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.

Dietary polyphenols and their relationship to the modulation of non-communicable chronic diseases and epigenetic mechanisms: A mini-review.

Chronic Non-Communicable Diseases (NCDs) have been considered a global health problem, characterized as diseases of multiple factors, which are developed throughout life, and regardless of genetics as a risk factor of important relevance, the increase in mortality attributed to the disease to environmental factors and the lifestyle one leads. Although the reactive species (ROS/RNS) are necessary for several physiological processes, their overproduction is directly related to the pathogenesis and aggravation of NCDs. In contrast, dietary polyphenols have been widely associated with minimizing oxidative stress and inflammation. In addition to their antioxidant power, polyphenols have also drawn attention for being able to modulate both gene expression and modify epigenetic alterations, suggesting an essential involvement in the prevention and/or development of some pathologies. Therefore, this review briefly explained the mechanisms in the development of some NCDs, followed by a summary of some evidence related to the interaction of polyphenols in oxidative stress, as well as the modulation of epigenetic mechanisms involved in the management of NCDs.

An MGMT Allelic Variant Can Affect Biochemical Relapse in Prostate Cancer Patients.

BACKGROUND/AIM: Prostate cancer (PCa) is one of the most frequent neoplasms in men around the world. In recent years, the search for new biomarkers with greater prognostic potential for PCa has intensified. This study aimed to evaluate single nucleotide polymorphisms (SNPs) and a combined panel of these polymorphisms in relation to biochemical recurrence in patients who were through prostatectomy, with an average of 7 years of follow-up. MATERIALS AND METHODS: Patients diagnosed with PCa (n=197) participated in this cohort study. Thirteen SNPs were analyzed: rs2279115 (BCL-2), rs26677604 (CASP3), rs1052571 (CASP9), rs11781886 (NKX3-1), rs2735343 (PTEN), rs2494750 (AKT1), rs2699887 (PI3KCA), rs3195676 (AMACR), rs17302090 (AR), rs2536 (mTOR), rs1695 (GSTP1), rs2308321 (MGMT) and rs1544410 (VDR). Variants were combined and four main panels were defined: cell death, cell survival, growth receptors, and metabolism. Genotyping was performed by real-time PCR. RESULTS: We did not observe any significant relation between the panels of variants analyzed, apart from the rare allele (G) of rs2308321 (MGMT) that was associated with a higher risk of recurrence (p=0.036) when compared to the prevalent (A) in the allelic model. CONCLUSION: This MGMT variant occurs in an exon, and it could potentially affect DNA repair and, therefore, the biochemical relapse of PCa patients.

Deep DNA sequencing of MGMT, TP53 and AGT in Mexican astrocytoma patients identifies an excess of genetic variants in women and a predictive biomarker.

PURPOSE: Astrocytomas are a type of malignant brain tumor with an unfavorable clinical course. The impact of AGT and MGMT somatic variants in the prognosis of astrocytoma is unknown, and it is controversial for TP53. Moreover, there is a lack of knowledge regarding the molecular characteristics of astrocytomas in Mexican patients. METHODS: We studied 48 Mexican patients, men and women, with astrocytoma (discovery cohort). We performed DNA deep sequencing in tumor samples, targeting AGT, MGMT and TP53, and we studied MGMT gene promoter methylation status. Then we compared our findings to a cohort which included data from patients with astrocytoma from The Cancer Genome Atlas (validation cohort). RESULTS: In the discovery cohort, we found a higher number of somatic variants in AGT and MGMT than in the validation cohort (10.4% vs < 1%, p < 0.001), and, in both cohorts, we observed only women carried variants AGT variants. We also found that the presence of either MGMT variant or promoter methylation was associated to better survival and response to chemotherapy, and, in conjunction with TP53 variants, to progression-free survival. CONCLUSIONS: The occurrence of AGT variants only in women expands our knowledge about the molecular differences in astrocytoma between men and women. The increased prevalence of AGT and MGMT variants in the discovery cohort also points towards possible distinctions in the molecular landscape of astrocytoma among populations. Our findings warrant further study.

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.

