Chao-Sung Chang

Epigenetic alteration of the SOCS1 gene in chronic myeloid leukaemia.

Summary.  The expression of the suppressor of cytokine signalling‐1 (SOCS1) protein is induced in response to stimulation by several cytokines. The induced SOCS1 inhibits the signalling pathway through the association with a variety of tyrosine kinase proteins. In this study, the mutation analyses, CpG island methylation status, and the expression of the SOCS1 gene in 112 chronic myeloid leukaemia (CML) samples, five leukaemia cell lines, and 30 normal controls were analysed. No genetic mutations of SOCS1 gene were noted in the CML samples. The SOCS1 gene was hypermethylated in 67% and 46% of the blastic and chronic phase CML samples respectively (P < 0·0001). However, there was no methylation of the SOCS1 gene in normal controls or CML in molecular remission. The methylation status of the SOCS1 gene is consistent with the results of the real‐time quantitative reverse transcription polymerase chain reaction and immunocytochemistry staining. Our results demonstrate that the SOCS1 gene silencing is caused by the methylation of CpG islands in CML and is reversed to an unmethylated status in molecular remission. As SOCS1 has universal activity to negatively regulate several cytokine signalling pathways, the loss of the negative regulation of cytokine signalling by the SOCS1 may play a role in the pathogenesis of CML progression.

Altered expression of circadian clock genes in human chronic myeloid leukemia.

Circadian clock genes use transcriptional-translational feedback loops to control circadian rhythms. Recent studies have demonstrated that expression of some circadian clock genes displays daily oscillation in peripheral tissues including peripheral blood and bone marrow. Circadian rhythms regulate various functions of human body, and the disruption of circadian rhythm has been associated with cancer development and tumor progression. However, the direct links between aberrant circadian clock gene expression and human disorders remain largely unknown. In this study, comparisons were made between the expression profiles of 9 circadian clock genes from peripheral blood mononuclear cells (PBMCs) and polymorphonuclear cells (PMNs) from 18 healthy volunteers. Peripheral blood (PB) total leukocytes from 54 healthy volunteers and 95 patients with chronic myeloid leukemia (CML) were also investigated. Similar expression profiles of all 9 circadian clock genes were observed in PBMCs and PMNs of healthy individuals. In PB total leukocytes of healthy individuals, the daily pattern of PER1, PER2, PER3, CRY1, CRY2, and CKIε expression level peaked at 0800 h, and BMAL1 peaked at 2000 h. Daily pattern expression of these 7 genes was disrupted in newly diagnosed pre—imatinib mesylate—treated and blast crisis—phase patients with CML. Partial daily pattern gene expression recoveries were observed in patients with CML with complete cytogenetic response and major molecular response. The expression of CLOCK and TIM did not show a time-dependent variation among the healthy and patients with CML. These results indicate a possible association of the disrupted daily patterns of circadian clock gene expression with the pathogenesis of CML.

Dihydropyrimidine dehydrogenase pharmacogenetics in the Taiwanese population.

Background/purpose5-Fluorouracil (5-FU) remains the most frequently used chemotherapy agent in various human cancers. Over 80% of the 5-FU administered is metabolized by dihydropyrimidine dehydrogenase (DPD) in the liver. However, mutations in the DPD gene have been found to be associated with low DPD activity causing severe complications. The aim of this study was to determine the frequency of 11 known mutations in Taiwanese subjects and the relationship between mutation and DPD level.MethodsSamples from a total of 300 subjects were investigated in this study. The PCR-RFLP method was used to identify 11 mutations of the DPYD gene, including 62G>A, 74A>G, 85T>C (DPYD*9A), 812delT, 1003G>T, 1156G>T, 1627A>G (DPYD*5), 1714C>G, 1897delC (DPYD*3), 2194G>A (DPYD*6), and IVS14+1G>A (DPYD*2A). DPD protein levels were determined using a DPD ELISA kit.ResultsFour mutations, including 74A>G, 85T>C (DPYD*9A), 1627A>G (DPYD*5), and 2194G>A (DPYD*6), were found in our 300 samples. The following mutations were not detected: 62G>A, 812delT, 1003G>T, 1156G>T, 1714C>G, 1897delC (DPYD*3), and IVS14+1G>A (DPYD*2A). The phenotype analysis by DPD protein level indicated that the 1627A>G (DPYD*5) mutation was not associated with the DPD protein level and might be a polymorphism in the DPD gene. The DPD level was also not correlated with gender.ConclusionNo significant correlations between these 11 mutations and DPD protein level were found indicating that examination of these mutations is insufficient to provide a high-value prediction of the 5-FU pharmacogenetic syndrome in Taiwanese. Genotype and phenotype analysis indicated the 1627A>G (DPYD*5) mutation to be a polymorphism.

