MGMT protein expression is a reliable predictive biomarker for temozolomide-containing chemotherapy in osteosarcoma.

The prognosis of patients with osteosarcoma who experience recurrence or progression (R/P) is extremely poor, and more effective and less toxic therapies are needed. In the current study, the clinical data of osteosarcoma patients who experienced R/P were retrospectively analyzed to verify the reliability of O-6-methylguanine-DNA methyltransferase (MGMT) protein expression or MGMT promoter methylation for predicting the response to off-label temozolomide (TMZ)-containing chemotherapy. Of the 30 evaluable patients, 9 (30%) showed no/low MGMT protein expression, whereas all 16 evaluable patients had unmethylated MGMT promoter irrespective of MGMT protein expression levels. Twenty-three patients received TMZ-containing chemotherapy for measurable lesions (n = 14) or as adjuvant therapy following resection of recurrent lesions (n = 9). Among 14 patients with radiologically measurable lesions, the objective response rate was higher in the MGMT no/low-expression group (50.0%) than in the MGMT intermediate/high-expression group with borderline significance (0%, p = 0.066). The 6-month progression-free survival (PFS) rate in patients with radiologically measurable lesions was significantly higher in the MGMT no/low-expression group (50.0%) than in the MGMT intermediate/high-expression group (0%, p = 0.036). In the multivariate analysis of the 23 patients receiving TMZ-containing chemotherapy, MGMT expression and disease status before TMZ-containing chemotherapy were significantly associated with PFS. No severe adverse effects were observed during TMZ-containing chemotherapy. MGMT protein expression, but not MGMT promoter methylation, could predict a favorable outcome in patients receiving TMZ-containing chemotherapy.

Unveiling the role of O(6)-methylguanine-DNA methyltransferase in cancer therapy: insights into alkylators, pharmacogenomics, and others.

Cancer chemotherapy is advancing as we understand how cellular mechanisms and drugs interact, particularly involving the enzyme MGMT, which repairs DNA damage that can cause cancer. This review examines MGMT's role in DNA repair, its impact on chemotherapy, and its complex interaction with radiation therapy. MGMT activity can both protect against mutations and cause drug resistance. Modulating MGMT could improve treatment efficacy and tailoring therapy to MGMT status may enhance patient outcomes. Understanding MGMT is crucial for developing precise cancer treatments and advancing patient care.

Prediction of O(6)-methylguanine-DNA methyltransferase promoter methylation status in IDH-wildtype glioblastoma using MRI histogram analysis.

To evaluate the utility of magnetic resonance imaging (MRI) histogram parameters in predicting O(6)-methylguanine-DNA methyltransferase promoter (pMGMT) methylation status in IDH-wildtype glioblastoma (GBM). From November 2021 to July 2023, forty-six IDH-wildtype GBM patients with known pMGMT methylation status (25 unmethylated and 21 methylated) were enrolled in this retrospective study. Conventional MRI signs (including location, across the midline, margin, necrosis/cystic changes, hemorrhage, and enhancement pattern) were assessed and recorded. Histogram parameters were extracted and calculated by Firevoxel software based on contrast-enhanced T1-weighted images (CET1). Differences and diagnostic performance of conventional MRI signs and histogram parameters between the pMGMT-unmethylated and pMGMT-methylated groups were analyzed and compared. No differences were observed in the conventional MRI signs between pMGMT-unmethylated and pMGMT-methylated groups (all p > 0.05). Compared with the pMGMT-methylated group, pMGMT-unmethylated showed a higher minimum, mean, Perc.01, Perc.05, Perc.10, Perc.25, Perc.50, and coefficient of variation (CV) (all p < 0.05). Among all significant CET1 histogram parameters, minimum achieved the best distinguishing performance, with an area under the curve of 0.836. CET1 histogram parameters could provide additional value in predicting pMGMT methylation status in patients with IDH-wildtype GBM, with minimum being the most promising parameter.

The interplay mechanism between IDH mutation, MGMT-promoter methylation, and PRMT5 activity in the progression of grade 4 astrocytoma: unraveling the complex triad theory.