Low MGMT digital expression is associated with a better outcome of IDH1 wildtype glioblastomas treated with temozolomide.

INTRODUCTION: Glioblastoma (GBM) is the deadliest primary brain tumor. The standard treatment consists of surgery, radiotherapy, and temozolomide (TMZ). TMZ response is heterogeneous, and MGMT promoter (MGMTp) methylation has been the major predictive biomarker. We aimed to describe the clinical and molecular data of GBMs treated with TMZ, compare MGMT methylation with MGMT expression, and further associate with patient's outcome. METHODS: We evaluate 112 FFPE adult GBM cases. IDH1 and ATRX expression was analyzed by immunohistochemistry, hotspot TERT promoter (TERTp) mutations were evaluated by Sanger or pyrosequencing, and MGMTp methylation was assessed by pyrosequencing and MGMT mRNA expression using the nCounter® Vantage 3D™ DNA damage and repair panel. RESULTS: Of the 112 GBMs, 96 were IDH1WT, and 16 were IDH1MUT. Positive ATRX expression was found in 91.6% (88/96) of IDHWT and 43.7% (7/16) of IDHMUT. TERTp mutations were detected in 70.4% (50/71) of IDHWT. MGMTp methylation was found in 55.5% (35/63) of IDHWT and 84.6% (11/13) of IDHMUT, and as expected, MGMTp methylation was significantly associated with a better response to TMZ. MGMT expression was inversely correlated with MGMTp methylation levels (- 0.506, p < 0.0001), and MGMT low expression were significantly associated with better patient survival. It was also observed that integrating MGMTp methylation and expression, significantly improved the prognostication value. CONCLUSIONS: MGMT mRNA levels evaluated by digital expression were associated with the outcome of TMZ-treated GBM patients. The combination of MGMT methylation and mRNA expression may provide a more accurate prediction of TMZ response in GBM patients.

MGMT-inhibitor in combination with TGF-ßRI inhibitor or CDK 4/6 inhibitor increases temozolomide sensitivity in temozolomide-resistant glioblastoma cells.

BACKGROUND: Glioblastoma (GB) remains an incurable and deadly brain malignancy that often proves resistant to upfront treatment with temozolomide. Nevertheless, temozolomide remains the most commonly prescribed FDA-approved chemotherapy for GB. The DNA repair protein methylguanine-DNA methyl transferase (MGMT) confers resistance to temozolomide. Unsurprisingly temozolomide-resistant tumors tend to possess elevated MGMT protein levels or lack inhibitory MGMT promotor methylation. In this study, cultured human temozolomide resistance GB (43RG) cells were introduced to the MGMT inhibitor O6-benzylguanine combined with temozolomide and either LY2835219 (CDK 4/6 inhibitor) or LY2157299 (TGF-ßRI inhibitor) seeking to overcome GB treatment resistance. METHODS: Treatment effects were assessed using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, western blot, cell viability, and cell cycle progression. RESULTS: Our in vitro study demonstrated that sequential treatment of O6-Benzylguanine with either LY2385219 or LY2157299-enhanced temozolomide enhanced sensitivity in MGMT+ 43RG cells. Importantly, normal human neurons and astrocytes remained impervious to the drug therapies under these conditions. Furthermore, LY2835219 has additional anti-proliferative effects on cell cycling, including induction of an RB-associated G (1) arrest via suppression of cyclin D-CDK4/6-Rb pathway. LY2157299 enhances anti-tumor effect by disrupting TGF-ß-dependent HIF-1α signaling and by activating both Smad and PI3K-AKT pathways towards transcription of S/G2 checkpoints. CONCLUSION: This study establishes the groundwork for the development of a combinatorial pharmacologic approach by using either LY2385219 or LY2157299 inhibitor plus O6-Benzylguanine to augment temozolomide response in temozolomide-resistant GB cells.

PARP­1 inhibition sensitizes temozolomide­treated glioblastoma cell lines and decreases drug resistance independent of MGMT activity and PTEN proficiency.

