Primary imatinib resistance in chronic myeloid leukemia patients in a developing country: BCR-ABL kinase domain mutations or BCR-ABL independent mechanisms?

Clinical resistance to imatinib (IM) in chronic myeloid leukemia (CML) carries adverse consequences. We investigated 22 CML patients who developed IM-resistance for BCR-ABL kinase domain (KD) mutations. The median follow-up for this study was 101.9 months (range: 22.2 to 176.5 months) and the estimated mean overall survival was 150.87 months (95% CI: 130.0 to 171.0). Five out of 22 patients tested positive for BCR-ABL KD mutations: 2 had T315I, 2 had E255K and 1 had V289F mutations. Of the remaining 17 patients who did not harbor BCR-ABL KD mutations, 11 patients received nilotinib while the rest continued on IM. All 17 achieved haematological remission but only 5 patients achieved complete cytogenetic remission, 4 of whom did so after switching to nilotinib. Our study shows that most of our IM-resistant patients do not test positive for BCR-ABL KD mutations by available testing methods and the role of second generation tyrosine kinase inhibitors remains undetermined. A critical analysis of the BCR-ABL KD mutations and the underlying mechanisms/ pathways of BCR-ABL independent IM-resistance along with potential treatments in the horizon will be discussed.

The prevalence and distribution of Brucella melitensis in goats in Malaysia from 2000 to 2009.

A study was conducted to describe the prevalence and distribution of zoonotic Brucella melitensis in goats in Peninsular Malaysia. Using serosurveillance data of the last decade (2000-2009) involving 119,799 goats and 3555 farms, the seroprevalence of brucellosis among goats was 0.91% (95% CI=0.86-0.96) and among farms was 7.09% (95% CI=6.27-7.98). The odds of brucellosis was significantly (P<0.05) higher in the later part of the decade, in larger herd size and among the states located in the peninsula as compared to eastern Malaysia. The infection was detected throughout Malaysia but at generally low seroprevalences with states like Perlis that border neighbouring countries having higher seroprevalence of brucellosis than other non-border states.

Prenatal diagnosis of aneuploidies in amniotic fluid by multiple ligation-dependent probe amplification (MLPA) analysis.

Prenatal diagnosis is essential in the new era of diagnosis and management of genetic diseases in obstetrics. Multiple ligation-dependent probe amplification (MLPA) is a recent technique for prenatal diagnosis for the relative quantification of 40 different nucleic acid sequences in one single reaction. We had utilized the MLPA technique in detecting aneuploidies in amniotic fluid samples from 25 pregnant women from the Obstetrics and Gynaecology Department UKMMC, versus the quantitative fluorescent polymerase chain reaction (QF-PCR) method. Conclusive results were obtained in 18 cases and all were concordant with that of the QF-PCR. All four cases of trisomies were correctly identified including one case with maternal cell contamination.

Nasal type NK/T-cell lymphoma - diagnosis and treatment difficulties.

Primary nasal lymphomas are rare. One of the most common cellular subtypes in the Asian population is natural killer (NK)/T-cell lymphoma (NKTL) with a high association of EBV. We report a case of a 42-year-old female, who presented with a worsening sore throat, odynophagia, dysphagia to solid food due to oropharyngeal ulcers and bilateral nasal blockage and recurrent fever for the past two weeks prior to admission. Physical examination revealed ulcers over the soft palate with nasopharyngeal slough. Computerized Tomography (CT) scan of the neck showed nasopharyngeal abscess with bilateral maxillary ethnoidal sinusitis. The diagnostic and management challenge is discussed.

Isolation and molecular characterization of Brucella melitensis from seropositive goats in Peninsula Malaysia.

A study was carried out to isolate Brucella melitensis using established bacteriological and PCR techniques in Brucella seropositive goats in farms in Selangor, Negeri Sembilan, Melaka and Pulau Pinang. Brucella melitensis was isolated from 7 of 134 reactors with the highest isolation from the vaginal swabs (57.14%) followed by the spleen (28.57%), uterine fluid (14.29%). No Brucella was isolated from the lymph nodes. PCR confirmed all the seven isolates as B. melitensis and isolates were phylogenetically related to other isolates from India, Iran, and Israel but most closely related to isolates from Singapore.

Prevalence of iron deficiency anaemia and thalassaemia trait among undergraduate medical students.