Background: The dysregulation of Isocitrate dehydrogenase (IDH) and the subsequent production of 2-Hydroxyglutrate (2HG) may alter the expression of epigenetic proteins in Grade 4 astrocytoma. The interplay mechanism between IDH, O-6-methylguanine-DNA methyltransferase (MGMT)-promoter methylation, and protein methyltransferase proteins-5 (PRMT5) activity, with tumor progression has never been described. Methods: A retrospective cohort of 34 patients with G4 astrocytoma is classified into IDH-mutant and IDH-wildtype tumors. Both groups were tested for MGMT-promoter methylation and PRMT5 through methylation-specific and gene expression PCR analysis. Inter-cohort statistical significance was evaluated. Results: Both IDH-mutant WHO grade 4 astrocytomas (n = 22, 64.7%) and IDH-wildtype glioblastomas (n = 12, 35.3%) had upregulated PRMT5 gene expression except in one case. Out of the 22 IDH-mutant tumors, 10 (45.5%) tumors showed MGMT-promoter methylation and 12 (54.5%) tumors had unmethylated MGMT. All IDH-wildtype tumors had unmethylated MGMT. There was a statistically significant relationship between MGMT-promoter methylation and IDH in G4 astrocytoma (p-value = 0.006). Statistically significant differences in progression-free survival (PFS) were also observed among all G4 astrocytomas that expressed PRMT5 and received either temozolomide (TMZ) or TMZ plus other chemotherapies, regardless of their IDH or MGMT-methylation status (p-value=0.0014). Specifically, IDH-mutant tumors that had upregulated PRMT5 activity and MGMT-promoter methylation, who received only TMZ, have exhibited longer PFS. Conclusions: The relationship between PRMT5, MGMT-promoter, and IDH is not tri-directional. However, accumulation of D2-hydroxyglutarate (2-HG), which partially activates 2-OG-dependent deoxygenase, may not affect their activities. In IDH-wildtype glioblastomas, the 2HG-2OG pathway is typically inactive, leading to PRMT5 upregulation. TMZ alone, compared to TMZ-plus, can increase PFS in upregulated PRMT5 tumors. Thus, using a PRMT5 inhibitor in G4 astrocytomas may help in tumor regression.

MGMT Methylation and Differential Survival Impact by Sex in Glioblastoma.

Introduction: Sex differences in glioblastoma (GBM) have been observed in incidence, genetic and epigenetic alterations, and immune response. These differences have extended to the methylation of the MGMT promoter, which critically impacts temozolomide resistance. However, the association between sex, MGMT methylation, and survival is poorly understood, which this study sought to evaluate. Methods: A retrospective cohort study was conducted and reported following STROBE guidelines, based on adults with newly diagnosed GBM who received their first surgical intervention at Cleveland Clinic (Ohio, USA) between 2012 and 2018. Kaplan-Meier and multivariable Cox proportional hazards models were used to analyze the association between sex and MGMT promoter methylation status on overall survival (OS). MGMT was defined as methylated if the mean of CpG 1-5 ≥ 12. Propensity score matching was performed on a subset of patients to evaluate the effect of individual CpG site methylation. Results: A total of 464 patients had documented MGMT methylation status with a mean age of 63.4 (range 19-93) years. A total of 170 (36.6%) were female, and 133 (28.7%) received gross total resection as a first intervention. A total of 42.5% were MGMT methylated, with females more often having MGMT methylation than males (52.1% vs. 37.4%, p = 0.004). In univariable analysis, OS was significantly longer for MGMT promoter methylated than un-methylated groups for females (2 yr: 36.8% vs. 11.1%; median: 18.7 vs. 9.5 months; p = 0.001) but not for males (2 yr: 24.3% vs. 12.2%; median: 12.4 vs. 11.3 months; p = 0.22, p for MGMT-sex interaction = 0.02). In multivariable analysis, MGMT un-methylated versus methylated promoter females (2.07; 95% CI, 1.45-2.95; p < 0.0001) and males (1.51; 95% CI, 1.14-2.00; p = 0.004) had worse OS. Within the MGMT promoter methylated group, males had significantly worse OS than females (1.42; 95% CI: 1.01-1.99; p = 0.04). Amongst patients with data on MGMT CpG promoter site methylation values (n = 304), the median (IQR) of CpG mean methylation was 3.0% (2.0, 30.5). Females had greater mean CpG methylation than males (11.0 vs. 3.0, p < 0.002) and higher per-site CpG methylation with a significant difference at CPG 1, 2, and 4 (p < 0.008). After propensity score matching, females maintained a significant survival benefit (18.7 vs. 10.0 months, p = 0.004) compared to males (13.0 vs. 13.6 months, p = 0.76), and the pattern of difference was significant (P for CpG-sex interaction = 0.03). Conclusions: In this study, females had higher mean and individual CpG site methylation and received a greater PFS and OS benefit by MGMT methylation that was not seen in males despite equal degrees of CpG methylation.