Information on the mechanisms that are associated with tumor resistance has the potential to provide the fundamental basis for novel therapeutic strategies. In glioblastoma (GBM), predictive biomarkers of cellular responses to temozolomide (TMZ) combined with poly­ADP­ribose polymerase inhibitor (PARPi) remain largely unidentified. In this context, the influence of MGMT (O6­methylguanine DNA methyltransferase) and PTEN (phosphatase and tensin homologue deleted on chromosome ten) has been studied in addition to the occurrence of synthetic lethality involving PTEN and PARPi. The present study investigated whether PARP­1 inhibition by NU1025 may increase the cytotoxicity of TMZ­induced lesions in GBM cells, and whether these mechanisms can be influenced by MGMT and PTEN status. The impact of PTEN deficiency in repair pathways, and the effects of PARP­1 inhibition and PTEN silencing, in terms of synthetic lethality, were also assessed. NU1025 combined with TMZ effectively sensitized TMZ­resistant cells (T98G PTEN­mutated and LN18 PTEN­wild­type) and TMZ­sensitive cells (U251MG PTEN­mutated), in contrast to NU1025 alone. However, the sensitizing effects were not observed in U87MG (PTEN­mutated) cells, suggesting that specific genetic alterations may influence the response to drug treatment. The sensitizing effects occurred independently of MGMT activity, which was evaluated in O6­BG­treated cells. PTEN silencing using small interfering (si)RNA did not sensitize PTEN­proficient cells to TMZ + NU1025, or NU1025 alone, indicating an absence of synthetic lethality. The responses to TMZ + NU1025 involved antiproliferative activity, G2/M arrest, double strand breaks and the induction of apoptosis. Following 20 days of recovery after three consecutive days of TMZ treatment, TMZ­resistant cells were observed. However, when TMZ was combined with NU1025, the viability of T98G and LN18 cells was extremely decreased, indicating a lethal drug combination. Therefore, independently of MGMT proficiency and PTEN status, TMZ combined with PARPi may be a promising strategy that can be used to overcome TMZ acquired resistance in GBM cells.

A co-formulation of interferons type I and II enhances temozolomide response in glioblastoma with unmethylated MGMT promoter status.

Temozolomide (TMZ) is a chemotherapeutic used for the treatment of glioblastoma. The MGMT repair enzyme (O'-(6)-methyl guanine-DNA-methyltransferase) promoter methylation is a predictive biomarker to TMZ response; interferons (IFNs) type I can downregulate MGMT expression improving survival in patients with unmethylated MGMT promoter. HeberFERON is a co-formulation of IFNs type I and II with higher antiproliferative effect over glioblastoma cell lines than individual IFNs. We investigated the proliferative response of patient-derived glioblastoma cultures to HeberFERON and its combination with TMZ in relation to MGMT promoter methylation and the regulation of MGMT transcript after HeberFERON treatment. Eleven glioblastoma-derived cultures, molecularly classified according to TCGA and MGMT promoter methylation, were assayed for proliferation inhibition with HeberFERON at low doses (1-25 IU/mL) [alone or combined with TMZ] or at higher doses (50-200 IU/mL) using CellTiter-Glo Luminescent Cell Viability Assay (Promega). Eight cultures were further treated with 100 IU/mL of HeberFERON for 72 h, total RNA purified (Qiagen) and converted to cDNA (Superscript III kit, Invitrogen) as quantitative PCR templates. Changes of MGMT&P53 transcripts level were monitored. Response of cultures to HeberFERON is variable, dose-dependent and apparently independent from TCGA classification and MGMT methylation status, based on the eight Classical cultures data. When combining HeberFERON with TMZ there was an increase in cell death for cultures, 2/4 with methylated and 5/5 with unmethylated MGMT promoter. In two out five cultures with unmethylated MGMT status, we observed a decrease of MGMT gene levels and an increase in P53 encoding gene levels. HeberFERON and TMZ combination should be further assayed in glioblastoma, mainly for those with unmethylated MGMT promoter.

Genosensor made with a self-assembled monolayer matrix to detect MGMT gene methylation in head and neck cancer cell lines.

