Methylation analysis of Rb, P16, MGMT, and hMLH-1in mucoepidermoid carcinomas. A pilot study

Mucoepidermoid carcinoma (MC) is the most common malignant epithelial neoplasm in the salivary glands. This neoplasm has varying proportions of mucous, epidermoid, intermediate, columnar, and clear cells. MCs have been associated with CRTC1-MAML2 genes; however, their pathogenesis is uncertain. Recently, epigenetic changes have been considered a possible aetiologic factor. To identify the methylation state of RB, P16, MGMT, and hMLH genes in the three severity grades of MC were used five MCs and one healthy minor salivary gland as a control group (CG) obtained from the Pathology and Oral Medicine Laboratory and analyzed using MS-PCR to compare the presence or absence of methylation in promotor regions. The Kruskal- Wallis test was performed, with p≤0.05 considered significant. CG was employed as the normalizer of methylation levels. All assays were performed in triplicate. The mean age of our population was 52.6±18.6 years old; the total population was female and included 2 low grade, 2 intermediate grade, and 1 high grade levels of severity. When comparing the methylation status of the three histopathological grades of MC against the control, statistically significant differences were observed in Rb-M, MGMT-M, and hMLH-1-NM for high-grade severity, with p values of 0.03, 0.05, and 0.04, respectively. Methylation is a possible mechanism for pathogenesis processing of high-grade MC. However, a larger sample population is necessary to validate this finding.

El carcinoma mucoepidermoide (CM) es la neoplasia epitelial maligna más frecuente de glándulas salivales. Esta neoplasia tiene proporciones variables de células mucosas, epidermoides, intermedias, cilíndricas y claras. Los CM se han asociado con los genes CRTC1-MAML2; sin embargo, su patogenia es incierta. Recientemente, los cambios epigenéticos se han considerado un posible factor etiológico. Para identificar el estado de metilación de los genes RB, P16, MGMT y hMLH en los tres grados de severidad de CM se utilizaron cinco CM y una glándula salival menor sana como grupo control (GC) obtenidos del Laboratorio de Patología y Medicina Oral y analizados mediante MS-PCR para comparar la presencia o ausencia de metilación en regiones promotoras. Se realizó la prueba de Kruskal-Wallis, considerándose significativa una p≤0,05. Se empleó GC como normalizador de los niveles de metilación. Todos los ensayos se realizaron por triplicado. La edad media de nuestra población fue de 52,6 ± 18,6 años; la población total era femenina e incluía 2 niveles de severidad de grado bajo, 2 de grado intermedio y 1 de alto grado. Al comparar el estado de metilación de los tres grados histopatológicos de CM contra el GC, se observaron diferencias estadísticamente significativas en Rb-M, MGMT-M y hMLH-1-NM para severidad de alto grado, con valores de p de 0.03, 0.05, y 0,04, respectivamente. La metilación es un posible mecanismo para el procesamiento de patogénesis de CM de alto grado. Sin embargo, se necesita una población de muestra más grande para validar este hallazgo.

The relationship between IDH, P53 mutations, MGMT methylation with characteristics in high grade glioma patients / La relación entre IDH, mutaciones P53, metilación de MGMT con las características de pacientes con glioma de alto grado

ABSTRACT Introduction: Some gene mutations in high grade glioma patients have many implications in prognosis and treatment response. Objectives: To describe the characteristics and associations of IDH, TP53 gene mutations and MGMT methylation status with some characteristics and treatment response in patients with high grade glioma. Methods: A descriptive, prospective, uncontrolled study was conducted, in 52 patients with high-grade glioma. Research variables include age, sex, Karnofsky score, the rate of IDH, P53 mutation, MGMT methylation; the relationship between genes mutation with some characteristics and response to treatment according to the RECIST classification. Results: For IDH gene mutation, grade III patients (23.1%) have a higher positive rate than grade IV (11.5 %); for P53 gene mutation, grade III patients (55.6 %) have a higher positive rate than grade IV (44.1 %); the rate of MGMT promoter methylation occurred in the study group of patients with the rate of 42.3 %. There is a relationship between IDH gene mutation with pathological results and malignancy in studied patients. Patients with the mutant expression of the IDH gene, p53, MGMT methylation status had better RECIST responses than patients without these expressions. Conclusion: High-grade glioma mainly occurs in men, over 40 years old. The presence of mutations in IDH, P53 genes, and MGMT methylation status was a beneficial factor for treatment response as assessed by RECIST.

