methylguanine-DNA Methyltransferase Promoter Research Articles

A Data-Driven Approach to Predicting 5-Aminolevulinic Acid-Induced Fluorescence and World Health Organization Grade in Newly Diagnosed Diffuse Gliomas.

BACKGROUND:A growing body of evidence has revealed the potential utility of 5-aminolevulinic acid (5-ALA) as a surgical adjunct in selected lower-grade gliomas. However, a reliable means of identifying which lower-grade gliomas will fluoresce has not been established.OBJECTIVE:To identify clinical and radiological factors predictive of intraoperative fluorescence in intermediate-grade gliomas. In addition, given that higher-grade gliomas are more likely to fluoresce than lower-grade gliomas, we also sought to develop a means of predicting glioma grade.METHODS:We investigated a cohort of patients with grade II and grade III gliomas who received 5-ALA before resection at a single institution. Using a logistic regression-based model, we evaluated 14 clinical and molecular variables considered plausible determinants of fluorescence. We then distilled the most predictive features to develop a model for predicting both fluorescence and tumor grade. We also explored the relationship between intraoperative fluorescence and diagnostic molecular markers.RESULTS:One hundered seventy-nine subjects were eligible for inclusion. Our logistic regression classifier accurately predicted intraoperative fluorescence in our cohort with 91.9% accuracy and revealed enhancement as the singular variable in determining intraoperative fluorescence. There was a direct relationship between enhancement on MRI and the likelihood of observed fluorescence. Observed fluorescence correlated with MIB-1 index but not with isocitrate dehydrogenase (IDH) status, 1p19q codeletion, or methylguanine DNA methyltransferase promoter methylation.CONCLUSION:We demonstrate a strong correlation between enhancement on preoperative MRI and the likelihood of visible fluorescence during surgery in patients with intermediate-grade glioma. Our analysis provides a robust method for predicting 5-ALA–induced fluorescence in patients with grade II and grade III gliomas.

The N6-Methylandenosine-Related Gene BIRC5 as a Prognostic Biomarker Correlated With Cell Migration and Immune Cell Infiltrates in Low Grade Glioma.

Gliomas account for 75% of all primary malignant brain tumors in adults and are associated with high mortality. Emerging evidence has demonstrated that baculoviral inhibitor of apoptosis repeat containing 5 (BIRC5) plays a critical role in cell apoptosis and the progression of diverse cancers. However, no studies have yet focused on the immunological function and mechanisms of upstream BIRC5 regulation in the progression of low-grade gliomas (LGG). Here, we evaluated BIRC5 expression and clinical characteristics in people with LGG using the Chinese Glioma Genome Atlas, The Cancer Genome Atlas, Gene Expression Omnibus, Rembrandt, and Gravendeel databases. We used Kaplan–Meier statistics and receiver operating characteristic (ROC) curves to analyze the prognostic value of BIRC5 in LGG. Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) enrichment terms were also explored to identify functional roles of BIRC5. The Tumor Immune Estimation Resource (TIMER) and Tumor Immune System Interaction (TISIDB) databases were used to examine the correlation between BIRC5 expression and immune cell infiltration in LGG. The Genomics of Drug Sensitivity in Cancer (GDSC) and Cancer Therapeutics Response Portal (CTRP) databases were used to examine the potential drugs targeting BIRC5. We used transwell and wound healing assays to determine the biological functions of BIRC5 in glioma cell migration. Our results demonstrated that BIRC5 was highly expressed in LGG and the expression level correlated with tumor grade, prognosis, histological subtype, isocitrate dehydrogenase 1 (IDH1) mutation, 1p/19q chromosomal co-deletion, chemotherapy status, and O[6]-methylguanine-DNA methyltransferase (MGMT) promoter methylation status. GO and KEGG analysis showed that BIRC5 is primarily involved in cell proliferation and immune response-related signaling pathways. We also found that BIRC5 was significantly correlated with m6A modification and diverse drug sensitivity. TIMER and TISIDB database analysis showed that BIRC5 expression is associated with infiltration of diverse immune cells and immune modulation in LGG. BIRC5 knockdown inhibited LGG cell migration. Collectively, our results demonstrate that BIRC5 is correlated with cell migration and immune infiltration in LGG and may be a useful prognostic biomarker.

