Adaptive fine-tuning based transfer learning for the identification of MGMT promoter methylation status.

BACKGROUND: Glioblastoma Multiforme (GBM) is an aggressive form of malignant brain tumor with a generally poor prognosis. O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation has been shown to be a predictive bio-marker for resistance to treatment of GBM, but it is invasive and time-consuming to determine methylation status. There has been effort to predict the MGMT methylation status through analyzing MRI scans using machine learning, which only requires pre-operative scans that are already part of standard-of-care for GBM patients. Purpose: To improve the performance of conventional transfer learning in the identification of MGMT promoter methylation status, we developed a 3D SpotTune network with adaptive fine-tuning capability. Using the pretrained weights of MedicalNet with the SpotTune network, we compared its performance with a randomly initialized network for different combinations of MR modalities. Methods: Using a ResNet50 as the base network, three categories of networks are created: 1) A 3D SpotTune network to process volumetric MR images, 2) a network with randomly initialized weights, and 3) a network pre-trained on MedicalNet. These three networks are trained and evaluated using a public GBM dataset provided by the University of Pennsylvania. The MRI scans from 240 patients are used, with 11 different modalities corresponding to a set of perfusion, diffusion, and structural scans. The performance is evaluated using 5-fold cross validation with a hold-out testing dataset. Results: The SpotTune network showed better performance than the randomly initialized network. The best performing SpotTune model achieved an area under the Receiver Operating Characteristic curve (AUC), average precision of the precision-recall curve (AP), sensitivity, and specificity values of 0.6604, 0.6179, 0.6667, and 0.6061 respectively. Conclusions: SpotTune enables transfer learning to be adaptive to individual patients, resulting in improved performance in predicting MGMT promoter methylation status in GBM using equivalent MRI modalities as compared to a randomly initialized network.

MGMT promoter methylation is a strong prognostic factor for survival after progression in high-grade gliomas.

BACKGROUND: High-grade gliomas (HGGs) have a rapid relapse and short survival. Studies have identified many clinical characteristics and biomarkers associated with progression-free survival (PFS) and over-survival (OS). However, there has not yet a comprehensive study on survival after the first progression (SAP). METHODS: From CGGA and TCGA, 319 and 308 HGGs were confirmed as the first progression. The data on clinical characteristics and biomarkers were analyzed in accordance with OS, PFS, and SAP. RESULTS: Analysis of 319 patients from CGGA, significant predictors of improved OS/PFS/SAP were WHO grade, MGMT promoter methylation, and Ki-67 expression in univariate analysis. Further multivariate analysis showed MGMT promoter methylation and Ki-67 expression were independent predictors. However, an analysis of 308 patients from TCGA found MGMT promoter methylation is the only prognostic marker. A longer SAP was observed in patients with methylated MGMT promoter after standard chemoradiotherapy. In our data, HGGs could be divided into low, intermediate, and high-risk groups for SAP by MGMT methylation and Ki-67 expression. CONCLUSIONS: Patients with MGMT promoter methylation have a prolonger SAP after standard chemoradiotherapy. HGGs could be divided into low, intermediate, and high-risk groups for SAP according to MGMT status and Ki-67 expression.

MEN1 Deficiency-Driven Activation of the ß-Catenin-MGMT Axis Promotes Pancreatic Neuroendocrine Tumor Growth and Confers Temozolomide Resistance.

O6-methylguanine DNA methyltransferase (MGMT) removes alkyl adducts from the guanine O6 position (O6-MG) and repairs DNA damage. High MGMT expression results in poor response to temozolomide (TMZ). However, the biological importance of MGMT and the mechanism underlying its high expression in pancreatic neuroendocrine tumors (PanNETs) remain elusive. Here, it is found that MGMT expression is highly elevated in PanNET tissues compared with paired normal tissues and negatively associated with progression-free survival (PFS) time in patients with PanNETs. Knocking out MGMT inhibits cancer cell growth in vitro and in vivo. Ectopic MEN1 expression suppresses MGMT transcription in a manner that depends on ß-Catenin nuclear export and degradation. The Leucine 267 residue of MEN1 is crucial for regulating ß-Catenin-MGMT axis activation and chemosensitivity to TMZ. Interference with ß-Catenin re-sensitizes tumor cells to TMZ and significantly reduces the cytotoxic effects of high-dose TMZ treatment, and MGMT overexpression counteracts the effects of ß-Catenin deficiency. This study reveals the biological importance of MGMT and a new mechanism by which MEN1 deficiency regulates its expression, thus providing a potential combinational strategy for treating patients with TMZ-resistant PanNETs.