RESUMEN Introducción: Algunas mutaciones genéticas en pacientes con glioma de alto grado tienen implicaciones en el pronóstico y respuesta al tratamiento. Objetivos: Describir las características y asociaciones de IDH, mutaciones del gen TP53 y estado de metilación de MGMT con algunas características y respuesta al tratamiento en pacientes con glioma de alto grado. Métodos: Se realizó un estudio descriptivo, prospectivo no controlado, en 52 pacientes con glioma de alto grado. Las variables investigadas fueron: edad, sexo, puntuación de Karnofsky, tasa de IDH, mutación P53, estado de metilación de MGMT, relación entre la mutación de genes con algunas características y la respuesta al tratamiento según la clasificación RECIST. Resultados: Mutación del gen IDH: los pacientes grado III (23,1 %) tienen una tasa positiva más alta que los grado IV (11,5 %). Mutación del gen P53: los grado III (55,6 %) tienen una tasa positiva más alta que los grado IV (44,1 %). La tasa de metilación del promotor de MGMT se produjo con una tasa del 42,3 %. Existe relación entre la mutación del gen IDH con los resultados patológicos y la malignidad. Los pacientes con la expresión mutante del gen IDH, p53, estado de metilación de MGMT tuvieron mejores respuestas RECIST. Conclusión: El glioma de alto grado se presenta principalmente en hombres, mayores de 40 años. La presencia de mutaciones en los genes IDH, P53 y el estado de metilación de MGMT fue un factor beneficioso para la respuesta al tratamiento según lo evaluado por RECIST.

DNA alkylation lesion repair: outcomes and implications in cancer chemotherapy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, represent a major form of DNA damage in cells. The repair of alkylation damage is critical in all cells because such damage is cytotoxic and potentially mutagenic. Alkylation chemotherapy is a major therapeutic modality for many tumors, underscoring the importance of the repair pathways in cancer cells. Several different pathways exist for alkylation repair, including base excision and nucleotide excision repair, direct reversal by methyl-guanine methyltransferase (MGMT), and dealkylation by the AlkB homolog (ALKBH) protein family. However, maintaining a proper balance between these pathways is crucial for the favorable response of an organism to alkylating agents. Here, we summarize the progress in the field of DNA alkylation lesion repair and describe the implications for cancer chemotherapy.

MMR-proficient and MMR-deficient colorectal cancer cells: 5-Fluorouracil treatment response and correlation to CD133 and MGMT expression

Cancer stem cells (CSCs) from colorectal cancer (CRC), characterized by CD133 expression, have beenassociated with 5-fluorouracile (5-FU) chemoresistance. DNA repair mechanisms, such as O6-alkylguanineDNA alkyltransferase (MGMT) and mismatch repair (MMR) systems, have also been correlated to 5-FUresistance in CRC. The aim of this study was to evaluate the modulation of CD133 and MGMT in MMRproficient and MMR-deficient CRC cells under 5-FU treatment and the effect of this drug in CSCs. CD133 andMGMT methylation status were determined in MMR-proficient (SW480 and HT29) and MMR-deficient (RKOand HCT116) cell lines by methylation-specific PCRs. SW480 and RKO were selected to determine modulation of CD133, MGMT and MMR expression after 5-FU treatment by qPCR. In addition, CD133, MGMTand MMR were analyze in SW480 and RKO CSCs. No association between promoter methylation and MGMTand CD133 expression was found. 5-FU treatment increased CD133 expression independently to MMR statusin SW480 and RKO and was able to increase hMLH1 expression in RKO, a MMR-deficient cell line. RKO/CSCs overexpressed CD133 and MMR (hMSH2 and hMSH6) while SW480/CSCs showed a significantincrease in CD133, MMR (hMLH1, hMSH2 and hMSH6) and MGMT, moreover 5-FU resistance thanparental cell lines. Thus, although CSCs 5-FU chemoresistance appears to be independently to MMR status,hMLH1 might play a key role in CSC response to 5-FU. New drugs exploding these differences could benefitthe prognostic of patients with CRC.

