Improved Therapeutic Effect against Leukemia by a Combination of the Histone Methyltransferase Inhibitor Chaetocin and the Histone Deacetylase Inhibitor Trichostatin A

SUV39H1 is a histone 3 lysine 9 (H3K9)-specific methyltransferase that is important for heterochromatin formation and the regulation of gene expression. Chaetocin specifically inhibits SUV39H1, resulted in H3K9 methylation reduction as well as reactivation of silenced genes in cancer cells. Histone deacetylase (HDAC) inhibitors inhibit deacetylases and accumulate high levels of acetylation lead to cell cycle arrest and apoptosis. In this study, we demonstrated that treatment with chaetocin enhanced apoptosis in human leukemia HL60, KG1, Kasumi, K562, and THP1 cells. In addition, chaetocin induced the expression of cyclin-dependent kinase inhibitor 2B (p15), E-cadherin (CDH1) and frizzled family receptor 9 (FZD9) through depletion of SUV39H1 and reduced H3K9 methylation in their promoters. Co-treatment with chaetocin and HDAC inhibitor trichostatin A (TSA) dramatically increased apoptosis and produced greater activation of genes. Furthermore, this combined treatment significantly increased loss of SUV39H1 and reduced histone H3K9 trimethylation responses accompanied by increased acetylation. Importantly, co-treatment with chaetocin and TSA produced potent antileukemic effects in leukemia cells derived from patients. These in vitro findings suggest that combination therapy with SUV39H1 and HDAC inhibitors may be of potential value in the treatment of leukemia.

Treatment outcome of all-trans retinoic acid/anthracycline combination chemotherapy and the prognostic impact of FLT3/ITD mutation in acute promyelocytic leukemia patients

BACKGROUND: All-trans retinoic acid (ATRA)/anthracycline chemotherapy is beneficial in newly diagnosed acute promyelocytic leukemia (APL); however, it is important to identify patients with high-risk disease to increase the cure rate. We investigated the outcome of ATRA/anthracycline chemotherapy and clinicobiological correlations of FLT3/ITD and NPM1 mutations in APL patients. METHODS: Induction therapy included oral ATRA (45 mg/m2/day) and idarubicin (12 mg/m2/day, intravenous, on days 2, 4, and 6). Patients achieving complete remission (CR) received 3 courses of ATRA combined with reinforced consolidation therapy. Mutations were analyzed using GeneScan and polymerasae chain reaction assays of bone marrow samples obtained from patients at diagnosis. RESULTS: Forty-five (84.9%) of 53 eligible patients achieved CR. The overall relapse rate was 8.9%, and the 3-year overall survival (OS) and leukemia-free survival (LFS) were 84.9+/-4.9% and 77.5+/-6.0%, respectively. The NPM1 mutation was not found in any patient, while the FLT3/ITD mutation was found in 10 (20.0%) patients. Of the FLT3/ITD+ patients, 80% belonged to the high-risk group, defined according to the presenting WBC and platelet counts. Among the patients who achieved CR, those who were FLT3/ITD+ had a higher relapse rate than those FLT3/ITD-. FLT3/ITD+ patients also had a significantly lower 3-year LFS than FLT3/ITD- patients. Multivariate analysis of the LFS showed that the FLT3/ITD mutation was independently associated with a shorter overall LFS, after adjusting for pretreatment risk stratification. CONCLUSION: This study investigated the clinical outcome of newly diagnosed APL patients treated with ATRA/anthracycline chemotherapy. Patients carrying the FLT3/ITD mutation had more aggressive clinical features and a poorer clinical outcome.

DNA Methylation Changes Following 5-azacitidine Treatment in Patients with Myelodysplastic Syndrome

DNA methyltransferase inhibitor, 5-azacitidine (AC) is effective in myelodysplastic syndromes (MDS) and can induce re-expression in cancer. We analyzed the methylation of 25 tumor suppressor genes in AC-treated MDS. Hypermethylation of CDKN2B, FHIT, ESR1, and IGSF4 gene was detected in 9/44 patients. In concordance with the clinical response, a lack of or decreased methylation in 4 patients with hematologic improvements and persistent methylation in 4 others with no response was observed. The mRNA expression of CDKN2B, IGSF4, and ESR1 was significantly reduced in MDS. Our results suggest that methylation changes contribute to disease pathogenesis and may serve as marker to monitor the efficacy of treatments.

