Abderrahim Khelif

Molecular cytogenetic characterization of variant Philadelphia translocations in chronic myeloid leukemia: genesis and deletion of derivative chromosome 9

The mechanisms for the formation of variant Philadelphia (Ph) translocations that occur in 5-10% of patients with chronic myeloid leukemia (CML) are not fully characterized. Studies on the prognosis of these variant translocations have yielded conflicting results, especially regarding imatinib outcome and the status of deletions on the derivative chromosome 9. To shed light on these controversial subjects, we sought to analyze all variant translocation cases presented at diagnosis and identified in our institution between the years 2001 and 2008. Of 336 CML patients who presented at diagnosis and were studied by conventional cytogenetics and fluorescence in situ hybridization (FISH), 25 patients (7.44%) exhibited variant Ph-rearrangements. All chromosomes could be implicated in variant Ph rearrangements, with 32 breakpoints defined. Their distribution was located preferentially in the CG-richest regions of the genome. Deletions on der(9) were observed in 15 of the 25 cases (60%), a greater proportion in typical Ph translocations (12-15%). Both one- and two-step mechanisms were encountered in our series, as well as multiple-step mechanisms, which originate more complex rearrangements. Higher prevalence was observed for the two-step mechanism (56%). Proper assessment of the prognostic significance of variant translocations requires better categorization of these translocations based on their mechanisms of genesis and 9q34 deletion status.

In vitro susceptibility to amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin of Aspergillus spp. isolated from patients with haematological malignancies in Tunisia

The resistance of Aspergillus species to antifungal is increasingly reported and the knowledge of the local epidemiology and antifungal susceptibility pattern is pivotal to define adequate treatment policies. Our study aimed to: 1) describe the in vitro antifungal susceptibility profile of the Aspergillus species isolated from patients with haematological malignancies in Tunisia; 2) compare the E-test and Sensititre Yeast-One assays for the detection of paradoxical growth and trailing effect, both phenotypes commonly exhibited by Aspergillus spp. upon exposure to caspofungin and 3) to evaluate the mortality rate in patients according to the causative Aspergillus species and the antifungal treatment.We tested amphotericin B, itraconazole, voriconazole, posaconazole and caspofungin against 48 Aspergillus isolates (17, A. niger; 18, A. flavus; 9, A. tubingensis; 1, A. westerdijkiae; and 1, A. ochraceus) with the E-test. Minimal inhibition concentrations were above the epidemiological cut-off values for amphotericin B in 67% of A. flavus strains; for caspofungin in 22% of A. flavus strains; and for itraconazole in 22% of A. tubingensis strains, voriconazole and posaconazole MICs were below the epidemiological cut-off values for all strains.When exposed to caspofungin, 42% of the strains exhibited trailing effect and 38% paradoxical growth. Trailing effect occurred in 61% of A. flavus strains and paradoxical growth in 62% of Aspergillus section Nigri strains. E-test and Sensititre Yeast-One assays were only fairly concordant for the detection of these phenotypes. Repeatability of both assays was high for trailing effect but poor for paradoxical growth. The relatively high frequency of amphotericin B resistant strains makes voriconazole best adapted as a first-line treatment of invasive aspergillosis from amphotericin B to voriconazole in this hospital.

Familial hematological malignancies: ASXL1 gene investigation

AbstractPurposeFamilial aggregation among patients with several hematological malignancies has been revealed. This emphasizes the importance of genetic factors. Only few genes predisposing to familial hematological malignancies have been reported until now due to the low occurrence. We have described in previous study PRF1 and CEBPA variants that might contribute to the background of genetic factors, which encourage us to extend our investigations to other cooperating genes. The aim of this study is to determine whether germline additional sex combs-like 1 (ASXL1) gene mutations may be involved?Methods/patientsIn this study, we investigated the candidate gene ASXL1 by direct sequencing in 88 unrelated Tunisian and French families with aggregated hematological malignancies.ResultsWe report a new p.Arg402Gln germline missense substitution in two related Tunisian patients which has not been previously described. We identified here this variant for the first time in non-Hodgkin lymphoma. The p.Arg402Gln variant was not found in 200 control chromosomes. In silico analysis has predicted potential deleterious effect on ASXL1 protein.ConclusionsFrom an extended candidate genes analyzed in the field of familial hematological malignancies, ASXL1 might be involved. This variant should be considered since a potential damaging effect was predicted by in silico analysis, with a view to develop functional assay in order to investigate the biological assessment.