lncRNA POU3F3 affects temozolomide-resistance in high-grade glioma cells via regulating MGMT expression / 中国肿瘤生物治疗杂志

@# Objective: To explore the mechanism of long non-coding RNA POU3F3 (lncRNAPOU3F3) affecting temozolomide (TMZ)-resistance in high-grade glioma cells via regulating MGMT expression. Methods: Sixty cases of tissues from patients treated at the Department of Neurosurgery, Peking University International Hospital during January 2016 and January 2018 were collected for this study, including 12 cases from brain trauma patients (normal group), 30 cases from primary high-grade glioma patients (primary onset group) and 18 cases from recurrent high-grade glioma patients (recurrence group, accepted surgery+TMZ already). U251 cells were induced with TMZ at the concentration of 1, 2, 4 and 8 μg/ml and maintained normal growth for a week to construct TMZ-resistant U251cell line (U251 TMZ-resistance, U251-TR); and the normal control group was treated with equal volume of physiological saline. Reverse transcription polymerase chain reaction (qPCR) and Wb were used to detect the mRNA and protein expressions of POU3F3 and MGMT (methylguanine DNA methyltransferase) in normal brain tissues and glioma cells. Lentivirus transfection was used to construct U251 cell line with stable POU3F3 interference (U251-TR siPOU3F3); CCK-8 was used to detect TMZ IC50 value (the half maximal inhibitory concentration) in each group of U251 cells, and Wb was used to detect the expression of MGMT protein in each group of cells. Results: Compared with the normal group and primaryonset group, the expression of POU3F3 in recurrence group was significantly increased (P<0.01). The TMZ IC50 of U251-TR cells was significantly higher than that of U251 cells (P<0.01), and The TMZ IC50 of U251-TR siPOU3F3 cells was significantly lower than that of U251-TR cellsbut higher than that of U251 cells (all P<0.01). The protein and mRNA expressions of POU3F3 and MGMT in U251-TR cells were significantly higher than that in U251 cells (P<0.01), while those expressions in U251-TR siPOU3F3 cells were significantly lower than those in U251-TR cells (P<0.01).Conclusion: lncRNAPOU3F3 is the key factor to promote TMZ resistance in human high-grade gliomas cells, which may exert certain guiding significance in the clinical treatment for TMZ resistance.

Appraisal of re-irradiation for the recurrent glioblastoma in the era of MGMT promotor methylation

Despite recent innovation in treatment techniques and subsequently improved outcomes, the majority of glioblastoma (GBL) have relapses, especially in locoregional areas. Local re-irradiation (re-RT) has been established as a feasible option for recurrent GBL of all ages with safety, tolerability, and effectiveness both in survival and quality of life regardless of fractionation schedule. To keep adverse effects under acceptable range, cumulative dose limit in equivalent dose at 2 Gy fractions by the linear-quadratic model at α/β = 2 for normal brain tissue (EQD2) with narrow margin should be observed and single/hypofractionated re-RT should be undertaken very carefully to recurrent tumor with large volume or adjacent to the brainstem. Promising outcome of re-operation (re-Op) plus re-RT (re-Op/RT) need to be validated and result from re-RT with temozolomide/bevacizumab (TMZ/BV) or new strategy is expected. Development of new-concept prognostic scoring or risk group is required to select patients properly and make use of predictive biomarkers such as O(6)-methylguanine-DNA methyltransferase (MGMT) promotor methylation that influence outcomes of re-RT, re-Op/RT, or re-RT with TMZ/BV.