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.

Challenges and future perspectives for the use of temozolomide in the treatment of SCLC.

Small-cell lung cancer (SCLC), accounting for 10-20 % of all lung tumors, represents the most aggressive high-grade neuroendocrine carcinoma. Most patients are diagnosed with extensive-stage SCLC (ES-SCLC), with brian metastases identified in âˆ¼ 80 % of cases during the disease cours, and the prognosis is dismal, with a 5-year survival rate of less than 5 %. Current available treatments in the second-line setting are limited, and topotecan has long been the only FDA-approved drug in relapsed or refractory ES-SCLC, until the recent approval of lurbinectedin, a selective inhibitor of RNA polymerase II. Temozolomide (TMZ) is an oral alkylating agent, which showed single-agent activity in SCLC, particularly among patients with O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation. Several studies have revealed the synergistic activity of temozolomide with poly-ADP-ribose polymerase (PARP) inhibitors, that prevent repair of TMZ-induced DNA damage. This review focuses on the rationale for the use of TMZ in ES-SCLC and provides an overview of the main trials that have evaluated and are currently investigating its role, both as a single-agent and in combinations, in relapse or refractory disease.

Unveiling the role of TAGLN2 in glioblastoma: From proneural-mesenchymal transition to Temozolomide resistance.

Glioblastoma (GBM) presents a daunting challenge due to its resistance to temozolomide (TMZ), a hurdle exacerbated by the proneural-to-mesenchymal transition (PMT) from a proneural (PN) to a mesenchymal (MES) phenotype. TAGLN2 is prominently expressed in GBM, particularly in the MES subtype compared to low-grade glioma (LGG) and the PN subtype. Our research reveals TAGLN2's involvement in PMT and TMZ resistance through a series of in vitro and in vivo experiments. TAGLN2 knockdown can restrain proliferation and invasion, trigger DNA damage and apoptosis, and heighten TMZ sensitivity in GBM cells. Conversely, elevating TAGLN2 levels amplifies resistance to TMZ in cellular and intracranial xenograft mouse models. We demonstrate the interaction relationship between TAGLN2 and ERK1/2 through co-immunoprecipitation (Co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) spectrometry analysis. Knockdown of TAGLN2 results in a decrease in the expression of p-ERK1/2, whereas overexpression of TAGLN2 leads to an increase in p-ERK1/2 expression within the nucleus. Subsequently, the regulatory role of TAGLN2 in the expression and control of MGMT has been demonstrated. Finally, the regulation of TAGLN2 by NF-κB has been validated through chromatin immunoprecipitation and ChIP-PCR assays. In conclusion, our results confirm that TAGLN2 exerts its biological functions by interacting with the ERK/MGMT axis and being regulated by NF-κB, thereby facilitating the acquisition of promoting PMT and increased resistance to TMZ therapy in glioblastoma. These results provide valuable insights for the advancement of targeted therapeutic approaches to overcome TMZ resistance in clinical treatments.

MGMT activated by Wnt pathway promotes cisplatin tolerance through inducing slow-cycling cells and nonhomologous end joining in colorectal cancer.

Chemotherapy resistance plays a pivotal role in the prognosis and therapeutic failure of patients with colorectal cancer (CRC). Cisplatin (DDP)-resistant cells exhibit an inherent ability to evade the toxic chemotherapeutic drug effects which are characterized by the activation of slow-cycle programs and DNA repair. Among the elements that lead to DDP resistance, O 6-methylguanine (O 6-MG)-DNA-methyltransferase (MGMT), a DNA-repair enzyme, performs a quintessential role. In this study, we clarify the significant involvement of MGMT in conferring DDP resistance in CRC, elucidating the underlying mechanism of the regulatory actions of MGMT. A notable upregulation of MGMT in DDP-resistant cancer cells was found in our study, and MGMT repression amplifies the sensitivity of these cells to DDP treatment in vitro and in vivo. Conversely, in cancer cells, MGMT overexpression abolishes their sensitivity to DDP treatment. Mechanistically, the interaction between MGMT and cyclin dependent kinase 1 (CDK1) inducing slow-cycling cells is attainted via the promotion of ubiquitination degradation of CDK1. Meanwhile, to achieve nonhomologous end joining, MGMT interacts with XRCC6 to resist chemotherapy drugs. Our transcriptome data from samples of 88 patients with CRC suggest that MGMT expression is co-related with the Wnt signaling pathway activation, and several Wnt inhibitors can repress drug-resistant cells. In summary, our results point out that MGMT is a potential therapeutic target and predictive marker of chemoresistance in CRC.