DNA methylation is involved in the oncogenesis of head and neck squamous cell carcinoma and could be used for early detection of cancer to increase the chances of cure, but unfortunately diagnosis is usually made at late stages of the disease. In this work we developed genosensors to detect DNA methylation of the MGMT gene in head and neck cancer cell lines. The probe for MGMT promoter methylation was immobilized on gold electrodes modified with 11-mercaptoundecanoic acid (11-MUA) self-assembled monolayers (SAM). Detection was performed with electrochemical impedance spectroscopy, with clear distinction between methylated and non-methylated DNA from head and neck cell lines. The genosensor is sensitive with a low detection limit of 0.24 × 10-12 mol L-1. In addition, the cell lines FaDu, JHU28 and SCC25 for the MGMT gene, could be distinguished from the HN13 cell line which has a high degree of MGMT methylation (97%), thus confirming the selectivity. Samples with different percentages of MGMT DNA methylation could be separated in multidimensional projections using the visualization technique interactive document mapping (IDMAP). The genosensor matrix and the immobilization procedures are generic, and can be extended to other DNA methylation biomarkers.

IKBKE enhances TMZ-chemoresistance through upregulation of MGMT expression in glioblastoma.

PURPOSE: Glioblastoma multiforme (GBM) is the most common and aggressive malignant type of brain tumor. Despite advances in diagnosis and therapy, the prognosis of patients with GBM has remained dismal. Multidrug resistance and high recurrence are two of the major challenges in successfully treating brain tumors. IKBKE (inhibitor of nuclear factor kappa-B kinase subunit epsilon) is a major oncogenic protein in tumors and can inhibit glioblastoma cell proliferation, migration, and tumorigenesis. Our study aimed to investigate the mechanism of IKBKE enhancing the resistance of glioma cells to temozolomide. METHODS: For the in vitro experiments, LN18 and U118 glioblastoma cells were treated with a combination of sh/oe-IKBKE lentivirus and TMZ. Cell proliferation was determined by the EdU assay and colony formation assays. Apoptosis was analyzed by the TUNEL assay. In vivo, LN18 NC and LN18 sh-IKBKE cells were implanted into the cerebrums of nude mice to detect the effect of combination therapy. The protein and mRNA levels were assayed by western blot, immunohistochemistry, and qRT-PCR. RESULTS: In this study, we demonstrated that IKBKE enhances the resistance of glioblastoma cells to temozolomide (TMZ) by activating the AKT/NF-κB signaling pathway to upregulate the expression of the DNA repair enzyme o6-methylguanine-dna methyltransferase (MGMT). In glioblastoma cells, IKBKE knockdown enhances apoptosis and suppresses cell proliferation, clone formation, and tumor development in vivo induced by TMZ. However, overexpression of IKBKE reduces the effects of TMZ. CONCLUSION: Our studies suggest that inhibition of IKBKE can enhance the therapeutic effect of TMZ on GBM in vitro and in vivo, providing new research directions and therapeutic targets for the treatment of GBM.

microRNA-181d associated with the methylation status of the MGMT gene in Glioblastoma multiforme cancer stem cells submitted to treatments with ionizing radiation and temozolomide.

Despite the increased understanding of the oncological mechanisms underlying Glioblastoma multiforme (GBM) pathophysiology, and recent advances in therapeutic strategies such as maximal surgical resection and post-operative radiotherapy with concomitant and adjuvant temozolomide chemotherapy, the prognosis for patients with brain tumors remains limited. Evidences indicate that the assessment of DNA methylation status in cancer stem cells would allow identifying molecules expressed in these cells, to lead to targeted elimination of this critical population from brain tumors, making the glioblastoma treatment more effective. This study aimed to analyze the role of microRNA-181d associated with the methylation status of the O6-methylguanine methyl transferase (MGMT) gene in Glioblastoma multiforme cancer stem cells subjected to treatment with temozolomide and ionizing radiation. Such responses were analyzed in terms of cell survival, evaluation of the MGMT gene methylation status by MS-HRM (Methylation-Sensitive High Resolution Melting), and analysis of miRNA-181d and MGMT gene expression by relative quantification of mRNA levels in cancer stem cells subjected to treatment with temozolomide and ionizing radiation, isolated or combined. We showed that ionizing radiation and temozolomide reduced the viability of cancer stem cells from GBM patients, as well as modified MGMT gene and miRNA-181d expression in cancer stem cells, suggesting that miRNA-181d interferes in the glioblastoma cancer stem cell response to treatment with temozolomide and ionizing radiation.

