Donor chimerism and bcr-abl gene status following non-myeloablative peripheral blood stem cell transplantation in chronic myeloid leukaemia patients in HUKM

Objective: This study was done to determine the relationship between donor chimerism and the presence of bcr-abl gene in chronic myeloid leukaemia (CML) patients post-transplantation. Methods: The study population consisted of all CML patients who had undergone non-myeloablative peripheral blood stem cell transplant in Hospital Universiti Kebangsaan Malaysia (HUKM) during the study period. All patients had their bone marrow aspiration done at diagnosis and day 30, 60, 100, 130, 160 and 190 post-transplantation. The samples were analysed for bcr-abl transcript as well as chimerism status. Results: A total of nine cases underwent non-myeloablative peripheral blood stem cell transplant. All patients were transplanted during the chronic phase. One patient was found to show mixed chimerism at day 30 post-transplant coinciding with bcr-abl transcript disappearance. Six patients showed that full donor chimerism correlated with bcr-abl transcript disappearance. In one patient, chronic myeloid leukaemia transformed into acute myeloid leukaemia. Another patient had a graft failure. Conclusion: This observational cohort study showed that full chimerism is required for disappearance of bcr-abl transcript but one case showed disappearance of bcr-abl transcript at day 30 while full chimerism was not achieved

ADAMTSL5 and CDH11: putative epigenetic markers for therapeutic resistance in acute lymphoblastic leukemia

Background and objectives: DNA hypermethylation has been linked to poor treatment outcome in childhood acute lymphoblastic leukemia (ALL). Genes differentially methylated in the chemoresponsive pre-B-ALL compared to chemoresistant pre-B-ALL cases provide potential prognostic markers. Methods: DNA methylation profiles of five B-ALL childhood patients who achieved morphological complete remission (chemoresponsive) and five B-ALL patients who did not (chemoresistant) after induction treatments as well as four normal controls were compared on 27 000 CpG sites microarray chips. Subsequently, methylation-specific polymerase chain reaction (MSP) on selected hypermethylated genes was conducted on an additional 37 chemoresponsive and 9 chemoresistant B-ALL samples and 2 normal controls. Results: Both methods were found to be highly correlated. Unsupervised principal component analysis showed that the chemotherapy-responsive and -resistant B-ALL patients could be segregated from one another. Selection of segregated genes at high stringency identified two potential genes (CDH11 and ADAMTSL5). MSP analysis on the larger cohort of samples (42 chemoresponsive, 14 chemoresistant B-ALL samples and 6 normal controls) revealed significantly higher rates of hypermethylation in chemoresistant samples for ADAMTSL5 (93 vs. 38%; p = 0.0001) and CDH11 (79% vs. 40%, p < 0.01). All control cases remained unmethylated. Conclusion: Chemoresistant B-ALL patients are associated with increased methylation in ADAMTSL5 and CDH11. These findings need to be validated in a larger group of patients, and the functional biological and prognostic significance of differential methylation needs to be studied further

Importance of clinical and morphological correlations in diagnosing langerhans cell histiocytosis

Langerhans cell histiocytosis (LCH) is a clonal histiocytic disorder. The variable clinical manifestations from isolated bone lesion to multisystem disease can cause difficulties and delay in diagnosis. We report a 2 years and 8 months-old girl who presented with a 2 weeks history of persistent fever and weight loss associated with progressive abdominal distension. Physical examination revealed pallor, bilateral proptosis, seaborrheic dermatitis over the scalp and hepatosplenomegaly. Skull X-ray demonstrated multiple lytic lesions at the base and the skull vault. Bone marrow morphology showed numerous abnormal Langerhans cells (LCs) and foamy macrophages. The trephine immunohistochemistry (IHC) stains for CD1a, S-100 and CD68 were inconclusive. The diagnosis of multisystem Langerhans cell histiocytosis (MS-LCH) in this patient was based on the clinical presentation, radiological and morphological analysis. She subsequently received chemotherapy and currently she is on maintenance therapy with a good clinical response. LCH is a rare disease and although the IHC was inconclusive, the correlation of clinical, radiological and morphological data are essential for the diagnosis

Differential expression patterns of leukaemia associated genes in leukaemia cell lines compared to healthy controls

