Aberrant DNA demethylation in promoter region and aberrant expression of mRNA of PAX4 gene in hematologic malignancies.

The PAX4 gene, a member of the paired box (PAX) gene family, is thought to be involved in regulating the fate of beta-cells in the mammalian pancreas. We observed the aberrant expression of PAX4 mRNA in 10 of 15 hematologic cell lines analyzed by RT-PCR. The restoration of PAX4 gene expression after treatment with the demethylating agent 5-aza-2'-deoxycitidine, as well as bisulfite sequencing analysis, indicated that gene overexpression was caused by DNA demethylation at the promoter region. Such DNA demethylation also was observed in primary lymphoma (20 out of 45 patients) on combined bisulfite restriction assay (COBRA). Forced expression of the PAX4 gene in the HEK293 and SHSY/610 cell lines conferred positive effects on cell growth. This profile of PAX4 thus corresponds to that of a candidate oncogene in hematologic malignancies.

Loss of O6-methylguanine-DNA methyltransferase protein expression is a favorable prognostic marker in diffuse large B-cell lymphoma.

Although aberrant promoter hypermethylation of O6-methylguanine-DNA methyltransferase (MGMT) is a favorable prognostic marker in patients with diffuse large B-cell lymphoma (DLBCL), MGMT protein expression has not been thoroughly examined. The aim of this study was to evaluate the clinical implication of MGMT protein expression and its correlation with promoter hypermethylation of the gene. We investigated MGMT protein expression by immunohistochemical analysis of 63 DLBCL patients who received cyclophosphamide as part of multidrug regimens. In addition, promoter methylation of the MGMT gene was analyzed by a methylation-specific polymerase chain reaction assay, and correlations with chemotherapeutic effect and prognosis were statistically evaluated. Immunohistochemical assay results for MGMT protein were negative in 30.2% of patients with newly diagnosed DLBCL. Immunostaining results were closely correlated with the methylation status of the promoter. Promoter DNA methylation of the gene was not detected in 34 (81.0%) of 42 tumor samples determined to be MGMT-positive DLBCL by immunostaining and was detected in 15 (88.2%) of 17 cases of MGMT-negative DLBCL. Overall survival (OS) and disease-free survival (DFS) rates were significantly higher in MGMT-negative patients than in MGMT-positive patients (5-year OS, 81.3% versus 56.6% [P = .0375]; 5-year DFS, 66.3% versus 39.9% [P = .0121]). The combined rate for complete response (CR) plus unconfirmed CR was significantly higher in MGMT-negative patients (15/19, 79.0%) than in MGMT-positive patients (25/44, 56.8%) (P = .0488). A multivariate analysis showed that absence of MGMT expression was an independent prognostic factor for OS (relative risk, 4.09; P = .0258). Lack of MGMT protein expression is associated with aberrant promoter DNA methylation and appears to be a useful marker for predicting the survival of DLBCL patients.

Promoter hypermethylation of the DNA-repair gene O6-methylguanine-DNA methyltransferase and p53 mutation in diffuse large B-cell lymphoma.

The gene for the DNA-repair enzyme O6-methylguanine-DNA methyltransferase (MGMT), which is closely related with cellular sensitivity to alkylating agents, is inactivated by promoter hypermethylation in several human cancers, including malignant lymphoma. Promoter hypermethylation of the MGMT gene is a favorable prognostic factor in diffuse large B-cell lymphoma (DLBCL). Although inactivation of the MGMT gene is closely related to p53 gene mutations in several cancers, the relationship between p53 gene mutation and MGMT inactivation in malignant lymphoma has not been thoroughly examined. We studied the correlation between MGMT hypermethylation and p53 mutation in DLBCL and their impacts on patient prognosis. In a retrospective cohort study, we used a methylation-specific polymerase chain reaction technique to analyze the methylation status of the promoter region of the MGMT gene in 116 DLBCL patients who received cyclophosphamide as part of multidrug combination chemotherapies. Denaturing high-performance liquid chromatography and direct sequencing were used to search for p53 gene mutations in exons 5 through 9 in 96 of the 116 samples. Disease-free survival and overall survival were estimated by the Kaplan-Meier method. Multivariate survival analyses were performed with the Cox proportional hazards model. Forty-five (38.8%) of 116 DLBCL patients showed MGMT promoter hypermethylation. The presence of MGMT hypermethylation was associated with better overall survival (P = .036). MGMT promoter hypermethylation was a prognostic factor that was independent of established prognostic factors, such as age, disease stage, serum lactic dehydrogenase level, and the number of extranodal disease sites (hazard ratio, 2.43; 95% confidence interval, 1.28-4.61; P = .007). p53 mutations were detected in 19 (19.8%) of 96 patients and were identified as a risk factor in the complete remission rate and overall survival (P = .0040, and P = .027, respectively). A correlation between MGMT hypermethylation and p53 mutation or p53 G:C-to-A:T mutation was not observed (P = .88, and P = .31, respectively). MGMT promoter hypermethylation and p53 mutation are useful prognostic markers in DLBCL. The impact of MGMT inactivation on p53 mutation in DLBCL is unclear.