Disulfiram, a Re-positioned Aldehyde Dehydrogenase Inhibitor, Enhances Radiosensitivity of Human Glioblastoma Cells In Vitro / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: Glioblastoma, the most common brain tumor in adults, has poor prognosis. The purpose of this study was to determine the effect of disulfiram (DSF), an aldehyde dehydrogenase inhibitor, on in vitro radiosensitivity of glioblastoma cells with different methylation status of O⁶-methylguanine-DNA methyltransferase (MGMT) promoter and the underlying mechanism of such effect. MATERIALS AND METHODS: Five human glioblastoma cells (U138MG, T98G, U251MG, U87MG, and U373MG) and one normal human astrocyte (NHA) cell were cultured and treated with DSF or 6MV X-rays (0, 2, 4, 6, and 8 Gy). For combined treatment, cells were treated with DSF before irradiation. Surviving fractions fit from cell survival based on colony forming ability. Apoptosis, DNA damage repair, and cell cycle distributionwere assayed bywestern blot for cleaved caspase-3, γH2AX staining, and flow cytometry, respectively. RESULTS: DSF induced radiosensitization in most of the glioblastoma cells, especially, in the cells with radioresistance as wildtype unmethylated promoter (MGMT-wt), but did not in normal NHA cell. DSF augmented or induced cleavage of caspase-3 in all cells after irradiation. DSF inhibited repair of radiation-induced DNA damage in MGMT-wt cells, but not in cells with methylated MGMT promoter. DSF abrogated radiation-induced G2/M arrest in T98G and U251MG cells. CONCLUSION: Radiosensitivity of glioblastoma cells were preferentially enhanced by pre-irradiation DSF treatment compared to normal cell, especially radioresistant cells such as MGMT-wt cells. Induction of apoptosis or inhibition of DNA damage repair may underlie DSF-induced radiosensitization. Clinical benefit of combining DSF with radiotherapy should be investigated in the future.

The clinical significance of MGMT promoter methylation in the treatment and progno-sis of glioma patients / 中国肿瘤临床

Adjuvant temozolomide-based chemotherapy has become the standard of care for most postoperative glioma patients. However, a large proportion of these patients do not respond to temozolomide. DNA repair enzyme O6-methylguanine-DNA methyl-transferase (MGMT) promoter methylation has emerged as an important molecular marker in patients with gliomas. It is associated with prognosis and resistance to alkylated drugs such as temozolomide. MGMT promoter methylation is the key mechanism of MGMT gene silencing, thereby inhibiting DNA repair and increasing the sensitivity of chemotherapy. We reviewed current data on the prog-nostic and predictive relevance of MGMT testing and clinical trials, summarized the clinical application of MGMT promoter methyla-tion, in order to provide reference for the individualized treatment of glioma patients.

Clinical and Prognostic Significance of O⁶-Methylguanine-DNA Methyltransferase Promoter Methylation in Patients with Melanoma: A Systematic Meta-Analysis

Tumor suppressor gene O⁶-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been reported in melanoma. However, the clinical and prognostic significance of MGMT promoter methylation in patients with melanoma remained to be determined. A systematic search was performed to identify eligible papers published. The overall odds ratios (ORs) or hazard ratios and their 95% confidence intervals were calculated. Final 12 eligible publications involving Caucasian population were performed in this study, including 1,071 metastatic melanoma patients, 154 primary melanoma patients, and 211 normal controls. MGMT promoter methylation was significantly higher in primary or metastatic melanoma than in normal controls (p < 0.05). No difference of MGMT promoter methylation was found in primary and metastatic melanoma (p=0.432). When metastatic melanoma was compared to normal controls, subgroup analysis showed the correlation between MGMT promoter methylation and different sample materials (tissue: OR=7.01, p < 0.001 and blood: OR=12.04, p=0.005). MGMT promoter methylation was not associated with response to drug therapy and the prognosis in overall survival and progression-free survival for multivariate analysis. Our results show that MGMT promoter methylation may be correlated with the increased risk of primary or metastatic melanoma. Based on blood samples, MGMT promoter methylation may become a noninvasive biomarker for the detection of metastatic melanoma. Further additional clinical studies are necessary.