Hematological Aspects of Dengue Fever

Fifty patients with dengue fever from Sept. 1987 to Jan. 1988 were studied for hematological features. The lowest blood counts values throughout the course of illness were Hb: 13.2 +/- 1.9 gm/dl, WBC: (2.77 +/- 1.63) x 10(3)/mm3, Platelet: (8.7 +/- 5.5) x 10(4)/mm3. Leukopenia (WBC less than 4000/mm3) was present in 38 (76%) of the cases and thrombocytopenia (platelet less than 10 x 10(4)/mm3) in 27 (54%) of the cases. Leukocytes reached nadir (1000-2000/mm3) at the 5th-6th day after fever onset, thrombocytes reached nadir (2.0-5.0 x 10(4)/mm3) at the 5th-7th day after fever onset. Bone marrow studies showed mild hypocellularity in the acute stage (less than 1 week) and normal cellularity in the convalescent stage (greater than 1 week). Megakaryocytes increased with various stages of maturation of megakaryocytes appearing in a majority of patients. Nuclear vacuolization of megakaryocytes could also be found. Bone marrow CFU-GM when performed within one week of illness showed no growth or low colony count, and was nearly normal after one week of fever onset. This study may suggest that leukopenia in dengue fever may be caused by virus-induced destruction or inhibition of myeloid progenitor cells. Thrombocytopenia may result from by destruction of peripheral platelet or bone marrow megakaryocytes by viruses which consequently reduce the platelet production.

Detection of the JAK2 V617F missense mutation by high resolution melting analysis and its validation.

BACKGROUND Janus kinase 2 (JAK2) is a tyrosine kinase involved in the cytokine signaling of several growth factors such as erythropoietin and thrombopoietin in normal and neoplastic cells. The G to T exchange at nucleotide 1849 in exon 14 of the JAK2 gene leads to a substitution of valine with phenylalanine at the amino acid position 617 (V617F) of the JAK2 protein. Currently, the occurrence of the JAK2 V617F mutation is well recognized in chronic myeloproliferative disorders (MPDs). METHODS We identified JAK2 V617F missense mutation in patients with MPD by high resolution melting (HRM) analysis. HRM analysis is a new gene scan tool that quickly performs the PCR and identifies sequences alterations without requiring post-PCR treatment. This study included 7 PV patients (41.1%), 6 ET patients (35.3%), and 4 myelofibrosis patients (23.5%). Additionally, our methodology was compared with amplification refractory mutation system (ARMS) assay. RESULTS Up to 5% of the JAK2 V617F mutation was successfully detected in patients with MPD using HRM analysis. Eleven out of 17 patients (64.7%) were positive for the presence of JAK2 V617F mutation. The prevalence of mutation in the different subtypes of MPDs was 85.7% in PV (6 of 7 patients), 66.7% in ET (4 of 6) and 5.9% in myelofibrosis (1 of 4). The results proved 100% comparable to those obtained by ARMS assay. CONCLUSIONS The HRM analysis is a rapid and effective technique for the detection of JAK2 V617F missense mutation.

Comparison of a Combination Ferrous Fumarate Product and a Polysaccharide Iron complex as Oral Treatments of Iron Deficiency Anemia: A Taiwanese Study

Despite efforts to improve iron supplements for iron deficiency anemia, there is no consensus on products that balance efficacy, safety and tolerability, and cost. Ferrous products are effective, but they are associated with more gastrointestinal side effects than ferric products. Ferric products tend to have lower absorption. We present results from a 12-week study that ran domized 72 people with uncomplicated iron deficiency anemia to receive a ferrous iron supplement (Ferall, a combination of ferrous fumarate with ascorbic acid, folic acid, and cyanocobalamin) or a ferric iron polysaccharide complex (Niferex, ferro-glycine sulfate) plus ascorbic acid. The ferrous product was significantly more effective, the primary and secondary endpoints including changes in levels of hemoglobin and serum ferritin. There was a slightly higher frequency of gastrointestinal side effects in patients taking the ferrous product, but both supplements were well tolerated. No participant withdrew from the study because of side effects. We concluded that the ferrous product is safe and effective for use in uncomplicated iron deficiency anemia. The lack of direct comparison between single-agent ferrous fumarate and the combination ferrous product limited interpretation of results in terms of possible effects due to other components, such as ascorbic acid.

β-thalassemia major evolution from β-thalassemia minor is associated with paternal uniparental isodisomy of chromosome 11p15

β-thalassemia major can be caused by homozygosity or compound heterozygosity for β-globin gene mutations (HBB gene). Most cases are inherited from parents who both have diseased alleles of the HBB gene. We report a patient with late-onset β-thalassemia major that evolved from β-thalassemia minor in which only one of her parents had the diseased HBB gene. To study the cause of β-thalassemia major in this patient, we performed the 100K single nucleotide polymorphism genotyping assay, fluorescence in situ hybridization, and DNA methylation analysis of the imprinting genes near the HBB gene. The results showed a loss of heterozygosity in the region of chromosome 11p14.3 to 11p15.5, which perfectly matched one allele of her father. Our study demonstrates that paternal uniparental isodisomy of chromosomal 11p15.5 is associated with the β-thalassemia major in this patient. Key words: β-thalassemia major, uniparental isodisomy, mosaicism.