Chronic Myeloid Leukemia as a Secondary Malignancy after Lymphoma in a Child. A Case Report and Review of the Literature

Background: Philadelphia chromosome-positive chronic myeloid leukemia (CML) in children is very rare. CML occurring as a secondary malignancy in individuals treated for diffuse large B-cell lymphoma (DLBCL) is also rare. Case Report: We present the case of a 5-year-old female patient who developed a right orbital mass that was diagnosed as DLBCL. 9 months after receiving treatment for DLBCL, she presented with a white cell count of 250,000/mm3. Peripheral blood and bone marrow (BM) evaluation revealed a myeloproliferative disorder. Cytogenetic and molecular studies demonstrated the presence of t(9;22). CML following DLBCL has not been previously described in the younger population. To our knowledge, this is the first report of a child who developed a CML as a second malignancy after DLBCL. Therapy-related CML and non-therapy-related secondary CML are discussed as potential explanations of this highly unusual clinical presentation. Conclusion: Hematological disorders such as CML may occur after lymphomas. With the increased use of BM cytogenetic studies during staging for lymphoid malignancies, future studies may be able to clarify the question of whether the CML clone in some of these patients existed before treatment for lymphoma.

Molecular study of CEPBA in familial hematological malignancies

Familial aggregation in patients with several haematological malignancies has been described, but the genetic basis for this familial clustering is not known. Few genes predisposing to familial haematological malignancies have been identified, among which RUNX1 and CEBPA have been described as predisposing genes to acute myeloid leukemia (AML). Recent studies on RUNX1 suggest that germline mutations in this gene predispose to a larger panel of familial haematological malignancies than AML. In order to strengthen this hypothesis, we have screened CEBPA for germline mutations in several families presenting aggregation of hematological malignancies (including chronic or acute, lymphoid or myeloid leukemias, Hodgkin’s or non Hodgkin’s lymphomas, and myeloproliferative or myelodysplastic syndromes) with or without solid tumours. Although no deleterious mutations were found, we report two novel and rare variants of uncertain significance. In addition, we confirm that the in frame insertion c.1175_1180dup (p.P194_H195dup) is a germline polymorphism.

A novel t(3;12)(q21;p13) translocation in a patient with accelerated chronic myeloid leukemia after imatinib and nilotinib therapy

The acquisition of secondary chromosomal aberrations in chronic myeloid leukemia (CML) patients with Philadelphia chromosome-positive (Ph+) karyotype signifies clonal evolution associated with the progression of the disease to its accelerated or blastic phase. Therefore, these aberrations have clinical and biological significance. T(3;12)(q26;p13), which is a recurrent chromosomal aberration observed in myeloid malignancies, is typically associated with dysplasia of megakaryocytes, multilineage involvement, short duration of any blastic phase, and extremely poor prognosis. We have identified a recurrent reciprocal translocation between chromosomes 3 and 12 with different breakpoint at bands 3q21 in the malignant cells from a 28-year-old man. The patient was initially diagnosed as having Ph+ CML in the chronic phase. The t(3;12)(q21;p13) translocation occurred 4 years after the patient was first diagnosed with CML while undergoing tyrosine kinase inhibitor therapy. We confirmed the t(3;12)(q21;p13) translocation via fluorescence in situ hybridization assay by using whole-chromosome paint probes for chromosomes 3 and 12. Our findings demonstrate that, similar to other recurrent translocations involving 3q26 such as t(3;3) and t(3;21), the t(3;12)(q21;p13) translocation is implicated not only in myelodysplastic syndrome and acute myeloid leukemia but also in the progression of CML. These findings extend the disease spectrum of this cytogenetic aberration.