Comparison of the effects of microfluidic paper-based immunoassay and traditional Western blot on the detection of target proteins / 基础医学与临床

Objective To achieve the goal of the qualitatively and relatively quantitatively analyze of the target pro-tein in a time-saving,labor-saving and reagents-saving way by the microfluidic paper-based immunoassay. Methods We chose MGMT as the target protein and compared the qualitative and semi-quantitative results of the MGMT ex-pression in the MCF7 cells which was treated with MGMT inhibitor, lomeguatrib, in both the traditional Western blot and the paper chip immunoassay. Results Microfluidic paper-based immunoassay can make the qualitative and relative quantitative detection on protein expression. Compared to the sensitivity of 1-5 ng of the traditional Western blot,the microfluidic paper-based immunoassay could detect as low as 10-25 pg of the protein. The sensitivity could be improved by 3 orders of magnitude. The entire operational duration took only 1 hour with less costed rea-gents being consumed. It maked the high-throughput protein detection as sensitive as the reverse phase protein as-says (RPPA) does. Conclusions The paper chip immunoassay could be performed qualitatively and semi-quanti-tatively to detect protein expression,and is more effective than that of traditional Western blot.

Comparison of the effects of microfluidic paper-based immunoassay and traditional Western blot on the detection of target proteins / 基础医学与临床

Objective To achieve the goal of the qualitatively and relatively quantitatively analyze of the target pro-tein in a time-saving,labor-saving and reagents-saving way by the microfluidic paper-based immunoassay. Methods We chose MGMT as the target protein and compared the qualitative and semi-quantitative results of the MGMT ex-pression in the MCF7 cells which was treated with MGMT inhibitor, lomeguatrib, in both the traditional Western blot and the paper chip immunoassay. Results Microfluidic paper-based immunoassay can make the qualitative and relative quantitative detection on protein expression. Compared to the sensitivity of 1-5 ng of the traditional Western blot,the microfluidic paper-based immunoassay could detect as low as 10-25 pg of the protein. The sensitivity could be improved by 3 orders of magnitude. The entire operational duration took only 1 hour with less costed rea-gents being consumed. It maked the high-throughput protein detection as sensitive as the reverse phase protein as-says (RPPA) does. Conclusions The paper chip immunoassay could be performed qualitatively and semi-quanti-tatively to detect protein expression,and is more effective than that of traditional Western blot.

MGMT and SPOCK2 Promoter Methylation in Diffuse Large B-Cell Lymphoma: A Study in Two Tertiary Health Centres in the East Coast of Malaysia / Jurnal Sains Kesihatan Malaysia

MGMT (O6 -Methylguanine-DNA Methyltransferase) suppresses tumor development by removing alkyl adduct, while SPOCK2 (SPARC/Osteonectin CWCV and Kazal-like domains proteoglycan) abolishes the inhibition of membrane-type matrix metalloproteinases (MT-MMP) which leads to angiogenesis. Hence, MGMT methylation may initiate malignant cells transformation. In contrast, SPOCK2 methylation is hypothesized not to be a common event in diffuse large B-cell lymphoma (DLBCL). In this study, we examined the methylation status of MGMT and SPOCK2 in DLBCL as in Malaysia the information is extremely lacking. A total of 88 formalin-fixed paraffin-embedded tissue of patients diagnosed with DLBCL from the year 2006 to 2013 were retrieved from Hospital Universiti Sains Malaysia, Kelantan and Hospital Tengku Ampuan Afzan, Pahang. Methylation-specific polymerase chain reaction (MSP) was used to examine the methylation status of both genes. Interestingly, methylation of MGMT was detected in all the 88 DLBCL samples, whereas SPOCK2 was found to be methylated in 83 of 88 (94.3%) DLBCL cases. Our study showed a remarkably high percentage of promoter methylation of both MGMT and SPOCK2 genes. Our finding also negates initial expectation that SPOCK2 methylation would be an uncommon event in the majority of DLBCL cases. This study has shown a very high percentage of promoter methylation of MGMT and SPOCK2 in the DLBCL cases studied by MSP, using archival lymphoma tissues. Nonetheless, additional research is needed to quantitatively evaluate MGMT and SPOCK2 methylation, and to analyse gene expression and/or protein expression in order to further understand the role of MGMT and SPOCK2 methylation in the pathogenesis of DLBCL.