DIAPH3 predicts survival of patients with MGMT-methylated glioblastoma.

Background: Glioblastoma is one of the most aggressive primary brain tumors, with a poor outcome despite multimodal treatment. Methylation of the MGMT promoter, which predicts the response to temozolomide, is a well-established prognostic marker for glioblastoma. However, a difference in survival can still be detected within the MGMT methylated group, with some patients exhibiting a shorter survival than others, emphasizing the need for additional predictive factors. Methods: We analyzed DIAPH3 expression in glioblastoma samples from the cancer genome atlas (TCGA). We also retrospectively analyzed one hundred seventeen histological glioblastomas from patients operated on at Saint-Luc University Hospital between May 2013 and August 2019. We analyzed the DIAPH3 expression, explored the relationship between mRNA levels and Patient's survival after the surgical resection. Finally, we assessed the methylation pattern of the DIAPH3 promoter using a targeted deep bisulfite sequencing approach. Results: We found that 36% and 1% of the TCGA glioblastoma samples exhibit copy number alterations and mutations in DIAPH3, respectively. We scrutinized the expression of DIAPH3 at single cell level and detected an overlap with MKI67 expression in glioblastoma proliferating cells, including neural progenitor-like, oligodendrocyte progenitor-like and astrocyte-like states. We quantitatively analyzed DIAPH3 expression in our cohort and uncovered a positive correlation between DIAPH3 mRNA level and patient's survival. The effect of DIAPH3 was prominent in MGMT-methylated glioblastoma. Finally, we report that the expression of DIAPH3 is at least partially regulated by the methylation of three CpG sites in the promoter region. Conclusion: We propose that combining the DIAPH3 expression with MGMT methylation could offer a better prediction of survival and more adapted postsurgical treatment for patients with MGMT-methylated glioblastoma.

Identification of TMZ resistance-associated histone post-translational modifications in glioblastoma using multi-omics data.

BACKGROUD: Glioblastoma multiforme (GBM) is among the most aggressive cancers, with current treatments limited in efficacy. A significant hurdle in the treatment of GBM is the resistance to the chemotherapeutic agent temozolomide (TMZ). The methylation status of the MGMT promoter has been implicated as a critical biomarker of response to TMZ. METHODS: To explore the mechanisms underlying resistance, we developed two TMZ-resistant GBM cell lines through a gradual increase in TMZ exposure. Transcriptome sequencing of TMZ-resistant cell lines revealed that alterations in histone post-translational modifications might be instrumental in conferring TMZ resistance. Subsequently, multi-omics analysis suggests a strong association between histone H3 lysine 9 acetylation (H3K9ac) levels and TMZ resistance. RESULTS: We observed a significant correlation between the expression of H3K9ac and MGMT, particularly in the unmethylated MGMT promoter samples. More importantly, our findings suggest that H3K9ac may enhance MGMT transcription by facilitating the recruitment of the SP1 transcription factor to the MGMT transcription factor binding site. Additionally, by analyzing single-cell transcriptomics data from matched primary and recurrent GBM tumors treated with TMZ, we modeled the molecular shifts occurring upon tumor recurrence. We also noted a reduction in tumor stem cell characteristics, accompanied by an increase in H3K9ac, SP1, and MGMT levels, underscoring the potential role of H3K9ac in tumor relapse following TMZ therapy. CONCLUSIONS: The increase in H3K9ac appears to enhance the recruitment of the transcription factor SP1 to its binding sites within the MGMT locus, consequently upregulating MGMT expression and driving TMZ resistance in GBM.

MGMT promoter methylation prediction based on multiparametric MRI via vision graph neural network.