A Genome-Wide Study of Moyamoya-Type Cerebrovascular Disease in the Korean Population

OBJECTIVE: Structural genetic variation, including copy-number variation (CNV), constitutes a substantial fraction of total genetic variability, and the importance of structural variants in modulating susceptibility is increasingly being recognized. CNV can change biological function and contribute to pathophysiological conditions of human disease. Its relationship with common, complex human disease in particular is not fully understood. Here, we searched the human genome to identify copy number variants that predispose to moya-moya type cerebrovascular disease. METHODS: We retrospectively analyzed patients who had unilateral or bilateral steno-occlusive lesions at the cerebral artery from March, 2007, to September, 2009. For the 20 subjects, including patients with moyamoya type pathologies and three normal healthy controls, we divided the subjects into 4 groups : typical moyamoya (n=6), unilateral moyamoya (n=9), progression unilateral to typical moyamoya (n=2) and non-moyamoya (n=3). Fragmented DNA was hybridized on Human610Quad v1.0 DNA analysis BeadChips (Illumina). Data analysis was performed with GenomeStudio v2009.1, Genotyping 1.1.9, cnvPartition_v2.3.4 software. Overall call rates were more than 99.8%. RESULTS: In total, 1258 CNVs were identified across the whole genome. The average number of CNV was 45.55 per subject (CNV region was 45.4). The gain/loss of CNV was 52/249, having 4.7 fold higher frequencies in loss calls. The total CNV size was 904,657,868, and average size was 993,038. The largest portion of CNVs (613 calls) were 1M-10M in length. Interestingly, significant association between unilateral moyamoya disease (MMD) and progression of unilateral to typical moyamoya was observed. CONCLUSION: Significant association between unilateral MMD and progression of unilateral to typical moyamoya was observed. The finding was confirmed again with clustering analysis. These data demonstrate that certain CNV associate with moyamoya-type cerebrovascular disease.

Prognostic significance of nucleophosmin mutations and FLT3 internal tandem duplication in adult patients with cytogenetically normal acute myeloid leukemia

BACKGROUND: Nucleophosmin (NPM1) gene and fms-like tyrosine kinase 3 gene-internal tandem duplication (FLT3-ITD) mutations are the most frequent mutations in patients with cytogenetically normal (CN)-AML. We analyzed the prognostic impact of these mutations and their interactions in adults with CN-AML. METHODS: NPM1 mutation (NPM1mut) and FLT3-ITD mutation (FLT3-ITD+) were analyzed by GeneScan and PCR assays of bone marrow samples obtained from 121 adult patients with CN-AML (age< or =60 years at diagnosis). RESULTS: The incidence of FLT3-ITD+ was higher in the NPM1mut group than in the wild-type NPM1 gene (NPM1wt) group. The patients were divided according to their mutation status into the NPM1mut/FLT3-ITD (isolated NPM1mut), NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD-, and NPM1wt/FLT3-ITD+ (isolated FLT3-ITD+) groups. The isolated NPM1mut group showed significantly better clinical outcomes in terms of relapse rate, 5-year relapse-free survival (RFS), and overall survival (OS) than the other groups. In contrast, the isolated FLT3-ITD+ group had a higher relapse rate and shorter RFS and OS than the other groups. The 5-year RFS rate was much higher among the patients who underwent allogeneic stem cell transplantation (alloSCT) than among those treated with high-dose cytarabine chemotherapy (HDAC) only as consolidation therapy in the isolated NPM1mut group and the NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD- group. CONCLUSION: Adult patients with CN-AML carrying isolated NPM1mut and isolated FLT3-ITD+ exhibit different clinical outcomes than those with NPM1mut/FLT3-ITD+ or NPM1wt/FLT3-ITD-. Although isolated NPM1mut leads to favorable clinical outcomes of CN-AML, the role of alloSCT in such patients remains to be considered.

DNA Methylation Profiles of MGMT, DAPK1, hMLH1, CDH1, SHP1, and HIC1 in B-Cell Lymphomas

BACKGROUND: This study was designed to examine the prevalence of aberrant promoter methylation in a selected panel of genes potentially involved in lymphoid tumors. METHODS: The promoter hypermethylation status of MGMT, DAPK1, hMLH1, CDH1, SHP1, and HIC1 was measured by methylation-specific PCR for 82 cases of B-cell lymphoma. Immunohistochemical staining using MGMT and SHP1 antibodies was conducted on 43 out of 82 cases. RESULTS: The number of MGMT aberrant methylations was lower in diffuse large B-cell lymphoma (DLBCL) than in other malignant lymphomas. The methylation of DAPK1 was frequently detected in follicular lymphoma (FL), marginal zone B-cell lymphoma (MZL) and DLBCL. With one exception, methylation of hMLH1 was not observed in B-cell lymphomas. The methylation frequency of CDH1, and HIC1 was similar in B-cell lymphomas. However, the methylation of SHP1 gene was more frequently observed in cases of FL, DLBCL, and MZL than in chronic lymphocytic lymphoma. MGMT and SHP1 promoter methylation were inversely correlated with the protein expression observed upon immunohistochemical staining. CONCLUSIONS: Aberrant promoter methylation of multiple genes occurs with variable frequency throughout the B-cell lymphomas, and methylation of hMLH1 is rarely observed in B-cell lymphomas.