Concurrent Chemoradiotherapy with Temozolomide Followed by Adjuvant Temozolomide for Newly Diagnosed Glioblastoma Patients: A Retrospective Multicenter Observation Study in Korea / Journal of the Korean Cancer Association, 대한암학회지

PURPOSE: The purpose of this study was to investigate the feasibility and survival benefits of combined treatment with radiotherapy and adjuvant temozolomide (TMZ) in a Korean sample. MATERIALS AND METHODS: A total of 750 Korean patients with histologically confirmed glioblastoma multiforme, who received concurrent chemoradiotherapy with TMZ (CCRT) and adjuvant TMZ from January 2006 until June 2011, were analyzed retrospectively. RESULTS: After the first operation, a gross total resection (GTR), subtotal resection (STR), partial resection (PR), biopsy alone were achieved in 388 (51.7%), 159 (21.2%), 96 (12.8%), and 107 (14.3%) patients, respectively. The methylation status of O6-methylguanine-DNA methyltransferase (MGMT) was reviewed retrospectively in 217 patients. The median follow-up period was 16.3 months and the median overall survival (OS) was 17.5 months. The actuarial survival rates at the 1-, 3-, and 5-year OS were 72.1%, 21.0%, and 9.0%, respectively. The median progression-free survival (PFS) was 10.1 months, and the actuarial PFS at 1-, 3-, and 5-year PFS were 42.2%, 13.0%, and 7.8%, respectively. The patients who received GTR showed a significantly longer OS and PFS than those who received STR, PR, or biopsy alone, regardless of the methylation status of the MGMT promoter. Patients with a methylated MGMT promoter also showed a significantly longer OS and PFS than those with an unmethylated MGMT promoter. Patients who received more than six cycles of adjuvant TMZ had a longer OS and PFS than those who received six or fewer cycles. Hematologic toxicity of grade 3 or 4 was observed in 8.4% of patients during the CCRT period and in 10.2% during the adjuvant TMZ period. CONCLUSION: Patients treated with CCRT followed by adjuvant TMZ had more favorable survival rates and tolerable toxicity than those who did not undergo this treatment.

The regulatory effect of 5-aza-2,-deoxycytidine in cervical cancer cells / 实用医学杂志

Objective To discuss the regulatory effect of 5-aza-2 ,-deoxycytidine on P16 and MGMT in cervical cancer cells. Methods After four kinds of cervical cancer cells (HeLa, SiHa, C33A and CaSki) were treated with 5-Aza-dC , MSP was used to detect the methylation variation of P16 and MGMT , and fluorogenic quantitative PCR and Western blot were employed for determination of P16 and MGMT expression. MTT and Annexin V-FITC/PI double staining were adopted for detection of cell proliferation and apoptosis. Results Both P16 and MGMT exhibited methylation in four kinds of cervical cancer cells , and after treatment with 5-Aza-dC ,their methylation levels were reversed. 5-Aza-dC was able to inhibit p16 and MGMT expression in the cervical cancer cells, and can also suppress cell proliferation and promote apoptosis. Conclusions Although methylation of P16 and MGMT are present in cervical cancer cells, their expression level was still high. Therefore, regulation of P16 and MGMT expression may be affected by other factors. 5-Aza-dC can suppress the growth of cervical cancer cells. Although 5-Aza-dC reverse the methylation levels of P16 and MGMT, it inhibits their gene expression. More experiments are needed to verify the hidden reasons and mechanisms.

