated, has been revealed. Patient Hb12 shows an acceptor splice substitution at the position 10507 in intron 4 (+2T > C). For Patient Hb14, a substitution of T to A at the position 31286 in exon 8, change the Cys 435 to Ser. Patient Hb16 present a substitution of G to A at the position 30932 in exon 8, which change Ala 317 to Thr. Replacement of a non-polar amino acid residue by a polar one is likely to affect the function, secretion or stability of the protein. Using PolyPhen these two mutations are predicted to be probably damaging with a score of 1.000 and 0.995 respectively. The question of whether these two candidate mutations Cys435Ser and Ala317Thr are pathogenic and alter the three-dimensional structure and function of F9 protein needs further investigation. However, as the latter mutations along with, Ala173Del, Ala317Thr and Cys435Ser occurred at amino acid residues highly conserved among different species, they may be involved in the F9 destabilization. Compared with previously published reports [5], we found that the two deletions identified in our patients bearing a severe disease. However, in our patient cohort the two nonsense mutations were associated with different phenotypes, severe and moderate disease respectively in patient Hb17(FIX:C level of 4) which is at variance with the majority of entries on the haemophilia B database for this mutation (which cite FIX:C and antigen levels of <1 for most of the 56 examples). Our observation in Hb17 is actually the exception for this particular mutation and for most nonsense mutations in general. In patient Hb3, we could not detect any mutations in the F9 gene using first dHPLC (no altered profile was observed) then sequencing. It is possible that this family might have pathological translocation, duplication or inversion in the factor IX gene leading to the disease. Further investigation is needed. To our knowledge this study is the first comprehensive molecular analysis of haemophilia B patients in Tunisia. Five novel mutations were identified and our data are globally in agreement with other reports in the international database. When requested, the data obtained from this study will be used for carrier testing and prenatal diagnosis. The identification of the mutations can also be used to estimate the risk of inhibitor development. It can also be valuable when planning future studies including gene therapy.

Expérience tunisienne dans la prise en charge des lymphomes agressifs de l’adulte : à propos de 337 patients

From January 1997 to December 2005, 337 patients with aggressive non Hodgkins lymphoma were treated with one of the two successive multicentric non randomized protocols established in Tunisia. The mean age was 53 years. Most patients had diffuse large cell lymphoma with B phenotype in 86% and T in 14%. The performance status was 2 or 3 in 34% of cases. The LDH were elevated in 74% of cases. Advanced disease (III or IV stage) was noted in 59% of cases and 10% had a tumoral mass greater than 10 cm. According to the international prognostic index (IPI) adjusted to age, we distinguish four groups: group 1 (0 factor and age < 70 years), group 2 (1-3 factors and age < or = 60 years), group 3 (1-3 factors and age between 61 and 70 years) and group 4 (1-3 factors and age > 70 years). The patients of group 1 (N = 47) received 3 courses of CHOP regimen followed by irradiation. The patients of group 2 (N = 160) received 4 courses of ACVBP regimen (+ rituximab for 21 patients) followed by consolidation (N = 92) or peripheral blood progenitor cell transplantation (N = 20). The patients of group 3 (N = 61) received 8 courses of CHOP regimen (+ rituximab for 20 patients). The patients of group 4 (N = 69) received 6 courses of mini-CEOP regimen (N = 48) or 6 courses CVP regimen (N = 21). The 4-year overall survival was 56% and the 4-year event free survival was 49%.