Gender differences in gliomas: From epidemiological trends to changes at the hormonal and molecular levels.

Gender plays a crucial role in the occurrence and development of cancer, as well as in the metabolism of nutrients and energy. Men and women display significant differences in the incidence, prognosis, and treatment response across various types of cancer, including certain sex-specific tumors. It has been observed that male glioma patients have a higher incidence and worse prognosis than female patients, but there is currently a limited systematic evaluation of sex differences in gliomas. The purpose of this study is to provide an overview of the association between fluctuations in sex hormone levels and changes in their receptor expression with the incidence, progression, treatment, and prognosis of gliomas. Estrogen may have a protective effect on glioma patients, while exposure to androgens increases the risk of glioma. We also discussed the specific genetic and molecular differences between genders in terms of the malignant nature and prognosis of gliomas. Factors such as TP53, MGMT methylation status may play a crucial role. Therefore, it is essential to consider the gender of patients while treating glioma, particularly the differences at the hormonal and molecular levels. This approach can help in the adoption of an individualized treatment strategy.

Prognosis of MGMT -Positive Gene in Patients with Brain Tumors of Grade III and Grade IV.

Sumit GoyalObjective To evaluate MGMT gene positivity is associated with better survival in patients diagnosed with brain tumor World Health Organization (WHO) grades III and IV Material and Methods Single-institute restrospective study. A total of 80 patients were enrolled, all underwent surgery either total or subtotal excision of the tumor and MGMT gene testing on tumor tissue by RT-PCR. All received adjuvant radiation (60 Gy/30 fractions, 5 fractions/week) with concurrent temozolomide (75 mg/m 2 ), followed by 12 cycles of adjuvant temozolomide (150 mg/m 2 1st cycle followed by 200 mg/m 2 ) with regular follow-up. Results A total of 80 patients, 75 underwent subtotal excision, 27 were WHO grade III vs. 48 WHO grade IV. Five underwent total excision 1 was WHO grade III vs. 4 WHO grade IV. The median PFS and OS in five patients in total excision in grade III patient was 9.0 and 20 compared with Grade IV, where the median PFS and OS was 8.8 and 17.8 months. Out of 75 patients in the subtotal group median PFS and OS, respectively, in Grade III group was 9.1 and 19.3 and, WHO grade IV with median PFS of 8.8 and OS of 18.8. Conclusion MGMT gene positivity is a prognostic factor in grade III and IV brain tumor.

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.

A novel strategy for predicting the efficacy of temozolomide treatment for metastatic pheochromocytomas/paragangliomas.

BACKGROUND: There are few studies on the efficacy of temozolomide (TMZ) in the treatment of Metastatic pheochromocytoma / paraganglioma (MPP) patients. And it remains unclear which MPP patients may benefit from TMZ treatment. METHODS: This was a prospective study. MPP patients were enrolled. Patients were treated with TMZ until disease progression or intolerable toxicities. The primary endpoints were disease control rate (DCR) and objective response rate (ORR). Secondary endpoints included biochemical response rate progression-free survival (PFS) and safety. We compared the difference between effective and ineffective groups, to explore which patients are more suitable for TMZ treatment. RESULTS: 62 patients with MPP were enrolled and tumor response were evaluated in 54 patients. The DCR was 83% (35/42), and the ORR was 24% (10/41) among the progressive patients. PFS was 25.2 ± 3.1 months. The most common adverse event was nausea (41/55). We found that 92.9% (13/14) of patients with MGMT methylation greater than 7% respond to treatment. For the patients with MGMT methylation less than 7%, Ki-67 index could be used to guide the use of TMZ in these patients. Among the patients with Ki-67 index less than 5%, 66% (8/12) patients showed respond to treatment, and only 33% (4/12) patients with Ki-67 index more than 5% showed respond to TMZ. CONCLUSIONS: This study indicated that TMZ is a potential choice for the treatment of MPP with the high ability on disease control and well tolerability. We recommended to MGMT methylation analysis test and Ki-67 index to guide TMZ application.

Prognostic Impact of TERT Promoter Mutations in Adult-Type Diffuse Gliomas Based on WHO2021 Criteria.