Prognostic Role of Methylation Status of the MGMT Promoter Determined Quantitatively by Pyrosequencing in Glioblastoma Patients

OBJECTIVE: This study investigated whether pyrosequencing can be used to determine the methylation status of the MGMT promoter as a clinical biomarker using relatively old archival tissue samples of glioblastoma. We also examined other prognostic factors for survival of glioblastoma patients. METHODS: The available study set included formalin-fixed paraffin-embedded (FFPE) tissue from 104 patients at two institutes from 1997 to 2012, all of which were diagnosed histopathologically as glioblastoma. Clinicopathologic data were collected by review of medical records. For pyrosequencing analysis, the PyroMark Q96 CpG MGMT kit (Qiagen, Hilden, Germany) was used to detect the level of methylation at exon 1 positions 17-39 of the MGMT gene, which contains 5 CpGs. RESULTS: Methylation of the MGMT promoter was detected in 43 (41.3%) of 104 samples. The average percentage methylation was 14.0+/-16.8% overall and 39.0+/-14.7% for methylated cases. There was no significant pattern of linear increase or decrease according to the age of the FFPE block (p=0.687). In multivariate analysis, age, performance status, extent of surgery, method of adjuvant therapy, and methylation status estimated by pyrosequencing were independently associated with overall survival. Additionally, patients with a high level of methylation survived longer than those with low methylation (p=0.016). CONCLUSION: In this study, the status and extent of methylation of the MGMT promoter analyzed by pyrosequencing were associated with overall survival in glioblastoma patients. Pyrosequencing is a quantitative method that overcomes the problems of MSP and a simple technique for accurate analysis of DNA sequences.

A Single Nucleotide Polymorphism in the MGMT Gene is a Novel Prognostic Biomarker in Patients with Metastatic Colorectal Cancer.

Background: Discovery of novel biomarkers of prognosis and drug response remains an elusive, yet critical goal. Thus, accurate and rapid screening of an array of pertinent mutations/SNPs is an essential step in cancer management. Methods: Using a high-throughput multiplex PCR microarray technique, we simultaneously screened the mutational status/SNP of 32 hotspots in multiple genes for metastatic colorectal cancer (mCRC) from 126 formalin fixed paraffin embedded samples from 78 patients. The efficacy of the technology was validated by cross-comparison with conventional Sanger sequencing and pyrosequencing. The clinical outcome was corroborated to the mutational status to determine prognostic and predictive significance of the 32 loci. Results: In a statistically robust multivariate model, patients with the TT genotype of the MGMT gene (rs1625649) enjoyed a significantly longer survival (61.8 months) when compared to those with GG or heterozygous GT genotype (29.3 months) [HR 0.30; 95% CI: 0.10-0.89, P= 0.03], with a 70% reduced risk of death from mCRC. Conclusion: The rs1625649 SNP within MGMT is a novel and potentially valuable prognostic biomarker for mCRC patients.

Primary Spinal Cord Oligodendroglioma with Postoperative Adjuvant Radiotherapy: A Case Report

Primary spinal cord oligodendrogliomas are rare tumors comprising two percent of all spinal cord tumors. Although a treatment guideline has yet to be established, maximal surgical resection is primary in the treatment of spinal cord oligodendrogliomas. Adjuvant radiotherapy has remained controversial, and it is unclear whether chemotherapy adds any benefit. In this case report, the authors present a 24-year-old male who had a seven-year history of left leg weakness and a radiating pain in both legs. Magnetic resonance image (MRI) showed an intramedullary mass at the T4-T8 level. He underwent subtotal removal of the tumor and pathologic diagnosis revealed a WHO grade II oligodendroglioma. The patient was treated with radiotherapy postoperatively and followed up with MRI annually. Clinical and radiological status of the patient had been stationary for four years after the surgery. The five-year follow-up MRI showed an increase in the size and extent of the residual tumor. Despite radiological progression, considering that symptoms and the performance status of the patient had remained unchanged, further treatment has not been performed. Given the clinical outcome of this patient, close observation after subtotal removal with adjuvant radiotherapy is one of the acceptable treatment options for WHO grade II spinal cord oligodendrogliomas.