Association of the Promoter Methylation and the rs12917 Polymorphism of MGMT with Formation of DNA Bulky Adducts and the Risk of Lung Cancer in Mexican Mestizo Population.

O6-Methylguanine-DNA methyltransferase (MGMT) is an enzyme that repairs the DNA damage caused by the tobacco habit, and low activity of this enzyme has been associated with a risk of lung cancer (LC). Our objective was to determine the association of the promoter methylation and the rs12917 polymorphism of MGMT with formation of DNA bulky adducts and the risk of LC in the Mexican Mestizo population. In this study are included 431 subjects. High-resolution melting analysis was used to determine the polymorphism MGMT rs12917 and methylation levels. DNA bulky adducts were determined by 32P-postlabeling. Our results showed that MGMT rs12917 and higher levels of methylation in the MGMT promoter are associated with the risk of LC. The levels of adducts are related with the phe/phe genotype and, only in the cases group, with the hypermethylation (>50%) of MGMT; however, this last association was not statistically significant.

Mifepristone Overcomes Tumor Resistance to Temozolomide Associated with DNA Damage Repair and Apoptosis in an Orthotopic Model of Glioblastoma.

The standard treatment for glioblastoma multiforme (GBM) is surgery followed by chemo/radiotherapy. A major limitation on patient improvement is the high resistance of tumors to drug treatment, likely responsible for their subsequent recurrence and rapid progression. Therefore, alternatives to the standard therapy are necessary. The aim of the present study was to evaluate whether mifepristone, an antihormonal agent, has a synergistic effect with temozolomide (used in standard therapy for gliomas). Whereas the mechanism of temozolomide involves damage to tumor DNA leading to apoptosis, tumor resistance is associated with DNA damage repair through the O6-methylguanine-DNA-methyltransferase (MGMT) enzyme. Temozolomide/mifepristone treatment, herein examined in Wistar rats after orthotopically implanting C6 glioma cells, markedly reduced proliferation. This was evidenced by a decreased level of the following parameters: a proliferation marker (Ki-67), a tumor growth marker (18F-fluorothymidine uptake, determined by PET/CT images), and the MGMT enzyme. Increased apoptosis was detected by the relative expression of related proteins, (e.g. Bcl-2 (B-cell lymphoma 2), Bax (bcl-2-like protein 4) and caspase-3). Thus, greater apoptosis of tumor cells caused by their diminished capacity to repair DNA probably contributed significantly to the enhanced activity of temozolomide. The results suggest that mifepristone could possibly act as a chemo-sensitizing agent for temozolomide during chemotherapy for GBM.

Distribution of tumor-infiltrating immune cells in glioblastoma.

AIM: Evaluation of features related to infiltrating immune cell level in glioblastoma. METHODS: Tumor-infiltrating lymphocytes (TILs) through H&E staining, and TILs (CD3, CD4, CD8 and CD20) and macrophage (CD68 and CD163) levels through immunohistochemistry were evaluated through digital analysis. RESULTS: CD68 (9.1%), CD163 (2.2%), CD3 (1.6%) and CD8 (1.6%) had the highest density. Higher CD4+ was associated with unmethylated MGMT (p = 0.016). Higher CD8+ was associated with larger tumoral size (p = 0.027). Higher CD163+ was associated with higher age (p = 0.044) and recursive partitioning analysis = 4. Women (p < 0.05), total resection (p < 0.05), MGMT-methylation (p < 0.001), radiotherapy (p < 0.001), chemotherapy (p < 0.001) and lower CD4+ (p < 0.05) were associated with longer overall survival. CONCLUSION: Macrophages are more frequent than TILs. Some subsets are associated with clinical features.

Delay in starting radiotherapy due to neoadjuvant therapy does not worsen survival in unresected glioblastoma patients.