Mutation in the telomerase reverse transcriptase promoter (TERTp )is commonly observed in various malignancies, such as central nervous system (CNS) tumors, malignant melanoma, bladder cancer, and thyroid carcinoma. These mutations are recognized as significant poor prognostic factors for these tumors. In this investigation, a total of 528 cases of adult-type diffuse gliomas diagnosed at a single institution were reclassified according to the 2021 WHO classifications of CNS tumors, 5th edition (WHO2021). The study analyzed clinicopathological and genetic features, including TERTp mutations in each tumor. The impact of known prognostic factors on patient outcomes was analyzed through Kaplan-Meier survival and Cox regression analysis. TERTp mutations were predominantly identified in 94.1% of oligodendrogliomas (ODG), followed by 66.3% in glioblastoma, IDH-wildtype (GBM-IDHwt), and 9.2% of astrocytomas, IDH-mutant (A-IDHm). When considering A-IDHm and GBM as astrocytic tumors (Group 1) and ODGs (Group 2), TERTp mutations emerged as a significant adverse prognostic factor (p = 0.013) in Group 1. However, within each GBM-IDHwt and A-IDHm, the presence of TERTp mutations did not significantly impact patient prognosis (p = 0.215 and 0.268, respectively). Due to the high frequency of TERTp mutations in Group 2 (ODG) and their consistent prolonged survival, a statistical analysis to evaluate their impact on overall survival was deemed impractical. When considering MGMTp status, the combined TERTp-mutated and MGMTp-unmethylated group exhibited the worst prognosis in OS (p = 0.018) and PFS (p = 0.034) of GBM. This study confirmed that the classification of tumors according to the WHO2021 criteria effectively reflected prognosis. Both uni- and multivariate analyses in GBM, age, MGMTp methylation, and CDKN2A/B homozygous deletion were statistically significant prognostic factors while in univariate analysis in A-IDHm, grade 4, the Ki-67 index and MYCN amplifications were statistically significant prognostic factors. This study suggests that it is important to classify and manage tumors based on their genetic characteristics in adult-type diffuse gliomas.

No benefit from TMZ treatment in GB with truly unmethylated MGMT promoter: Reanalysis of the CE.6 and the pooled Nordic/NOA-08 trials in elderly GB patients.

BACKGROUND: The treatment of elderly/ frail patients with glioblastoma is a balance between avoiding undue toxicity, while not withholding effective treatment. It remains debated, whether these patients should receive combined chemo-radiotherapy with temozolomide (RT/TMZ➜TMZ) regardless of the O6-methylguanine DNA methyltransferase gene promoter (MGMTp) methylation status. MGMT is a well-known resistance factor blunting the treatment effect of TMZ, by repairing the most genotoxic lesion. Epigenetic silencing of the MGMTp sensitizes glioblastoma to TMZ. For risk adapted treatment, it is of utmost importance to accurately identify patients, who will not benefit from TMZ treatment. METHODS: Here, we present a reanalysis of the clinical trials CE.6 and the pooled NOA-08 and Nordic trials in elderly glioblastoma patients that compared RT to RT/TMZ➜TMZ, or RT to TMZ, respectively. For 687 patients with available MGMTp methylation data, we applied a cutoff discerning truly unmethylated glioblastoma, established in a pooled analysis of four clinical trials for glioblastoma, with RT/TMZ➜TMZ treatment, using the same quantitative methylation specific MGMTp PCR assay. RESULTS: When applying this restricted cutoff to the elderly patient population, we confirmed that glioblastoma with truly unmethylated MGMTp derived no benefit from TMZ treatment. In the Nordic/NOA-08 trials RT was better than TMZ, suggesting little or no benefit from TMZ. CONCLUSION: For evidence-based treatment of glioblastoma patients validated MGMTp methylation assays should be used that accurately identify truly unmethylated patients. Respective stratified management of patients will reduce toxicity without compromising outcome and allow testing of more promising treatment options.

Second surgery for relapsed glioblastoma: an observational study on criteria for patient selection in real life.

Aim: There is little consensus on salvage management of glioblastoma after recurrence, for lack of evidence. Materials & methods: A retrospective study of treatments in patients with recurrent glioblastoma. Results: Surgery at recurrence was related to better overall survival (OS) and progression-free survival (PFS). Surgery at recurrence, Karnofsky index, MGMT methylation status, younger age at diagnosis and number of chemotherapy cycles were positive factors for OS and PFS. The benefit of OS was relevant for a second surgery performed at least 9 months after the first one. Systemic treatments after the second surgery were linked to an improved PFS. Conclusion: Younger age, Karnofsky index, MGMT methylation status and a median time between surgeries ≥9 months may be criteria for eligibility for surgery at recurrence.