Fatal anaphylactic reaction to intravenous infusion of Ondansetron: a report of two cases

Ondansetron is a selective 5 hydroxy-tryptamine 3 (5-HT3) receptor antagonist widely used as an effective antiemetic drug especially for the prevention of chemotherapy-induced nausea and vomiting [1]. The efficacy of ondansetron has been reported in several studies and it is reported to be a well-tolerated drug [2]. The most common side effects reported to ondansetron are headaches, diarrhea, constipation, fever and dizziness [3-5]. Anaphylactic reaction to ondansetron is rarely reported [6,7]. Abstract

GATA2 gene analysis in several forms of hematological malignancies including familial aggregations

The genetic predisposition to familial hematological malignancies has been previously reported highlighting inherited gene mutations. Several genes have been reported but genetic basis remains not well defined. In this study, we extended our investigation to a potential candidate GATA2 gene which was analyzed by direct sequencing in 119 cases including familial aggregations with a variety of hematological malignancies and sporadic acute leukemia belonging to Tunisian and French populations. We reported a deleterious p.Arg396Gln GATA2 mutation in one patient diagnosed with both sporadic acute myeloid leukemia (AML) and breast cancer. We also reported several GATA2 variations in familial cases. The absence of deleterious mutations in this large cohort of familial aggregations of hematological malignancies may strengthen the hypothesis that GATA2 mutations are an important predisposing factor, although as a secondary genetic event, required for the development of overt malignant disease.

Microsatellite Typing of Aspergillus flavus Strains in a Tunisian Onco-hematology Unit

Aspergillus flavus is the most common species associated with invasive aspergillosis in Tunisia. The molecular epidemiology of the species is poorly documented. We used five highly discriminative microsatellite markers for the genotyping of clinical and hospital environmental A. flavus strains to assess whether IA could be hospital-acquired in the onco-hematology unit of the Farhat Hached teaching hospital of Sousse, Tunisia. The genotyping of 18 clinical isolates, collected from sputa of 17 acute leukemia patients, and 81 isolates, collected in these patients’ hospital environment and food, identified 57 isolates that were grouped in 10 clones, each of them including 2–17 isolates. The remaining 42 isolates showed a unique genotype. Two main transmission scenarios were observed: (1) the same clone was isolated from different patients; (2) the same clone was isolated from a patient, its hospital environment and/or food. These findings strongly suggest the occurrence of hospital-acquired A. flavus infection/colonization in the investigated onco-hematology unit.

Chemiluminescent detection of clonal immunoglobulin and T cell receptor gene rearrangements in Tunisian lymphoid malignancies, leukemias and lymphomas.

Clonal rearrangement of antigen receptor genes is commonly used to characterize the lymphoproliferative diseases. In order to perform molecular characterization in the diagnostics and monitoring of lymphoid malignancies, leukemias and lymphomas in Tunisia, we have introduced the use of chemiluminescent probes for immunoglobulin (IG) and T cell receptor (TR) gene rearrangement detection employing the Southern blot method. The chemiluminescent and radioactive detection methods tested with alkaline phosphatase and 32P labelled probes, respectively, were used for the IG and TR gene rearrangement characterization. Our results show the same pattern of rearrangement. Moreover, the chemiluminescent signal is detected faster and it is as sensitive as the radioactive one. We report the optimized conditions for using IGH, IGK, IGL, TRB and TRG probes in non radioactive detection. We have applied the chemiluminescent Southern blot method to analyze examples of Tunisian leukemias and lymphomas. The results allowed the assessment of clonality and the T or B cell lineage of these cases. The use of non radioactive probes makes chemiluminescent Southern blot detection reliable, safe and sensitive. As the use of radioactivity is not common in our laboratories and the licensing requirements needed for its use prohibitive, the chemiluminescent technique will be of great help for detection and characterization of molecular markers in lymphoid malignancies in Tunisia.