PURPOSE: We retrospectively examined the potential effect on overall survival (OS) of delaying radiotherapy to administer neoadjuvant therapy in unresected glioblastoma patients. PATIENTS AND METHODS: We compared OS in 119 patients receiving neoadjuvant therapy followed by standard treatment (NA group) and 96 patients receiving standard treatment without neoadjuvant therapy (NoNA group). The MaxStat package of R identified the optimal cut-off point for waiting time to radiotherapy. RESULTS: OS was similar in the NA and NoNA groups. Median waiting time to radiotherapy after surgery was 13 weeks for the NA group and 4.2 weeks for the NoNA group. The longest OS was attained by patients who started radiotherapy after 12 weeks and the shortest by patients who started radiotherapy within 4 weeks (12.3 vs 6.6 months) (P = 0.05). OS was 6.6 months for patients who started radiotherapy before the optimal cutoff of 6.43 weeks and 19.1 months for those who started after this time (P = 0.005). Patients who completed radiotherapy had longer OS than those who did not, in all 215 patients and in the NA and NoNA groups (P = 0.000). In several multivariate analyses, completing radiotherapy was a universally favorable prognostic factor, while neoadjuvant therapy was never identified as a negative prognostic factor. CONCLUSION: In our series of unresected patients receiving neoadjuvant treatment, in spite of the delay in starting radiotherapy, OS was not inferior to that of a similar group of patients with no delay in starting radiotherapy.

MGMT promoter methylation in Peruvian patients with glioblastoma.

PURPOSE: O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation predicts the outcome and response to alkylating chemotherapy in glioblastoma. The aim of this study is to evaluate the prevalence of MGMT methylation in Peruvian glioblastoma cases. PATIENTS AND METHODS: We evaluated retrospectively 50 cases of resected glioblastoma during the period 2008-2013 at Instituto Nacional de Enfermedades Neoplasicas in Peru. Samples consisted of paraffin embedded and frozen tumour tissue. MGMT-promoter methylation status and the expression level of MGMT gene were evaluated by methylation-specific PCR and real-time PCR, respectively. RESULTS: Unmethylated, methylated and partially methylated statuses were found in 54%, 20% and 26% of paraffin-embedded samples, respectively. Methylation status was confirmed in the Virgen de la Salud Hospital and frozen samples. There was an association between the status of MGMT-promoter methylation and the level of gene expression (p = 0.001). Methylation was associated with increased progression-free survival (p = 0.002) and overall survival (OS) (p < 0.001). CONCLUSION: MGMT-promoter methylation frequency in Peruvian glioblastoma is similar to that reported in other populations and the detection test has been standardised.

MGMT methylation assessment in glioblastoma: MS-MLPA versus human methylation 450K beadchip array and immunohistochemistry.

PURPOSE: The MGMT gene encodes a DNA repair enzyme that counteracts with chemotherapy efficiency, specifically with alkylating agents such as temozolomide (TMZ). It is well established that MGMT methylation should be screened as a predictive marker for TMZ in glioblastoma, and we thus aimed to determine a reliable and practical diagnostic method of MGMT methylation detection. PATIENTS AND METHODS: 55 glioblastomas were investigated for MGMT methylation status using methylation-specific multiplexed ligation probe amplification (MS-MLPA), illumina human methylation 450K BeadChip array (HM450 K) analysis, and compared to MGMT protein expression by immunohistochemistry (IHC) staining. The methylation status of promoter, intron and all MGMT CpG targeted sites were separately correlated to patient's survival. RESULTS: In addition to MS-MLPA and 450 K concordance, our results showed significantly higher overall survival (OS) of patients receiving TMZ and presenting MGMT methylated promoter (mean OS = 21.5 months, p = 0.046). Including all glioblastoma cases and regardless of chemotherapy, MS-MLPA showed significant survival difference between MGMT methylated and unmethylated cases (mean OS = 13, p = 0.021). CONCLUSION: We concluded that in glioblastoma, MGMT promoter methylation predicts TMZ sensitivity. This current comparative analysis leads to consider that MS-MLPA is a valuable as HM450 K array for MGMT methylation status screening.

The GSTM1null (deletion) and MGMT84 rs12917 (Phe/Phe) haplotype are associated with bulky DNA adduct levels in human leukocytes.