BACKGROUND: Anaemia is a global health problem including Malaysia. In adults, anaemia may affect work productivity. Iron deficiency anaemia and thalassaemia are common causes of anaemia in Malaysia. However, there is scarcity of data on national prevalence of iron deficiency anaemia and thalassaemia, especially in young adults. This cross sectional study was performed to determine the prevalence of iron deficiency anaemia and thalassaemia among medical students of Universiti Kebangsaan Malaysia Medical Centre (UKMMC). MATERIALS AND METHODS: Blood samples collected in EDTA tubes were analyzed for haemoglobin level and red cell parameters such as MCV, MCH and red cell counts. Samples with abnormal red cell indices were sent for analysis of RBC morphology, iron status, haemoglobin analysis and DNA analysis. RESULTS: A total of 400 samples were available for this study. Fifty-eight (14.5%) students had hypochromic microcytic red cell indices in which 44 (11%) showed thalassaemia red cell indices while 14 (3.5%) had iron deficiency red cell indices which were finally confirmed by serum iron/TIBC analysis. Amongst those suspected to have thalassaemia, 12 (27.3%) were confirmed as alpha thalassaemia trait (αα/--(SEA)), 11 (25%) as Haemoglobin-E trait, 8 (18.2%) as beta thalassaemia trait and 2 (4.5%) as Haemoglobin Constant Spring (αα/α(CS)α). However, eleven students (25%) with thalassaemia red cell indices could not be confirmed with the common thalassaemia primers available, thus causes have yet to be established. CONCLUSION: Our prevalence of thalassaemia was high and thus we opine that better screening methods should be adopted.

A study of JAK2 (V617F) gene mutation in patients with chronic myeloproliferative disorders.

BACKGROUND AND AIMS: Chronic myeloproliferative diseases (MPDs) are heterogenous group of haematological malignant disorders. It is now a well recognized fact that the JAK2 (V617F) mutation occurs in majority of the patients with polycythaemia vera (PV) and half of those with myelofibrosis and essential thrombocythaemia. The presence of JAK2 (V617F) mutation is considered an important criterion for the exclusion of secondary-reactive from clonal disorders. In the present uni-institutional study, we analyzed the JAK2 (V617F) mutation status in the ethnic Malay and Chinese patients who were diagnosed as MPDs. MATERIALS AND METHODS: The study was performed on known cases of chronic MPDs either at diagnosis or during the follow-up. A total of 45 cases were studied with informed consent. The allele specific PCR, ARMS-PCR and RQ-PCR methods were used. RESULTS: The frequency of the JAK2 (V617F) mutation varied between the MPD subtypes, with the mutation being most frequent in PV (95.8%) and 39% showed homozygous mutant allele. The mutation was detected in 52.9% cases of ET, of which 36.4% were homozygous for the mutant allele and 1 case of MF was homozygous for the mutant allele. CONCLUSION: Screening for the mutation in all cases suspected of chronic MPD could be beneficial in differentiating patients with reactive erthrocytosis or thrombocytosis from the true clonal MPDs especially polycythaemia vera.

Molecular study and genotype/phenotype correlation of ß Thalassemia in Malaysia.

INTRODUCTION: To study the ß-gene mutations spectrum, the genotype/phenotype correlation, the modulatory effect of co-inherited factors such as α-gene mutations and of Xmn1 polymorphism in a large cohort of Malaysian patients. METHODS: A total of 264 cases clinically diagnosed as Thalassemia major (TM) (111), Thalassemia intermedia (21), HbE-ß Thalassemia (131), and 1 HbE homozygous were studied. The detection of α and ß gene mutations and characterization of Xmn1 polymorphism were performed by multiplex PCR, amplification refractory mutation system (ARMS), DNA sequencing, and restriction fragment length polymorphism (RFLP)-PCR. RESULTS: A total of 19 ß Thalassemia mutations were characterized. CD26 and CD41/42 were the most common found in the Malay and Chinese population, respectively. The sensitivity of the clinical diagnosis for ß TM, thalassemia intermedia, and HbE/ß thalassemia was 94.0%, 15.2%, and 89.2%, respectively. Patients with Xmn1 heterozygosity [+/-] required less frequent transfusion compared with those without the polymorphism. Co-inheritance of α-thalassemia alleviates the severity of HbE-ß thalassemia in our cohort. CONCLUSION: Molecular analysis should be used for a better diagnosis and management of ß thalassemia.