Purpose: Glioblastoma (GBM) is aggressive and malignant. The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter in GBM tissue is considered an important biomarker for developing the most effective treatment plan. Although the standard method for assessing the MGMT promoter methylation status is via bisulfite modification and deoxyribonucleic acid (DNA) sequencing of biopsy or surgical specimens, a secondary automated method based on medical imaging may improve the efficiency and accuracy of those tests. Approach: We propose a deep vision graph neural network (ViG) using multiparametric magnetic resonance imaging (MRI) to predict the MGMT promoter methylation status noninvasively. Our model was compared to the RSNA radiogenomic classification winners. The dataset includes 583 usable patient cases. Combinations of MRI sequences were compared. Our multi-sequence fusion strategy was compared with those using single MR sequences. Results: Our best model [Fluid Attenuated Inversion Recovery (FLAIR), T1-weighted pre-contrast (T1w), T2-weighted (T2)] outperformed the winning models with a test area under the curve (AUC) of 0.628, an accuracy of 0.632, a precision of 0.646, a recall of 0.677, a specificity of 0.581, and an F1 score of 0.661. Compared to the winning models with single MR sequences, our ViG utilizing fused-MRI showed a significant improvement statistically in AUC scores, which are FLAIR (p=0.042), T1w (p=0.017), T1wCE (p=0.001), and T2 (p=0.018). Conclusions: Our model is superior to challenge champions. A graph representation of the medical images enabled good handling of complexity and irregularity. Our work provides an automatic secondary check pipeline to ensure the correctness of MGMT methylation status prediction.

Quality assessment of the MRI-radiomics studies for MGMT promoter methylation prediction in glioma: a systematic review and meta-analysis.

OBJECTIVES: To evaluate the methodological quality and diagnostic accuracy of MRI-based radiomic studies predicting O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status in gliomas. METHODS: PubMed Medline, EMBASE, and Web of Science were searched to identify MRI-based radiomic studies on MGMT methylation in gliomas published until December 31, 2022. Three raters evaluated the study methodological quality with Radiomics Quality Score (RQS, 16 components) and Transparent Reporting of a Multivariable Prediction Model for Individual Prognosis Or Diagnosis (TRIPOD, 22 items) scales. Risk of bias and applicability concerns were assessed with QUADAS-2 tool. A meta-analysis was performed to estimate the pooled area under the curve (AUC) and to assess inter-study heterogeneity. RESULTS: We included 26 studies, published from 2016. The median RQS total score was 8 out of 36 (22%, range 8-44%). Thirteen studies performed external validation. All studies reported AUC or accuracy, but only 4 (15%) performed calibration and decision curve analysis. No studies performed phantom analysis, cost-effectiveness analysis, and prospective validation. The overall TRIPOD adherence score was between 50% and 70% in 16 studies and below 50% in 10 studies. The pooled AUC was 0.78 (95% CI, 0.73-0.83, I2 = 94.1%) with a high inter-study heterogeneity. Studies with external validation and including only WHO-grade IV gliomas had significantly lower AUC values (0.65; 95% CI, 0.57-0.73, p < 0.01). CONCLUSIONS: Study RQS and adherence to TRIPOD guidelines was generally low. Radiomic prediction of MGMT methylation status showed great heterogeneity of results and lower performances in grade IV gliomas, which hinders its current implementation in clinical practice. CLINICAL RELEVANCE STATEMENT: MGMT promoter methylation status appears to be variably correlated with MRI radiomic features; radiomic models are not sufficiently robust to be integrated into clinical practice to accurately predict MGMT promoter methylation status in patients with glioma before surgery. KEY POINTS: • Adherence to the indications of TRIPOD guidelines was generally low, as was RQS total score. • MGMT promoter methylation status prediction with MRI radiomic features provided heterogeneous diagnostic accuracy results across studies. • Studies that included grade IV glioma only and performed external validation had significantly lower diagnostic accuracy than others.

MGMT in TMZ-based glioma therapy: Multifaceted insights and clinical trial perspectives.

Temozolomide (TMZ) is the most preferred and approved chemotherapeutic drug for either first- or second-line chemotherapy for glioma patients across the globe. In glioma patients, resistance to treatment with alkylating drugs like TMZ is known to be conferred by exalted levels of MGMT gene expression. On the contrary, epigenetic silencing through MGMT gene promoter methylation leading to subsequent reduction in MGMT transcription and protein expression, is predicted to have a response favoring TMZ treatment. Thus, MGMT protein level in cancer cells is a crucial determining factor in indicating and predicting the choice of alkylating agents in chemotherapy or choosing glioma patients directly for a second line of treatment. Thus, in-depth research is necessary to achieve insights into MGMT gene regulation that has recently enticed a fascinating interest in epigenetic, transcriptional, post-transcriptional, and post-translational levels. Furthermore, MGMT promoter methylation, stability of MGMT protein, and related subsequent adaptive responses are also important contributors to strategic developments in glioma therapy. With applications to its identification as a prognostic biomarker, thus predicting response to advanced glioma therapy, this review aims to concentrate on the mechanistic role and regulation of MGMT gene expression at epigenetic, transcriptional, post-transcriptional, and post-translational levels functioning under the control of multiple signaling dynamics.