Effects of 5-Aza-Cde on DNA Methylation and Expression of hMLHl and MGMT Gene in Lung Cancer Cell Line A549/DDP / 现代检验医学杂志

Objective To investigate the effects of 5-Aza-2′-deoxycytidine (5-Aza-Cde)on DNA methylation and expression of hMLH1 and MGMT gene in the human lung cancer cell line A549/DDP.Methods A549/DDP cells were cultured with RPMI 1 640 medium and were treated with 5 μmol/L DNA methyhransferase inhibitor 5-Aza-Cde.Methylation-specific pol-ymerase chain reaetioll (MSP)was used to detect the promoter methylation state of the hMLH1 and MGMT gene.RT-PCR was used to detect the mRNA expression of hMLH1 and MGMT before and after treatment with 5-Aza-Cde,respectively. Results Before treatment with 5-Aza-Cde,hMLH1 and MGMT expressions were absent,and promoter hypermethylation of the hMLH1 and MGMT gene were detected in A549 cells.After treatment with 5-Aza-Cde,the promoter region of the hM-LH1 and MGMT gene exhibited a demethylation state,and their mRNA expressions were increased.Conclusion Promoter hypermethyhtion is amajor mechanism of hMLH1 and MGMT gene silencing in human lung cancer cells,and can be reversed by the demethylating agent 5-Aza-Cde,which can regulate the expressions of the hMLH1 and MGMT gene.

Relationship between promoter methylation & tissue expression of MGMT gene in ovarian cancer.

Background & objectives: Epigenetic alterations, in addition to multiple gene abnormalities, are involved in the genesis and progression of human cancers. Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumour suppressor genes. O6-methyguanine-DNA methyltransferase (MGMT) is a DNA repair gene that removes mutagenic and cytotoxic adducts from the O6-position of guanine induced by alkylating agents. MGMT promoter hypermethylation and reduced expression has been found in some primary human carcinomas. We studied DNA methylation of CpG islands of the MGMT gene and its relation with MGMT protein expression in human epithelial ovarian carcinoma. Methods: A total of 88 epithelial ovarian cancer (EOC) tissue samples, 14 low malignant potential (LMP) tumours and 20 benign ovarian tissue samples were analysed for MGMT promoter methylation by nested methylation-specific polymerase chain reaction (MSP) after bisulphite modification of DNA. A subset of 64 EOC samples, 10 LMP and benign tumours and five normal ovarian tissue samples were analysed for protein expression by immunohistochemistry. Results: The methylation frequencies of the MGMT gene promoter were found to be 29.5, 28.6 and 20 per cent for EOC samples, LMP tumours and benign cases, respectively. Positive protein expression was observed in 93.8 per cent of EOC and 100 per cent in LMP, benign tumours and normal ovarian tissue samples. Promoter hypermethylation with loss of protein expression was seen only in one case of EOC. Interpretation & conclusions: Our results suggest that MGMT promoter hypermethylation does not always reflect gene expression.

The study on the relationship between methylation of MGMT, hMSH2 gene promoters and protein ex-pression in glioma patients / 实用肿瘤学杂志

Objective To investigate the relevance between protein expression and methylation of MG-MT and hMSH2 in glioma patimts.Methods Immunohistochemical and methylation specific PCR were adopted respectively to test on 275 cases of glioma patients for the protein expression and methylation situation of MGMT and hMSH2.Results The negative protein expression rate of MGMT and hMSH 2 in the tissue of brain golima were 47.2% and 62.5% respectively;the occurrence of methylation in gene promoter region were accordingly 41.8% and 22.4%.Statistical analysis revealed that MGMT promoter methylation in peripheral blood gene groups was related with the protein negative expression of tumor tissue (P0.05).Conclusion The meth-ylation of MGMT is a common molecular situation in the generation of brain glioma ,which may be connected with that of tumor.However,hMSH2 promoter methylation might not the main reason for inactivation of hMSH 2 pro-tein,there may be other important factors affecting its expression .