Isolation and molecular characterization of Brucella melitensis from seropositive goats in Peninsula Malaysia

A study was carried out to isolate Brucella melitensis using established bacteriological and PCR techniques in Brucella seropositive goats in farms in Selangor, Negeri Sembilan, Melaka and Pulau Pinang. Brucella melitensis was isolated from 7 of 134 reactors with the highest isolation from the vaginal swabs (57.14%) followed by the spleen (28.57%), uterine fluid (14.29%). No Brucella was isolated from the lymph nodes. PCR confirmed all the seven isolates as B. melitensis and isolates were phylogenetically related to other isolates from India, Iran, and Israel but most closely related to isolates from Singapore.

Real-time quantification for BCR-ABL transcripts in chronic myeloid leukaemia patients in UKMMC, Malaysia.

Molecular pathogenesis of chronic myeloid leukemia (CML) is well established and molecular monitoring for patients with CML has become an important practice in the management of patients on imatinib therapy. In the present study, we report the use of RQ-PCR method for detection of BCR-ABL fusion gene for our CML cases. We performed a two-step RQ-PCR on bone marrow aspirates or peripheral blood of 37 CML patients. Quantitative expression of BCR-ABL fusion gene was carried out relative to the expression of a housekeeping gene as endogenous control to compensate for uneven cell numbers, RNA quality, or variations in reverse transcription efficiencies. Twenty-four of these patients were pre-treated with hydroxyurea or alpha interferon prior to the imatinib therapy. Their BCR-ABL fusion gene levels were monitored for 18 months. All samples processed were evaluable. The PCR amplification efficiency of the ABL gene is 90.5% (0.2158) and the BCR-ABL gene, 93.4% (0.1573).

G6PD enzyme activity in normal term Malaysian neonates and adults using a OSMMR2000-D kit with Hb normalization.

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is one of the commonest causes of neonatal jaundice in Malaysia. Screening of cord blood for G6PD deficiency by the semiquantitative fluorescent spot test (FST) is performed in Malaysia but this test can miss cases of partial G6PD deficiency. The OSMMR-D kit assay measures G6PD activity and hemoglobin (Hb) concentration, allowing direct expression of results in U/gHb. We evaluated this method and established the normal range for G6PD activity in normal term neonates and adults. EDTA blood from 94 neonates and 295 adults (age 15-59 years old) with normal Hb and FST were selected. The normal means for G6PD activity for neonates and adults were 12.43 +/- 2.28 U/gHb and 9.21 +/- 2.6 U/gHb, respectively; the reference ranges for normal G6PD activity in neonates and adults were 10.15-14.71 U/gHb and 6.61-11.81 U/gHb respectively. There were no significant differences in mean normal G6PD activity between the Malays and Chinese racial groups or between genders. The upper and lower limit cut-off points for partial deficiency in neonates were 7.4 U/gHb (60% of the normal mean) and 2.5 U/gHb (20% of the normal mean), respectively. For adults, the upper and lower limit cut-off points for partial deficiency in adults were 5.52 U/gHb (60% of the normal mean) and 1.84 U/gHb (20% of the normal mean), respectively. The quantitation of G6PD enzymes using this OSMMR-D kit with Hb normalization was simple since the Hb was analyzed simultaneously and the results were reproducible with a CV of less than 5%.

Non-secretory multiple myeloma with diagnostic challenges.

Non-secretory multiple myeloma (NSMM) is a rare variant of the classic form of multiple myeloma (MM). In NSMM, no monoclonal gammopathy can be detected in serum or urine by conventional techniques, making the diagnosis more difficult. We describe a 71-year-old man who had been diagnosed and treated for granulocytic sarcoma one year prior to his recent problems of progressive low-back pain of two months duration. Skeletal X-rays showed diffuse osteolytic lesions with multiple pathological fractures but there was no monoclonal gammopathy in the serum or urine. The biopsy of the lytic lesion on the upper part of the femur showed infiltration by abnormal plasma cells. A diagnosis of NSMM was made and he was treated with chemotherapy. The early diagnostic difficulty and the challenges faced regarding the case are discussed.

Juvenile myelomonocytic leukaemia: a case series.