AI-driven estimation of O6 methylguanine-DNA-methyltransferase (MGMT) promoter methylation in glioblastoma patients: a systematic review with bias analysis.

BACKGROUND: Accurate and non-invasive estimation of MGMT promoter methylation status in glioblastoma (GBM) patients is of paramount clinical importance, as it is a predictive biomarker associated with improved overall survival (OS). In response to the clinical need, recent studies have focused on the development of non-invasive artificial intelligence (AI)-based methods for MGMT estimation. In this systematic review, we not only delve into the technical aspects of these AI-driven MGMT estimation methods but also emphasize their profound clinical implications. Specifically, we explore the potential impact of accurate non-invasive MGMT estimation on GBM patient care and treatment decisions. METHODS: Employing a PRISMA search strategy, we identified 33 relevant studies from reputable databases, including PubMed, ScienceDirect, Google Scholar, and IEEE Explore. These studies were comprehensively assessed using 21 diverse attributes, encompassing factors such as types of imaging modalities, machine learning (ML) methods, and cohort sizes, with clear rationales for attribute scoring. Subsequently, we ranked these studies and established a cutoff value to categorize them into low-bias and high-bias groups. RESULTS: By analyzing the 'cumulative plot of mean score' and the 'frequency plot curve' of the studies, we determined a cutoff value of 6.00. A higher mean score indicated a lower risk of bias, with studies scoring above the cutoff mark categorized as low-bias (73%), while 27% fell into the high-bias category. CONCLUSION: Our findings underscore the immense potential of AI-based machine learning (ML) and deep learning (DL) methods in non-invasively determining MGMT promoter methylation status. Importantly, the clinical significance of these AI-driven advancements lies in their capacity to transform GBM patient care by providing accurate and timely information for treatment decisions. However, the translation of these technical advancements into clinical practice presents challenges, including the need for large multi-institutional cohorts and the integration of diverse data types. Addressing these challenges will be critical in realizing the full potential of AI in improving the reliability and accessibility of MGMT estimation while lowering the risk of bias in clinical decision-making.

Combined Lanreotide Autogel and Temozolomide Treatment of Progressive Pancreatic and Intestinal Neuroendocrine Tumors: The Phase II SONNET Study.

BACKGROUND: In advanced neuroendocrine tumors (NET), antiproliferative treatment options beyond somatostatin analogs remain limited. Temozolomide (TMZ) has shown efficacy in NET alone or combined with other drugs. MATERIALS AND METHODS: SONNET (NCT02231762) was an open, multicenter, prospective, phase II study to evaluate lanreotide autogel 120 mg (LAN) plus TMZ in patients with progressive advanced/metastatic grade 1/2 gastroenteropancreatic (GEP) NET or of unknown primary. Patients could be enrolled at first-line or higher therapy line. The primary endpoint was disease control rate ([DCR], rate of stable disease [SD], partial [PR], and complete response [CR]) at 6 months of LAN and TMZ. Patients with nonfunctioning (NF) NET without progression at 6 months were randomized to 6-month LAN maintenance or watch and wait, patients with functioning (F)-NET with clinical benefit (PR, SD) continued on LAN. RESULTS: Fifty-seven patients were recruited. The majority of patients received the study drug at second or higher treatment line and had an NET G2. DCR at 6 months LAN and TMZ was 73.5%. After 6 months of further LAN maintenance, 54.5% of patients with F-NET and 71.4% with NF-NET had SD or PR vs 41.7% with NF-NET on observation only. LAN and TMZ were effective in all subgroups analyzed. At 12 months of follow-up, median progression-free survival was 11.1 months. Median serum chromogranin A decreased except in NF-NET on observation. O6-methylguanine DNA methyltransferase promoter methylation appeared to better reflect TMZ response than loss of gene expression. During combination therapy, the most frequent treatment-emergent adverse events grade 3/4 reported were nausea (14%), thrombocytopenia (12.3%), and neutropenia (8.8%). Four deaths were reported resulting from severe adverse events not considered related to study medication. CONCLUSIONS: LAN plus TMZ is a treatment option for patients with progressive GEP-NET with more aggressive biological profile showing a manageable safety profile.