[DAD, new intensive chemotherapy for patients with myeloma: a preliminary report].

A new intensive chemotherapy regimen, DAD, composed of doxorubicin, melphalan and dexamethasone, was given to 17 patients with multiple myeloma. The end point of this regimen was to obtain a deep posttreatment nadir in the M-protein levels so as to increase the chance of plateau attainment which would be associated with prolonged survival in each patient. It was noteworthy that all the 17 evaluable patients achieved a partial response. Nine of the 17 (52.9%) attained a plateau. Ten of the 17 patients (58.8%) obtained a deep posttreatment nadir in their M-protein levels (IgG < 2,000 mg/dl, IgA < 1,000 mg/dl, BJP = 0 g/dl/day), and six of them reached a plateau phase, which was not significantly more frequent than those who did not obtain a deep posttreatment nadir in their M-protein levels (three of seven reached plateau phase). The median survival of the 17 patients (37.6 months) was significantly prolonged compared with that of patients treated with our previous chemotherapy regimens, VMCP (22.5 months) and MMPP (23.5 months), and was comparable to that of MMCP (29.5 months).

Molecular response to nilotinib in a patient with imatinib-intolerant e19a2-positive chronic myeloid leukemia.

The BCR-ABL1 fusion gene is the molecular marker of chronic myeloid leukemia (CML). The e19a2 transcript is a rare variant associated with various clinical presentations and courses of CML. We herein present a case of e19a2-positive CML who was intolerant to initial treatment with imatinib and successfully responded to subsequent nilotinib therapy. She achieved a major molecular response and has since be able to sustain it. According to the literature, achieved molecular response by imatinib monotherapy has not yet been reported in e19a2-positive CML patients. Second generation tyrosine kinase inhibitors may therefore be a more effective treatment for e19a2-positive CML patients.

Expanded distribution of the T315I mutation among hematopoietic stem cells and progenitors in a chronic myeloid leukemia patient during imatinib treatment.

T315I mutation of the ABL-kinase domain in chronic myeloid leukemia (CML) confers resistance to imatinib (IM) as well as second-generation tyrosine kinase inhibitors (TKIs). We report a chronic-phase CML patient undergoing IM treatment, who showed the overt existence of the T315I mutation after 15 months. We retrospectively analyzed the distribution of the T315I mutation using the invader assay and direct DNA sequencing among FACSAria-sorted populations from bone marrow cells: total mononuclear cells (TMC), hematopoietic stem cells (HSC)/Thy-1(+), HSC/Thy-1⁻, common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), at 0, 3, 6, 9, and 12 months after IM treatment. T315I was barely detectable by 12 months in TMC, but detectable in 19.2% of HSC/Thy-1⁻ and 46.4% of MEP at diagnosis, and finally expanded into all populations. These results suggest that the monitoring of gene mutations in HSC and progenitors at diagnosis might be helpful for the early detection of TKI-resistant CML patients and facilitate appropriate therapeutic decision.

Azithromycin-related agranulocytosis in an elderly man with acute otitis media.

Azithromycin (AZM) is widely used for respiratory tract infections and otitis media because of its activity against Haemophilus influenzae and atypical pathogens, and its ease of administration. Although leukopenia is the one of the most frequent AZM-related laboratory abnormalities in children, agranulocytosis has not been reported in adults. Here, we present the case of an 81-year-old man with agranulocytosis following AZM-treatment for acute otitis media. He developed febrile neutropenia and granulocyte colony-stimulating factor and cefepim were administered. All his symptoms and absolute neutrophil counts were recovered within 7 days after admission. Physicians must be vigilant in identifying drug-induced neutropenia in AZM-treated patients because early detection can decrease the severity and prevent mortality.

Aberrant DNA methylation of p57(KIP2) gene in the promoter region in lymphoid malignancies of B-cell phenotype.

The cyclin-dependent kinase inhibitor p57(KIP2) is thought to be a potential tumor suppressor gene (TSG). The present study examines this possibility. We found that the expression of p57(KIP2) gene is absent in various hematological cell lines. Exposing cell lines to the DNA demethylating agent 5-aza-2'-deoxycytidine restored p57(KIP2) gene expression. Bisulfite sequencing analysis of its promoter region showed that p57(KIP2) DNA was completely methylated in cell lines that did not express the p57(KIP2) gene. Thus, DNA methylation of its promoter might lead to inactivation of the p57(KIP2) gene. DNA methylation of this region is thought to be an aberrant alteration, since DNA was not methylated in normal peripheral blood mononuclear cells or in reactive lymphadenitis. Methylation-specific polymerase chain reaction analysis found frequent DNA methylation of the p57(KIP2) gene in primary diffuse large B-cell lymphoma (54.9%) and in follicular lymphoma (44.0%), but methylation was infrequent in myelodysplastic syndrome and adult T-cell leukemia (3.0% and 2.0%, respectively). These findings directly indicate that the profile of the p57(KIP2) gene corresponds to that of a TSG.