CTNI-21. PHASE 2 STUDY OF VAL-083 AND RADIOTHERAPY IN NEWLY DIAGNOSED MGMT-UNMETHYLATED GBM

Abstract Approximately 60% of glioblastoma multiforme (GBM) patients possess an unmethylated methylguanine DNA-methyltransferase (MGMT) gene promoter, which confers a limited clinical response to standard-of-care treatment with temozolomide (TMZ), resulting in shorter median survival when compared to patients with a methylated MGMT promoter. VAL-083 is a novel bi-functional DNA targeting agent that induces interstrand DNA cross-links at N7-guanine, leading to DNA double-strand breaks and ultimately cell death. VAL-083 circumvents MGMT-mediated TMZ resistance in vitro and in vivo. A Phase 2 study has been conducted to evaluate efficacy and safety of VAL-083 when administered concurrently with radiation therapy (RT) in newly diagnosed MGMT unmethylated GBM. The study was conducted in 2 stages: Stage 1 was a dose-escalation phase to confirm the dose of VAL-083 in this setting. Patients received VAL-083 at 20, 30, or 40 mg/m2/day x 3 days every 21 days along with standard radiation treatment (RT) (2 Gy/day, 5 days/week for 6 weeks). At the end of this stage, 30 mg/m2/day of VAL-083 in combination with RT was generally safe and well-tolerated. Stage 2 was an expansion phase to enroll up to 20 additional patients at the 30 mg/m2/day of VAL-083 in combination with RT. All patients have been enrolled, with a total of 29 patients in the study, and 25 patients receiving 30 mg/m2/day VAL-083. All 29 patients have completed treatment and patients are in the follow-up phase of the study. Consistent with our prior experience, myelosuppression was the most common adverse event. As of March 2021, 22/29 (75.9%) subjects had disease progression. The median progression free survival for all patients enrolled was 9.3 (95%CI: 6.4-12.0) months. Sixteen (16/29; 55.2%) patients had died, and median overall survival for all patients enrolled was 19.6 (95%CI: 14.0-22.4) months. Further safety and efficacy updates will be presented at the meeting. Clinicaltrials.gov identifier: NCT03050736.

P14.09 BORTEM-17: A Phase IB/II Single-Arm, Control Non-Randomized, Multicentre, Open Label Clinical Trial for Recurrent Glioblastoma with unmethylated MGMT promoter (NCT03643549)

BACKGROUNDGlioblastoma (GBM) is the most malignant primary brain tumor in adults where median survival in unselected patients is approximately 10 months. There is no standard treatment for patients who progress on temozolomide and patients are best treated within investigational clinical protocols. Patients harbouring tumours with functional O6 methylguanine DNA methyltransferase (MGMT) DNA repair enzyme have particularly poor prognosis with median overall survival of 12.7 months, compared to 21.7 months for patients with hypermethylated MGMT promoter. The pre-clinical studies have shown that Bortezomib depletes the MGMT enzyme, restoring the tumour ´s susceptibility to Temozolomide, if the chemotherapy is administered in the precise schedule when the MGMT enzyme is depleted. Additionally, Bortezomib shows an antitumour effect by blocking autophagy flux. Based on the promising pre-clinical results, a non-randomized, open label phase IB/II clinical trial was designed. The primary endpoints include assessment of safety of Bortezomib administered with Temozolomide for phase IB and median progression free survival, overall survival as well as progression free rate at 6 months.MATERIAL AND METHODSRecurrent glioblastoma patients with unmethylated MGMT promotor, progressing at least 12 weeks after completion of postoperative radiotherapy, with adequate organ function, performance status Karnofsky 70 or better and radiologically measurable lesions are screened for study inclusion. The experimental treatment consists of Bortezomib 1.3mg/m2 administered IV on days 1, 4, 7, during each 4-week chemotherapy cycle with per oral Temozolomide at 200mg/m2 5 days/week every 4 weeks starting on day 3. Study group will be compared to historical controls on conventional management. The sample size was calculated to 63 patients, ten of them were included in the phase IB.RESULTSThe phase IB of the trial was completed in 2019 and the combination of Temozolomide and Bortezomib was shown to be safe and well tolerated. Until April 2021 a total number of 23 patients were included into the trial. The patients are treated at 4 different referral university hospitals in Norway. A clinical treatment benefit with both radiological tumor volume response and stable disease were observed. The patient inclusion in the trial is delayed due COVID-19. The majority of observed side effects are mild or moderate. The grade 3 or 4 adverse effects included thrombocytopenia, ataxia, muscle weakness, delirium and hyperglycemia. Patients that progressed under the treatment received another line of therapy according to the institutional practice.CONCLUSIONA combination of Bortezomib and Temozolomide administered in a defined time sequence to achieve sensitization of glioblastoma to alkylating agent is safe and feasible and may represent a novel treatment option for patients with this devastating disease.