Evaluation of mutations on O6-methylguanine methyl transferase structure and its interactions: molecular dynamics simulation study.

O6-methylguanine DNA methyl transferase (MGMT) is a significant vehicle for the cellular clearance of alkyl lesions, particularly the methyl group of the O-6 and O-4 positions of guanine and thymine, respectively. Many publications have studied the correlation between polymorphisms in MGMT and susceptibility to various cancers. In the present study, we investigated the consequence of L84F, common single-nucleotide polymorphism, K125E, site-specific mutagenesis, and L84F/K125E on conformation, stability, and behavior of MGMT in the free form and interaction with proliferating cell nuclear antigen (PCNA) and DNA as partners in the biochemical network by using molecular dynamics simulation method. Our results showed that all free variants of MGMT differed from the native form. However, among all free variants of MGMT, the L84F/K125E variant exhibited similar properties compared with the wild-type. In contrast, in complex modes, only amino acid residues of the L84F variant are involved in the interactions with PCNA and DNA somewhat differently relative to the wild-type. Furthermore, L84F SNP showed the highest binding free energy compared to other variants and native forms. These alterations in the amino acids and binding free energy of L84F relative to the native are the reasons for changing its region connection compared to the native form. Therefore, we propose conducting further investigations into the impact of inhibitors or chemotherapeutic agents to assess their effectiveness on MGMT variants compared to the wild-type, aiming to reduce the cost of cancer treatment that will depend on inhibiting native MGMT protein.Communicated by Ramaswamy H. Sarma.

Circular DNAzyme-Switched CRISPR/Cas12a Assay for Electrochemiluminescent Response of Demethylase Activity.

DNA nanostructure provides powerful tools for DNA demethylase activity detection, but its stability has been significantly challenged. By virtue of circular DNA with resistance to exonuclease degradation, herein, the circular DNAzyme duplex with artificial methylated modification was constructed to identify the target and output the DNA activators to drive the CRISPR/Cas12a, constructing an "on-off-on" electrochemiluminescence (ECL) biosensor for monitoring the activity of the O6-methylguanine-DNA methyltransferase (MGMT). Specifically, the circular DNAzyme duplex consisted of the chimeric RNA-DNA substrate ring with double activator sequences and two single-stranded DNAzymes, whose catalytic domains were premodified with the methyl groups. When the MGMT was present, the methylated DNAzymes were repaired and restored the catalytic activity to cleave the chimeric RNA-DNA substrates, followed by the output of DNA activators to initiate the CRISPR/Cas12a. Subsequently, the ECL signals of silver nanoparticle-modified SnO2 nanospheres (Ag@SnO2) were recovered by releasing the ferrocene-labeled quenching probes (Fc-DNA) from the electrode surface because of the trans-cleavage activity of CRISPR/Cas12a, thus achieving the specific and sensitive ECL detection of MGMT from 2.5 × 10-4 to 2.5 × 102 ng/mL with a low limit (9.69 × 10-5 ng/mL). This strategy affords novel ideas and insights into research on how to project stable nucleic acid probes to detect DNA demethylases beyond traditional methods.

Proteomic and metabolomic analyses illustrate the mechanisms of expression of the O6 -methylguanine-DNA methyltransferase gene in glioblastoma.