[Box: see text].

Enhancing Temozolomide (TMZ) chemosensitivity using CRISPR-dCas9-mediated downregulation of O6-methylguanine DNA methyltransferase (MGMT).

PURPOSE: Glioblastoma (GBM) stands out as the most prevalent and aggressive intracranial tumor, notorious for its poor prognosis. The current standard-of-care for GBM patients involves surgical resection followed by radiotherapy, combined with concurrent and adjuvant chemotherapy using Temozolomide (TMZ). The effectiveness of TMZ primarily relies on the activity of O6-methylguanine DNA methyltransferase (MGMT), which removes alkyl adducts from the O6 position of guanine at the DNA level, thereby counteracting the toxic effects of TMZ. METHOD: In this study, we employed fusions of catalytically-inactive Cas9 (dCas9) to DNA methyltransferases (dCas9-DNMT3A) to selectively downregulation MGMT transcription by inducing methylation at MGMT promoter and K-M enhancer. RESULT: Our findings demonstrate a significant reduction in MGMT expression, leading to intensified TMZ sensitivity in the HEK293T cell line. CONCLUSION: This study serves as a proof of concept for the utilization of CRISPR-based gene suppression to overcome TMZ resistance and enhance the lethal effect of TMZ in glioblastoma tumor cells.

Chitosan-PLGA mucoadhesive nanoparticles for gemcitabine repurposing for glioblastoma therapy.

Glioblastoma (GBM) is a highly deadly brain tumor that does not respond satisfactorily to conventional treatment. The non-alkylating agent gemcitabine (GEM) has been proposed for treating GBM. It can overcome MGMT protein-mediated resistance, a major limitation of conventional therapy with the alkylating agent temozolomide (TMZ). However, GEM's high systemic toxicity and poor permeability across the blood-brain barrier (BBB) pose significant challenges for its delivery to the brain. Thus, mucoadhesive poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) coated with chitosan (CH), suitable for intranasal GEM delivery, were proposed in this work. A central composite design (CCD) was implemented for NPs optimization, and NPs with appropriate characteristics for intranasal administration were obtained. in vitro studies revealed that the NPs possess excellent mucoadhesive properties and the ability to selectively release GEM in the simulated tumor tissue environment. in vitro studies using two human GBM cell lines (U215 and T98G) revealed the NPs' ability to promote GEM's antiproliferative activity to sensitize cells to the effect of TMZ. The findings of this work demonstrate that the developed CH-GEM-NPs are suitable delivery systems for GEM, both as a single therapy and as a chemosensitizer to the GBM gold standard therapy.

'The Reports of My Death Are Greatly Exaggerated'-Evaluating the Effect of Necrosis on MGMT Promoter Methylation Testing in High-Grade Glioma.

(1) Background: MGMT (O-6-methylguanine-DNA methyltransferase) promoter methylation remains an important predictive biomarker in high-grade gliomas (HGGs). The influence of necrosis on the fidelity of MGMT promoter (MGMTp) hypermethylation testing is currently unknown. Therefore, our study aims to evaluate the effect of varying degrees of necrosis on MGMTp status, as determined by pyrosequencing, in a series of primary and recurrent HGGs; (2) Methods: Within each case, the most viable blocks (assigned as 'true' MGMTp status) and the most necrotic block were determined by histopathology review. MGMTp status was determined by pyrosequencing. Comparisons of MGMTp status were made between the most viable and most necrotic blocks. (3) Results: 163 samples from 64 patients with HGGs were analyzed. MGMTp status was maintained in 84.6% of primary and 78.3% of recurrent HGGs between the most viable and necrotic blocks. A threshold of ≥60% tumor cellularity was established at which MGMTp status was unaltered, irrespective of the degree of necrosis. (4) Conclusions: MGMTp methylation status, as determined by pyrosequencing, does not appear to be influenced by necrosis in the majority of cases at a cellularity of at least 60%. Further investigation into the role of intratumoral heterogeneity on MGMTp status will increase our understanding of this predictive marker.

Temperature-Wise Calibration Increases the Accuracy of DNA Methylation Levels Determined by High-Resolution Melting (HRM).