P14.65 Survival in a consecutive series of 467 glioblastoma patients: impact of prognostic factors and recurrent treatment at two independent institutions

Abstract BACKGROUND Glioblastoma (GBM) is an aggressive primary brain tumor with median overall survival (OS) of less than one year in unselected adult patients. There is no standard therapy at recurrence. We aimed to evaluate OS in a consecutive series of GBM patients from Norway’s two largest regional health authorities, compare the effect of physicians’ choice of antineoplastic treatment upon recurrence and identify prognostic and predictive factors. MATERIAL AND METHODS Clinicopathological data from n=467 patients with histologically confirmed GBM diagnosed and treated at Haukeland and Oslo university hospitals from January 2015 to December 2017 was retrospectively collected. Data included tumor location, methylation status of the methylguanine-DNA methyltransferase (MGMT) promoter and mutation of the isocitrate dehydrogenase (IDH) genes, patient age and sex, extent of tumor resection at primary diagnosis, and treatment at first, second and third tumor recurrences. Cox-proportional hazards regression with pairwise analyses adjusted for multiple testing with Scheffé’s post-hoc test were used to adjust effect of multiple risk factors on mortality. RESULTS Median OS was 12.1 months and 21.4 % and 6.8 % of patients were alive at 2 and 5 years, respectively. Treatment at recurrence varied between institutions but did not impact OS (p=0.201). Median time to progression was 8.2 months. Age, MGMT promoter methylation, tumor location and extent of tumor resection were all independent prognostic factors for OS. Patients receiving radiotherapy to 60 Gray with concomitant and adjuvant temozolomide at primary diagnosis had best outcome with median OS of 16.1 months and 9.3% were alive at 5 years. At first recurrence patients eligible for gammaknife/stereotactic radiosurgery (GK/SRS) or surgery, alone or combined with chemotherapy, had superior survival compared to chemotherapy alone (p<0.0001 and p=0.014, respectively). On Scheffé’s post-hoc analyses only GK/SRS was superior to chemotherapy (p=0.01). At second and third recurrence none of the antineoplastic strategies came across as superior or inferior to each other using Cox. On Scheffé’s post-hoc analyses chemotherapy alone and combined with bevacizumab were superior at second recurrence (p=0.008 and p=0.042, respectively) and chemotherapy combined with bevacizumab was superior at third recurrence (p=0.043), compared to no antineoplastic treatment. Our retrospective study is limited by small sample sizes and heterogeneous treatment groups, especially at second and third recurrences, as well as patient selection. CONCLUSION Recurrence treatment differed between the two institutions but there was no difference in OS. Our findings underline the lack of standard therapy upon GBM recurrence and the urgent need for novel therapeutic strategies.