Introduction: The phenotype and genotype of cancer cells portray hallmarks of cancer which may have clinical value. Cancer cell lines are ideal models to study and confirm these characteristics. We previously established two subtracted cDNA libraries with differentially expressed genes from an acute myeloid leukaemia patient with poor prognosis (PP) and good prognosis (GP). Objective: To compare gene expression of the leukaemia associated genes with selected biological characteristics in leukaemia cell lines and normal controls. Methodology: Expression of 28 PP genes associated with early fetal/embryonic development, HOX-related genes, hematopoiesis and aerobic glycolysis/hypoxia genes and 36 GP genes involved in oxidative phosphorylation, protein synthesis, chromatin remodelling and cell motility were examined in B-lymphoid (BV173, Reh and RS4;11) and myeloid (HL-60, K562) leukaemia cell lines after 72h in culture as well as peripheral blood mononuclear cells from healthy controls (N=5) using semi-quantitative polymerase chain reaction (PCR) method. Cell cycle profiles were analysed on flow cytometry while MTT cytotoxicity assay was used to determine drug resistance to epirubicin. Results: Genes expressed significantly higher in B-lymphoid leukaemia cell lines compared to healthy controls were mostly of the GP library i.e. oxidative phosphorylation (3/10), protein synthesis (4/11), chromatin remodelling (3/3) and actin cytoskeleton genes (1/5). Only two genes with significant difference were from the PP library. Cancer associated genes, HSPA9 and PSPH (GP library) and BCAP31 (PP library) were significantly higher in the B-lymphoid leukemia cell lines. No significant difference was observed between myeloid cell lines and healthy controls. This may also be due heterogeneity of cell lines studied. PBMC from healthy controls were not in cell cycle. G2/M profiles and growth curves showed B-lymphoid cells just reaching plateau after 72 hour culture while myeloid cells were declining. IC50 values from cytotoxicity assay revealed myeloid cell lines had an average 13-fold higher drug resistance to epirubicin compared to B-lymphoid cell lines. Only CCL1, was expressed at least two-fold higher in myeloid compared to B-lymphoid cell lines. In contrast, MTRNR2, EEF1A1, PTMA, HLA-DR, C6orf115, PBX3, ENPP4, SELL, and IL3Ra were expressed more than 2-fold higher in B-lymphoid compared to myeloid cell lines studied here. Conclusion: Thus, B-lymphoid leukaemia cell lines here exhibited active, proliferating characteristics closer to GP genes. Higher expression of several genes in B-lymphoid compared to myeloid leukaemia cell lines may be useful markers to study biological differences including drug resistance between lineages

Positive TPMT genotype-phenotype correlation underscores importance of TPMT genotyping for personalized thiopurine dosing

This study explored TPMT genotype-phenotype correlation in a group of acute lymphoblastic leukemia (ALL) patients to investigate the potential of TPMT genotyping for personalized thiopurine dosing. Genotyping for G238C (TPMT*2), G460A (TPMT*3B) and A719G (TPMT*3C) loci was determined in 89 subjects via PCR, while TPMT activity was measured using HPLC. TPMT*3C was the only mutant allele detected in 4 heterozygous carriers. These patients had significantly lower (23.0 nmol/g Hb/h) TPMT activity compared to wildtype patients (51.0 nmol/g Hb/h) (p = 0.003). Positive correlation between TPMT genotype and phenotype projects the possibility of using TPMT genotyping as a guide prior thiopurine drug administration.

Reverse micelle liquid-liquid extraction of a pharmaceutical product

Reverse micelle extraction has received considerable attention in recent years due to its ability to selectively solubilise solutes from an aqueous phase, and in the case of biomolecules to maintain their biological activities. The apparent success of research on protein extraction from the aqueous phase using reverse micelle provides motivation to study the solubilisation of antibiotic. The objective of this study is to investigate the extraction of antibiotic (penicillin G is chosen as model antibiotic) from aqueous solution (forward extraction) and from the reverse micelle to a new aqueous solution (backward extraction). Sodium di(2-ethylhexyl)sulfosuccinate (AOT) is chosen as the surfactant and isooctane as the organic solvent. The UV-Vis spectrophotometer is used to determine the mass of penicillin G in solution after the extraction process. The extraction is expected to be influenced by the initial penicillin G concentration, the salt type and concentration in the aqueous phase, pH, and surfactant concentration. It is expected that as penicillin is an interfacially active compound that will interacts with AOT surfactant, the interfacial association will be dependent on both pH and surfactant concentration