AIM: Glioblastoma (GBM) has been reported to be the most common high-grade primary malignant brain tumor in clinical practice and has a poor prognosis. O6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation has been related to prolonged overall survival (OS) in GBM patients after temozolomide treatment. METHODS: Proteomics and metabolomics were combined to explore the dysregulated metabolites and possible protein expression alterations in white matter (control group), MGMT promoter unmethylated GBM (GBM group) or MGMT promoter methylation positive GBM (MGMT group). RESULTS: In total, 2745 upregulated and 969 downregulated proteins were identified in the GBM group compared to the control group, and 131 upregulated and 299 downregulated proteins were identified in the MGMT group compared to the GBM group. Furthermore, 131 upregulated and 299 downregulated metabolites were identified in the GBM group compared to the control group, and 187 upregulated and 147 downregulated metabolites were identified in the MGMT group compared to the GBM group. The results showed that 94 upregulated and 19 downregulated proteins and 20 upregulated and 16 downregulated metabolites in the MGMT group were associated with DNA repair. KEGG pathway enrichment analysis illustrated that the dysregulated proteins and metabolites were involved in multiple metabolic pathways, including the synthesis and degradation of ketone bodies, amino sugar and nucleotide sugar metabolism. Moreover, integrated metabolomics and proteomics analysis was performed, and six key proteins were identified in the MGMT group and GBM group. Three key pathways were recognized as potential biomarkers for recognizing MGMT promoter unmethylated GBM and MGMT promoter methylation positive GBM from GBM patient samples, with areas under the curve of 0.7895, 0.7326 and 0.7026, respectively. CONCLUSION: This study provides novel mechanisms to understand methylation in GBM and identifies some biomarkers for the prognosis of two different GBM types, MGMT promoter unmethylated or methylated GBM, by using metabolomics and proteomics analyses.

Enhancing temozolomide antiglioma response by inhibiting O6-methylguanine-DNA methyltransferase with selected phytochemicals: in silico and in vitro approach.

The aim of our study was to investigate the potential of rutin, catechin, dehydrozingerone, naringenin, and quercetin, both alone and in combination with temozolomide, to inhibit the expression of O6-methylguanine-DNA methyltransferase (MGMT) in glioma cells. MGMT has been shown to be a major cause of temozolomide resistance in glioma. Our study used both in silico and in vitro methods to assess the inhibitory activity of these phytochemicals on MGMT, with the goal of identifying the most effective combination of compounds for reducing temozolomide resistance. After conducting an initial in silico screening of natural compounds against MGMT protein, five phytochemicals were chosen based on their high docking scores and favorable binding energies. From the molecular docking and simulation studies, we found that quercetin showed a good inhibitory effect of MGMT with its high binding affinity. C6 glioma cells showed increased cytotoxicity when treated with the temozolomide and quercetin combination. It was understood from the isobologram and combination index plot that the drug combination showed a synergistic effect at the lowest dose. Quercetin when combined with temozolomide significantly decreased the MGMT levels in C6 cells in comparison with the other drugs as estimated by ELISA. The percentage of apoptotic cells increased significantly in the temozolomide-quercetin group indicating the potency of quercetin in decreasing the resistance of temozolomide as confirmed by acridine orange/ethidium bromide staining. Our experiment hence suggests that temozolomide resistance can be reduced by combining the drug with quercetin which will serve as an effective therapeutic target for glioblastoma treatment. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03821-7.

DDX5 (p68) orchestrates ß-catenin, RelA and SP1 mediated MGMT gene expression in human colon cancer cells: Implication in TMZ chemoresistance.

DDX5 (p68) upregulation has been linked with various cancers of different origins, especially Colon Adenocarcinomas. Similarly, across cancers, MGMT has been identified as the major contributor of chemoresistance against DNA alkylating agents like Temozolomide (TMZ). TMZ is an emerging potent chemotherapeutic agent across cancers under the arena of drug repurposing. Recent studies have established that patients with open MGMT promoters are prone to be innately resistant or acquire resistance against TMZ compared to its closed conformation. However, not much is known about the transcriptional regulation of MGMT gene in the context of colon cancer. This necessitates studying MGMT gene regulation which directly impacts the cellular potential to develop chemoresistance against alkylating agents. Our study aims to uncover an unidentified mechanism of DDX5-mediated MGMT gene regulation. Experimentally, we found that both mRNA and protein expression levels of MGMT were elevated in response to p68 overexpression in multiple human colon cancer cell lines and vice-versa. Since p68 cannot directly interact with the MGMT promoter, transcription factors viz., ß-catenin, RelA (p65) and SP1 were also studied as reported contributors. Through co-immunoprecipitation and GST-pull-down studies, p68 was established as an interacting partner of SP1 in addition to ß-catenin and NF-κB (p50-p65). Mechanistically, luciferase reporter and chromatin-immunoprecipitation assays demonstrated that p68 interacts with the MGMT promoter via TCF4-LEF, RelA and SP1 sites to enhance its transcription. To the best of our knowledge, this is the first report of p68 as a transcriptional co-activator of MGMT promoter and our study identifies p68 as a novel and master regulator of MGMT gene expression.