Prevalence of Oral Microbes in the Saliva of Oncological Patients

This study examined the prevalence of oral microbes in the saliva of oncological patients and healthy subjects. PCR was used to assess the frequency of oral microbes including 3 cariogenic bacteria, 5 periodontopathic bacteria and 4 Candida species in the saliva of 104 oncological patients and 52 healthy subjects. Among these microorganims, Streptococcus mutans, Fusobacterium nucleatum and Candida albicans were most frequently detected in both groups. There were no significant differences in the prevalence of cariogenic bacteria between the patient and healthy groups, whereas significant differences in the frequency of Porphyromonas gingivalis and Tannerella forsythia were observed between the two groups (p < 0.05). The prevalence of all five periodontopathogens was higher in the healthy group than in the patient group. The prevalence of C. albicans in patients was significantly higher than that of healthy group (p < 0.05). In conclusion, there were significant differences in the prevalence of P. gingivalis, T. forsythia and C. albicans between the oncological patient group and healthy group.

Low Frequency and Variability of FLT3 Mutations in Korean Patients with Acute Myeloid Leukemia

FLT3 mutations are common genetic changes, and are reported to have prognostic significance in acute myeloid leukemia (AML). The FLT3 internal tandem duplication (ITD) and the D835 activating mutation in the tyrosine kinase domain (TKD) were analyzed by polymerase chain reaction (PCR) in the genomic DNA of Korean patients with AML at diagnosis and during follow-up. There were 226 patients with AML enrolled between March 1996 and August 2005. The incidence of ITD and TKD at diagnosis was 13% (29/226) and 3% (6/226). When compared to Western and other Asian patients with AML, Korean patients had a lower frequency by about two-thirds of ITD and TKD. Among the non-M3 cases (N=203), the patients with an ITD had a significantly shorter event-free survival when compared with those without an ITD (p=0.0079). Among 54 relapsed patients, 9 patients had the FLT3 ITD at diagnosis. Six patients demonstrated a reappearance of the ITD and 3 patients remained negative at relapse. One patient, among 45 patients who relapsed, had a negative baseline ITD but acquired a de novo ITD at relapse. There were 101 samples from 93 patients in remission; they were all negative for an ITD. Among 34 patients who failed to achieve a remission, five patients had a persistent ITD and one patient had a de novo ITD. These results support the concept of resistance of FLT3 ITD leukemic clones to chemotherapy. Therefore, effective therapy with FLT3 targeting agents may improve the prognosis of non-M3 AML patients with the FLT3 mutation.

FLT3 Internal Tandem Duplication in Acute Myeloid Leukemia with Normal Karyotype

BACKGROUND: The presence of FLT3 internal tandem dupulication (FLT3/ITD) in patients with acute myeloid leukemia (AML) with normal karyotype was investigated in order to evaluate its clinical and prognostic significance. METHODS: The FLT3/ITD was studied by PCR assay in bone marrow samples obtained from 123 patients at diagnosis. Ninety patients who received intensive induction chemotherapy were evaluated. RESULTS: Of total 123 patients, forty-seven (38.2%) demonstrated the aberrant FLT3/ITD. Patients with FLT3/ITD had significantly higher leukocyte counts at presentation than did patients without FLT3/ITD (P=0.04). By multivariate analysis, the FLT3/ITD was an independent prognostic factor of leukemic-free survival (LFS) (P=0.01) in AML patients with normal karyotype. CONCLUSION: This study demonstrated that the presence of the FLT3/ITD was a significant factor for poor prognosis in AML patients with normal karyotype.

Biphenotypic Acute Leukemia with BCR-ABL mRNA Transcript b3a2 Type: A Case Report with Review of the Literature / 대한진단검사의학회지

Biphenotypic acute leukemia (BAL) is a subtype of leukemia of ambiguous lineage in the World Health Organization classification system. About one third of the cases have the Philadelphia chromosome, and some cases are associated with other structural abnormalities involving 11q23. BAL is known to have a poor prognosis in both children and adults. According to the previously reported BAL cases with positive BCR-ABL fusion gene, most of the BCR-ABL mRNA transcript type was e1a2. So, we describe here a 30-year-old adult BAL case with the karyotype 46,XY,t(9;22)(q34;q11.2) resulting in a very rare b3a2 type of BCR-ABL mRNA transcript.