SUV on dual-phase FDG PET/CT correlates with the Ki-67 proliferation index in patients with newly diagnosed non-Hodgkin lymphoma.

Purpose PET using 18F-FDG integrated with CT is beneficial for staging patients with non-Hodgkin lymphoma (NHL). The Ki-67 index is used to assess the proliferation potential of tumor cells. The aim of this study was to evaluate the correlation of the Ki-67 index in tissue samples with the SUV at different sites on dual-phase FDG PET/CT of patients with newly diagnosed NHL. Materials and Methods From September 2009 to March 2011, patients with newly diagnosed NHL who had received dual-phase FDG PET/CT for staging and biopsy samples that were evaluated for the Ki-67 expression were enrolled. The SUVmax of the biopsy site, the tumorous lesion sites, and 3 different bone marrow sites (right iliac crest, sternum, and L1) were measured. The SUVmean of the liver and spleen were also measured. Results There were a total of 27 patients in this study. Significant correlations were observed between the Ki-67 index and the SUVmax of the right iliac crest in patients with early-stage disease (stage I and II) patients, the SUVmax of the biopsy and whole-body lesion sites in patients with late-stage disease (stage III and IV), and the retention index of SUVmax of the right iliac crest in patients whose bone marrow were involved by lymphoma cells. Conclusions For patients with newly diagnosed NHL, the significant correlation between the Ki-67 index and the SUV in this study suggests that dual-phase FDG PET/CT may be used as a noninvasive measurement of tumor proliferation.

Cytomegalovirus infection and disease after allogeneic hematopoietic stem cell transplantation: experience in a center with a high seroprevalence of both CMV and hepatitis B virus

Cytomegalovirus (CMV) infection and disease are important concerns after allogeneic hematopoietic stem cell transplantation (allo-HSCT). The similarity of hepatitis B virus (HBV) and CMV with regards to their chronic viral persistence and potential reactivation at the time of impaired cellular immunity has raised clinicians’ interest in the occurrence and association between them among patients receiving allo-HSCT; however, only limited data have been obtained from a high seroprevalence region of both CMV and HBV. We monitored 117 adult allo-HSCT patients with both CMV polymerase chain reaction and pp65 antigenemia assay weekly until day 100. In 91.8% of our cases, donors and recipients were both CMV seropositive, and 13.7% of the patients were positive for HBV surface antigen. The incidences of CMV infection and disease were 45.3% and 6.8%, respectively. Grade II–IV acute graft-versus-host disease and anti-thymocyte globulin-containing conditioning regimen were associated with an increased risk of CMV infection in a multivariate analysis (hazard ratio 3.02, 95% CI 1.68–5.42, p < 0.001 and hazard ratio 5.29, 95% CI 2.57–10.8, p < 0.001). No survival disadvantage was found in patients who developed CMV infection (p = 0.699) and CMV disease (p = 0.093). No clinically significant HBV reactivation was found, and the underlying HBV infection in donors or recipients before allo-HSCT did not increase the risk of CMV infection and CMV disease and did not influence survival after allo-HSCT.

Association of Endostatin D104N with Leukemia

The bone marrow and/or peripheral blood from 126 patients with acute myeloid leukemia (AML), 57 with chronic myeloid leukemia (CML), 91 with acute lymphocytic leukemia (ALL), and 178 normal controls were analyzed using a polymerase chain reaction‐restriction fragment length polymorphism (RFLP) assay to evaluate the association of the endostatin polymorphisms D104N (nucleotide 4349G→A) with leukemia. In the 178 normal Taiwanese, the allele frequency of 4349G was 98% (348/356) and that of 4349A was 2% (8/356). The frequencies of homozygous 4349G (104D/D) and heterozygous 4349G/A (104D/N) were 95. 5% (170/178) and 4.5% (8/178), respectively. However, no individuals were homozygous 4349A (104N/N). Among the leukemia patients, 124/126 with AML (98.4%), 55/57 with CML (94.9%), and 89/91 with ALL (97.9%) were homozygous 4349G. In addition, 2/126 with AML (1.6%), 2/57 with CML (5.1%), and 2/91 with ALL (2.1%) were heterozygous 4349G/A. No patients were homozygous 4349A. Similar frequencies of endostatin polymorphisms were observed in leukemic patients and normal controls. This suggests that the endostatin polymorphism is not associated with the risk of leukemia.