Stereotactic Radiosurgery for Contrast-Enhancing Satellite Nodules in Recurrent Glioblastoma: A Rare Case Series From a Single Institution.

Introduction Glioblastoma (GBM) is the most common malignant adult brain tumor and is invariably fatal. The standard treatment for GBM involves resection where possible, followed by chemoradiation per Stupp's protocol. We frequently use stereotactic radiosurgery (SRS) as a single-fraction treatment for small (volume ≤ 1cc) nodular recurrent GBM to the contrast-enhancing target on T1 MRI scan. In this paper, we aimed to evaluate the safety and efficacy of SRS for patients with contrast-enhancing satellite nodules in recurrent GBM. Methods This retrospective study analyzed the clinical and radiological outcomes of five patients who underwent CyberKnife (Accuray Inc., Sunnyvale, California) SRS at the institute between 2013 and 2022. Results From 96 patients receiving SRS for GBM, five (four males, one female; median age 53) had nine distinct new satellite lesions on MRI, separate from their primary tumor beds. Those nine lesions were treated with a median margin dose of 20 Gy in a single fraction. The three-, six, and 12-month local tumor control rates were 77.8%, 66.7%, and 26.7%, respectively. Median progression-free survival (PFS) was seven months, median overall survival following SRS was 10 months, and median overall survival (OS) was 35 months. Interestingly, the only lesion that did not show radiological progression was separate from the T2-fluid attenuated inversion recovery (FLAIR) signal of the main tumor. Conclusion Our SRS treatment outcomes for recurrent GBM satellite lesions are consistent with existing findings. However, in a unique case, a satellite nodule distinct from the primary tumor's T2-FLAIR signal and treated with an enlarged target volume showed promising control until the patient's demise. This observation suggests potential research avenues, given the limited strategies for 'multicentric' GBM lesions.

The impact of survivorship bias in glioblastoma research.

Despite advances in the therapy of Central Nervous System (CNS) malignancies, treatment of glioblastoma (GB) poses significant challenges due to GB resistance and high recurrence rates following post-operative radio-chemotherapy. The majority of prognostic and predictive GB biomarkers are currently developed using tumour samples obtained through surgical interventions. However, the selection criteria adopted by different neurosurgeons to determine which cases are suitable for surgery make operated patients not representative of all GB cases. Particularly, geriatric and frail individuals are excluded from surgical consideration in some cancer centers. Such selection generates a survival (or selection) bias that introduces limitations, rendering the patients or data chosen for downstream analyses not representative of the entire community. In this review, we discuss the implication of survivorship bias on current and novel biomarkers for patient selection, stratification, therapy, and outcome analyses.

Differences in clinical outcomes based on molecular markers in glioblastoma patients treated with concurrent tumor-treating fields and chemoradiation: exploratory analysis of the SPARE trial.

BACKGROUND: Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Despite enormous research efforts, GBM remains a deadly disease. The standard-of-care treatment for patients with newly diagnosed with GBM as per the National Cancer Comprehensive Cancer Network (NCCN) is maximal safe surgical resection followed by concurrent chemoradiation and maintenance temozolomide (TMZ) with adjuvant tumor treating fields (TTF). TTF is a non-pharmacological intervention that delivers low-intensity, intermediate frequency alternating electric fields that arrests cell proliferation by disrupting the mitotic spindle. TTF have been shown in a large clinical trial to improve patient outcomes when added to radiation and chemotherapy. The SPARE trail (Scalp-sparing radiation with concurrent temozolomide and tumor treating fields) evaluated adding TTF concomitantly to radiation and chemotherapy. METHODS: This study is an exploratory analysis of the SPARE trial looking at the prognostic significance of common GBM molecular alterations, namely MGMT, EGFR, TP53, PTEN and telomerase reverse transcriptase (TERT), in this cohort of patients treated with concomitant TTF with radiation and chemotherapy. RESULTS: As expected, MGMT promoter methylation was associated with improved overall survival (OS) and progression-free survival (PFS) in this cohort. In addition, TERT promoter mutation was associated with improved OS and PFS in this cohort as well. CONCLUSIONS: Leveraging the molecular characterization of GBM alongside advancing treatments such as chemoradiation with TTF presents a new opportunity to improve precision oncology and outcomes for GBM patients.