High-resolution melting (HRM) is a cost-efficient tool for targeted DNA methylation analysis. HRM yields the average methylation status across all CpGs in PCR products. Moreover, it provides information on the methylation pattern, e.g., the occurrence of monoallelic methylation. HRM assays have to be calibrated by analyzing DNA methylation standards of known methylation status and mixtures thereof. In general, DNA methylation levels determined by the classical calibration approach, including the whole temperature range in between normalization intervals, are in good agreement with the mean of the DNA methylation status of individual CpGs determined by pyrosequencing (PSQ), the gold standard of targeted DNA methylation analysis. However, the classical calibration approach leads to highly inaccurate results for samples with heterogeneous DNA methylation since they result in more complex melt curves, differing in their shape compared to those of DNA standards and mixtures thereof. Here, we present a novel calibration approach, i.e., temperature-wise calibration. By temperature-wise calibration, methylation profiles over temperature are obtained, which help in finding the optimal calibration range and thus increase the accuracy of HRM data, particularly for heterogeneous DNA methylation. For explaining the principle and demonstrating the potential of the novel calibration approach, we selected the promoter and two enhancers of MGMT, a gene encoding the repair protein MGMT.

Evaluation of the clinical use of MGMT methylation in extracellular vesicle-based liquid biopsy as a tool for glioblastoma patient management.

Glioblastoma (GB) is a devastating tumor of the central nervous system characterized by a poor prognosis. One of the best-established predictive biomarker in IDH-wildtype GB is O6-methylguanine-DNA methyltransferase (MGMT) methylation (mMGMT), which is associated with improved treatment response and survival. However, current efforts to monitor GB patients through mMGMT detection have proven unsuccessful. Small extracellular vesicles (sEVs) hold potential as a key element that could revolutionize clinical practice by offering new possibilities for liquid biopsy. This study aimed to determine the utility of sEV-based liquid biopsy as a predictive biomarker and disease monitoring tool in patients with IDH-wildtype GB. Our findings show consistent results with tissue-based analysis, achieving a remarkable sensitivity of 85.7% for detecting mMGMT in liquid biopsy, the highest reported to date. Moreover, we suggested that liquid biopsy assessment of sEV-DNA could be a powerful tool for monitoring disease progression in IDH-wildtype GB patients. This study highlights the critical significance of overcoming molecular underdetection, which can lead to missed treatment opportunities and misdiagnoses, possibly resulting in ineffective therapies. The outcomes of our research significantly contribute to the field of sEV-DNA-based liquid biopsy, providing valuable insights into tumor tissue heterogeneity and establishing it as a promising tool for detecting GB biomarkers. These results have substantial implications for advancing predictive and therapeutic approaches in the context of GB and warrant further exploration and validation in clinical settings.

Accurate and comprehensive evaluation of O6-methylguanine-DNA methyltransferase promoter methylation by nanopore sequencing.

AIMS: The methylation status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter region is essential in evaluating the prognosis and predicting the drug response in patients with glioblastoma. In this study, we evaluated the utility of using nanopore long-read sequencing as a method for assessing methylation levels throughout the MGMT CpG-island, compared its performance to established techniques and demonstrated its clinical applicability. METHODS: We analysed 165 samples from CNS tumours, focusing on the MGMT CpG-island using nanopore sequencing. Oxford Nanopore Technologies (ONT) MinION and PromethION flow cells were employed for single sample or barcoded assays, guided by a CRISPR/Cas9 protocol, adaptive sampling or as part of a whole genome sequencing assay. Methylation data obtained through nanopore sequencing were compared to results obtained via pyrosequencing and methylation bead arrays. Hierarchical clustering was applied to nanopore sequencing data for patient stratification. RESULTS: Nanopore sequencing displayed a strong correlation (R2 = 0.91) with pyrosequencing results for the four CpGs of MGMT analysed by both methods. The MGMT-STP27 algorithm's classification was effectively reproduced using nanopore data. Unsupervised hierarchical clustering revealed distinct patterns in methylated and unmethylated samples, providing comparable survival prediction capabilities. Nanopore sequencing yielded high-confidence results in a rapid timeframe, typically within hours of sequencing, and extended the analysis to all 98 CpGs of the MGMT CpG-island. CONCLUSIONS: This study presents nanopore sequencing as a valid and efficient method for determining MGMT promotor methylation status. It offers a comprehensive view of the MGMT promoter methylation landscape, which enables the identification of potentially clinically relevant subgroups of patients. Further exploration of the clinical implications of patient stratification using nanopore sequencing of MGMT is warranted.