Juvenile myelomonocytic leukaemia (JMML), previously known as juvenile chronic myeloid leukaemia (JCML) is a rare, myelodysplastic - myeloproliferative disease typically presenting in early childhood. This disorder is difficult to distinguish from other myeloproliferative syndrome such as chronic myeloid leukaemia (CML) because of the similarities in their clinical and bone marrow findings. However, because of its unique biological characteristics such as absolute monocytosis with dysplasia, absence of Philadelphia chromosome or BCR-ABL fusion protein, hypergammaglobulinaemia and raised fetal haemoglobin level, this disorder does not satisfy the criteria for inclusion in the CML or chronic myelomonocytic leukaemia (CMML) group, as seen in adult patients. We describe a series of three patients with JMML, who had almost similar clinical and laboratory findings, and discuss the difficulty in the classification and treatment of the disease.

Assessment of P-gp and MRP1 activities using MultiDrugQuant Assay Kit: a preliminary study of correlation between protein expressions and its functional activities in newly diagnosed acute leukaemia patients.

Multidrug resistance (MDR) is believed to be responsible for poor response of patients towards chemotherapy particularly patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL). The best-characterized resistance mechanism is the one mediated by permeability-glycoprotein (P-gp) encoded by MDR1 gene, which is responsible for drug efflux. We studied P-gp and multidrug resistance-associated protein 1 (MRP1) expression and functional activities in 43 newly diagnosed acute leukemia cases (19 paediatric ALL cases and 24 adult AML cases). The expression and functional activities were examined using flow cytometry and MultiDrugQuant assay kit (involving calcein AM uptake and efflux). P-gp and MRP1 expression and its functional activities were observed in 68.4% of paediatric ALL. In adult AML cases, all cases expressed MRP1 and its functional activities but only 58.3% were positive for P-gp and its functional activities. We were able to show a significant correlation between the expression of the multidrug resistant protein (P-gp and MRP1) and their functional activity in adult AML and paediatric ALL samples.

Rapid detection of the UGT1A1 single nucleotide polymorphism G211A using real-time PCR with Taqman minor groove binder probes.

The UGT1A1 Taqman MGB probe single nucleotide polymorphism (SNP) genotyping assay was developed to detect nucleotide 211 of the UDP-glucoronocyltransferase 1A1 (UGT1A1) gene. Defects in this enzyme interfere with process of conjugation of bilirubin and cause unconjugated hyperbilirubinemia. Variation at nucleotide 211 in the coding region of the UGT1A1 gene has been shown to be prevalent in Japanese and Chinese. Using an ABI sequence detection system (SDS) 7000, an allele-specific real-time PCR-based genotyping method was established to detect nucleotide G211A. Cord blood from 125 infants without hyperbilirubinemia (controls) were compared with cord blood from 74 infants (cases) with severe hyperbilirubinemia (total serum bilirubin > 300 micromol/L). Homozygous variation of the UGT1A1 gene at nucleotide 211(A/A) is significantly more common in cases (14.9%) than in controls (0.8%) (P<0.001). Direct sequencing from 20 randomly selected samples showed eight samples with homozygous wild type, seven with homozygous variant, and five samples were heterozygous. The result from this assay was in complete concordance with the DNA sequencing result and clearly discriminate wild-type (G/G), homozygous variant (A/A), and heterozygous (G/A). This assay is rapid and robust for screening of SNP G211A to determine if this polymorphism plays a role in causing severe neonatal jaundice in the local context.

Expression of multidrug resistance (MDR) proteins and in vitro drug resistance in acute leukemias.

The expression of the multidrug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 +/- 2.320) compared to adult AML samples (0.021 +/- 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 +/- 0.529) compared to childhood ALL samples (0.100 +/- 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 +/- 1.760) and ara-C (1.310 +/- 2.390), which was higher compared to childhood AML samples (0.129 +/- 0.214 and 0.210 +/- 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.

Transient abnormal myelopoeisis in newborns with Down syndrome.

Transient abnormal myelopoeisis (TAM) is a haematological phenomenon commonly seen in newborns with Down syndrome. Although the majority show spontaneous resolution, this condition should not be dismissed too readily as there have been associated fatalities. Furthermore, even for those who do show spontaneous resolution, a significant percentage will develop acute megakaryoblastic leukaemia within the next few years of life. We report a series of four patients with TAM who presented with hepatosplenomegaly and leucocytosis detected on preliminary investigations.