Tobacco smoke and air pollutants contain carcinogens, such as polycyclic aromatic hydrocarbons (PAHs) and tobacco specific nitrosamines (TSNA), that are substrates of metabolizing enzymes generating reactive metabolites that can bind to DNA. Variation in the activity of these enzymes may modify the extent to which these metabolites can interact with DNA. We compared the levels of bulky DNA adducts in blood leukocytes from 93 volunteers living in Mexico City with the presence of 13 single nucleotide polymorphisms (SNPs) in genes related to PAH and TSNA metabolism (AhR rs2044853, CYP1A1 rs1048943, CYP1A1 rs1048943, CYP1A1 rs1799814, EPHX1 rs1051740, EPHX1 rs2234922, GSTM1 null, GSTT1 null and GSTP1 rs947894), DNA repair (XRCC1 rs25487, ERCC2 rs13181 and MGMT rs12917) and cell cycle (TP53 rs1042522). (32)P-postlabeling analysis was used to quantify bulky DNA adduct formation. Genotyping was performed using PCR-RFLP. The mean levels of bulky DNA adducts were 8.51±3.66 adducts/10(8) nucleotides (nt) in smokers and 8.38±3.59 adducts/10(8) nt in non-smokers, being the difference not statistically significant. Without taking into account the smoking status, GSTM1 null individuals had a marginally significant lower adduct levels compared with GSTM1 volunteers (p=0.0433) and individuals heterozygous for MGMT Leu/Phe had a higher level of bulky adducts than those who were homozygous wild type (p=0.0170). A multiple regression analysis model showed a significant association between the GSTM1 (deletion) and MGMT rs12917 (Phe/Phe) haplotype and the formation of DNA adducts in smokers (R(2)=0.2401, p=0.0215). The presence of these variants conferred a greater risk for higher adduct levels in this Mexican population.

MGMT and MLH1 methylation in Helicobacter pylori-infected children and adults.

AIM: To evaluate the association between Helicobacter pylori (H. pylori) infection and MLH1 and MGMT methylation and its relationship with microsatellite instability (MSI). METHODS: The methylation status of the MLH1 and MGMT promoter region was analysed by methylation specific methylation-polymerase chain reaction (MSP-PCR) in gastric biopsy samples from uninfected or H. pylori-infected children (n = 50), from adults with chronic gastritis (n = 97) and from adults with gastric cancer (n = 92). MLH1 and MGMT mRNA expression were measured by real-time PCR and normalised to a constitutive gene (ß actin). MSI analysis was performed by screening MSI markers at 4 loci (Bat-25, Bat-26, D17S250 and D2S123) with PCR; PCR products were analysed by single strand conformation polymorphism followed by silver staining. Statistical analyses were performed with either the χ(2) test with Yates continuity correction or Fisher's exact test, and statistical significance for expression analysis was assessed using an unpaired Student's t-test. RESULTS: Methylation was not detected in the promoter regions of MLH1 and MGMT in gastric biopsy samples from children, regardless of H. pylori infection status. The MGMT promoter was methylated in 51% of chronic gastritis adult patients and was associated with H. pylori infection (P < 0.05); this region was methylated in 66% of gastric cancer patients, and the difference in the percentage of methylated samples between these patients and those from H. pylori-infected chronic gastritis patients was statistically significant (P < 0.05). MLH1 methylation frequencies among H. pylori-infected and non-infected chronic gastritis adult patients were 13% and 7%, respectively. We observed methylation of the MLH1 promoter (39%) and increased MSI levels (68%) in samples from gastric cancer patients in comparison to samples from H. pylori-infected adult chronic gastritis patients (P < 0.001 and P < 0.01, respectively). The frequency of promoter methylation for both genes was higher in gastric cancer samples than in H. pylori-positive chronic gastritis samples (P < 0.05). The levels of MLH1 and MGMT mRNA were significantly reduced in chronic gastritis samples that were also hypermethylated (P < 0.01). CONCLUSION: In summary, MGMT and MLH1 methylation did not occur in earlier-stage H. pylori infections and thus might depend on the duration of infection.