<i>ADAMTSL5</i> and <i>CDH11</i>: putative epigenetic markers for therapeutic resistance in acute lymphoblastic leukemia

<p><b>Background and objectives:</b> DNA hypermethylation has been linked to poor treatment outcome in childhood acute lymphoblastic leukemia (ALL). Genes differentially methylated in the chemoresponsive pre-B-ALL compared to chemoresistant pre-B-ALL cases provide potential prognostic markers.</p> <p><b>Methods:</b> DNA methylation profiles of five B-ALL childhood patients who achieved morphological complete remission (chemoresponsive) and five B-ALL patients who did not (chemoresistant) after induction treatments as well as four normal controls were compared on 27 000 CpG sites microarray chips. Subsequently, methylation-specific polymerase chain reaction (MSP) on selected hypermethylated genes was conducted on an additional 37 chemoresponsive and 9 chemoresistant B-ALL samples and 2 normal controls.</p> <p><b>Results:</b> Both methods were found to be highly correlated. Unsupervised principal component analysis showed that the chemotherapy-responsive and -resistant B-ALL patients could be segregated from one another. Selection of segregated genes at high stringency identified two potential genes (<i>CDH11</i> and <i>ADAMTSL5</i>). MSP analysis on the larger cohort of samples (42 chemoresponsive, 14 chemoresistant B-ALL samples and 6 normal controls) revealed significantly higher rates of hypermethylation in chemoresistant samples for <i>ADAMTSL5</i> (93 vs. 38%; <i>p</i> = 0.0001) and <i>CDH11</i> (79% vs. 40%, <i>p</i> < 0.01). All control cases remained unmethylated.</p> <p><b>Conclusion:</b> Chemoresistant B-ALL patients are associated with increased methylation in ADAMTSL5 and CDH11. These findings need to be validated in a larger group of patients, and the functional biological and prognostic significance of differential methylation needs to be studied further.</p

Cohort profile: The Malaysian Cohort (TMC) project: a prospective study of non-communicable diseases in a multi-ethnic population

The Malaysian Cohort study was initiated in 2005 by the Malaysian government. The topdown approach to this population-based cohort study ensured the allocation of sufficient funding for the project which aimed to recruit 100 000 individuals aged 35–70 years. Participants were recruited from rural and urban areas as well as from various socioeconomic groups. The main objectives of the study were to identify risk factors, to study gene-environment interaction and to discover biomarkers for the early detection of cancers and other diseases. At recruitment, a questionnaire-based interview was conducted, biophysical measurements were performed and biospecimens were collected, processed and stored. Baseline investigations included fasting blood sugar, fasting lipid profile, renal profile and full blood count. From April 2006 to the end of September 2012 we recruited a total of 106 527participants. The baseline prevalence data showed 16.6% participants with diabetes, 46.5% with hypertension, 44.9% with hypercholesterolaemia and 17.7% with obesity. The follow-up phase commenced in June 2013. This is the most comprehensive and biggest cohort study in Malaysia, and has become a valuable resource for epidemiological and biological research. For information on collaboration and also data access, investigators can contact the project leader at ([email protected])

Cohort Profile: The Malaysian Cohort (TMC) project: a prospective study of non-communicable diseases in a multi-ethnic population

The Malaysian Cohort study was initiated in 2005 by the Malaysian government. The top-down approach to this population-based cohort study ensured the allocation of sufficient funding for the project which aimed to recruit 100 000 individuals aged 35–70 years. Participants were recruited from rural and urban areas as well as from various socioeconomic groups. The main objectives of the study were to identify risk factors, to study gene-environment interaction and to discover biomarkers for the early detection of cancers and other diseases. At recruitment, a questionnaire-based interview was conducted, biophysical measurements were performed and biospecimens were collected, processed and stored. Baseline investigations included fasting blood sugar, fasting lipid profile, renal profile and full blood count. From April 2006 to the end of September 2012 we recruited a total of 106 527participants. The baseline prevalence data showed 16.6% participants with diabetes, 46.5% with hypertension, 44.9% with hypercholesterolaemia and 17.7% with obesity. The follow-up phase commenced in June 2013. This is the most comprehensive and biggest cohort study in Malaysia, and has become a valuable resource for epidemiological and biological research. For information on collaboration and also data access, investigators can contact the project leader at ([email protected])