Assessing donor chimerism using flow cytometry in paroxysmal nocturnal haemoglobinuria after stem cell transplantation--a case report.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired haemopoietic stem cell disorder arising from somatic mutation of the X-linked PIG-A gene which leads to deficiency of the glycosylphosphatidylinositol (GP1) membrane anchor proteins such as CD 59 (MIRL: membrane inhibitor of reactive lysis) and CD 55 (DAF: decay accelerating factor). Allogeneic peripheral blood stem cell transplant (PBSCT) is a curative mode of treatment in symptomatic PNH patients. Assessment of donor chimerism for PBSCT can be performed by various methods including short tandem repeat loci (STR) and variable number of tandem repeats (VNTR). Flow cytometry, which is much cheaper and faster, also can be used to assess engraftment in patients with PNH. Engrafted patients will show the presence of CD 55 and CD 59 on their red cells and white cells. We describe here the usefulness of flow cytometry in the assessment of donor chimerism following allogeneic PBSCT, in a case of PNH.

Comparison of myeloperoxidase detection by flow cytometry using two different clones of monoclonal antibodies.

INTRODUCTION: Myeloperoxidase (MPO) is present in azurophilic granules which appear in the promyelocyte stage of differentiation and is expressed in granulomonocytic cells. MPO is usually detected by cytochemistry. The demonstration of peroxidase in at least 3% of bone marrow blasts defines an acute leukaemia as acute myeloblastic leukaemia (AML). MPO is important in distinguishing acute myeloblastic leukaemia (AML) from acute lymphoblastic leukaemia (ALL). It is difficult to diagnose AML with minimal evidence of myeloid differentiation (AML- M0) by conventional light microscopy. However, these AML-M0 blasts can be detected by monoclonal antibodies. Anti-MPO recognizes the enzymatically inactive precursor forms of MPO. There are a few commercially available monoclonal antibodies against MPO. In this study, we evaluated two monoclonal antibodies against MPO from different commercial sources. METHODS: Anti-MPO were purchased from Dako (Denmark) and Becton Dickinson, BD (California, USA). MPO detection was done using the permeabilisation-staining technique, followed by analysis with flow cytometer (FASCalibur, California, USA). RESULTS: 63 cases of acute leukaemias (38 ALL and 25 AML) were studied. Anti-MPO by Dako showed that 12/38 (31.6%) of ALL cases were positive, but all these cases were clear-cut negative for anti-MPO from BD. 24/38 (63.2%) of these ALL cases were associated with aberrant expression of myeloid antigens. However, only 8/24 (33.3%) cases with aberrant myeloid antigen expression showed positive reaction to anti-MPO (Dako). 23/25 (92%) of AML showed concordance results for both anti-MPO by Dako and BD. CONCLUSION: Anti-MPO is a useful and reliable marker for the diagnosis of AML. However, this study had demonstrated that results vary with the monoclonal antibody used in ALL cases. Anti-MPO (Dako) had shown false positive result in 31.6% of ALL cases whereas anti-MPO (BD) had shown consistent negative result in ALL cases.

Allogeneic haemopoietic stem cell transplantation using non-myeloablative conditioning--a local experience.

Allogeneic haemopoietic stem cell transplantation was initially considered as a means of delivering supralethal doses of chemotherapy with or without total body irradiation for the treatment of malignancy. However, it has become clear that this mode of therapy does not eradicate the malignancy in many patients and its benefit is largely due to the immune mediated graft versus malignancy effect. This has led to development of alternative strategy to utilize a less intensive preparative regimen pre-transplantation that provides sufficient immunosuppression to achieve engraftment of an allogeneic stem cell graft, thus allowing the evolution of a graft versus malignancy effect post-transplantation. Since September 1999, we had carried out 10 cases of allogeneic peripheral blood stem cell transplantation: one case of aplastic anaemia, four cases of acute myeloid leukemia (AML) in first remission, and five cases of chronic myeloid leukemia (CML) in chronic phase. The preparative regimen was non-myeloablative comprising Fludarabine with Cyclophosphamide or Busulphan. Recovery from transplantation was rapid with no or brief period of neutropenia or thrombocytopenia. Engraftment was established by determining donor's short tandem repeats in the recipient's bone marrow at day 30, 60 and 100 post-transplantation. Seven cases (70%) show partial or complete donor's chimerism by day 30 indicating successful engraftment. No treatment mortality was noted at day 100. Graft versus host disease was generally limited. Up to the date of reporting, two patients with CML had graft failure, one was successfully re-transplanted later. Two patients with AML had since relapsed and passed away. The others remain alive and well. The cost of transplantation on average was estimated to be about a quarter of that using a myeloablative regimen. It appears that this treatment strategy is a promising approach for the management of blood disorders.