18F-FET PET Uptake Characteristics of Long-Term IDH-Wildtype Diffuse Glioma Survivors

Simple SummaryIDH-wildtype (IDHwt) gliomas represent a tumor entity with poor overall survival. Only rare cases have an overall survival over several years. Dynamic and static 18F-FET PET is recommended as valuable complementary tool for glioma imaging in gliomas. This study shows that, besides molecular genetic prognosticators, long survival (≥36 months survival) in IDHwt gliomas is associated with a longer time-to-peak and smaller volume on 18F-FET PET at initial diagnosis compared to glioma patients with a short-term survival (≤15 months survival). 18F-FET uptake intensity and MRI-derived tumor size do not differ in patients with long-term survival compared to patient with a short-term survival.Background: IDHwt diffuse gliomas represent the tumor entity with one of the worst clinical outcomes. Only rare cases present with a long-term survival of several years. Here we aimed at comparing the uptake characteristics on dynamic 18F-FET PET, clinical and molecular genetic parameters of long-term survivors (LTS) versus short-term survivors (STS): Methods: Patients with de-novo IDHwt glioma (WHO grade III/IV) and 18F-FET PET prior to any therapy were stratified into LTS (≥36 months survival) and STS (≤15 months survival). Static and dynamic 18F-FET PET parameters (mean/maximal tumor-to-background ratio (TBRmean/max), biological tumor volume (BTV), minimal time-to-peak (TTPmin)), diameter and volume of contrast-enhancement on MRI, clinical parameters (age, sex, Karnofksy-performance-score), mode of surgery; initial treatment and molecular genetics were assessed and compared between LTS and STS. Results: Overall, 75 IDHwt glioma patients were included (26 LTS, 49 STS). LTS were significantly younger (p < 0.001), had a higher rate of WHO grade III glioma (p = 0.032), of O(6)-Methylguanine-DNA methyltransferase (MGMT) promoter methylation (p < 0.001) and missing Telomerase reverse transcriptase promoter (TERTp) mutations (p = 0.004) compared to STS. On imaging, LTS showed a smaller median BTV (p = 0.017) and a significantly longer TTPmin (p = 0.008) on 18F-FET PET than STS, while uptake intensity (TBRmean/max) did not differ. In contrast to the tumor-volume on PET, MRI-derived parameters such as tumor size as well as all other above-mentioned parameters did not differ between LTS and STS (p > 0.05 each). Conclusion: Besides molecular genetic prognosticators, a long survival time in IDHwt glioma patients is associated with a longer TTPmin as well as a smaller BTV on 18F-FET PET at initial diagnosis. 18F-FET uptake intensity as well as the MRI-derived tumor size (volume and maximal diameter) do not differ in patients with long-term survival.

Cost-effectiveness of concomitant and adjuvant temozolomide for glioblastoma patients with unmethylated O6-methylguanine-DNA methyltransferase promoter regions in the United States.

2058 Background: Promoter region methylation of the O6-methylguanine-DNA methyltransferase (MGMT) gene has emerged as a predictive factor for longer overall (OS) and progression-free (PFS) survival response to concomitant and adjuvant temozolomide (TMZ) chemotherapy in glioblastoma. Recently, several investigators have suggested the small survival benefit of TMZ for unmethylated patients is insufficient to warrant the increased risk of adverse events and that these patients should instead be enrolled in clinical trials. This study informs the debate by estimating the incremental costs and benefits associated with TMZ use in methylated and unmethylated patients based on available published evidence. Methods: Published OS and PFS Kaplan-Meier curves were digitized, parameterized, and extrapolated to ten years. Partitioned Survival Analysis trees were constructed in TreeAge Pro 2020. Costs of TMZ, RT, and inpatient hospitalization were included, among others. It was assumed that all patients receive surgery prior to beginning radiation, and that RT and TMZ (six adjuvant cycles) dosing was standard. Published utilities associated with the progression-free and post-progression health states, subject to each treatment, were used. A 3% annual discount rate and a willingness-to-pay of $150,000/quality-adjusted life year (QALY) were assumed. Probabilistic sensitivity analysis (PSA) was conducted. Results: In methylated patients, RT + TMZ versus RT is associated with $67,622 in additional costs and 10.91 additional quality-adjusted life months (QALMs). In unmethylated patients, RT + TMZ versus RT is associated with $9,203 in additional costs and 2.24 additional QALMs. The implied incremental cost effectiveness ratios (ICERs) support TMZ use in both populations and suggest it is more favorable for unmethylated patients ($74,378/QALY and $49,302/QALY for methylated and unmethylated patients, respectively). However, the incremental net monetary benefit (INMB) associated with TMZ use is substantially smaller for unmethylated patients ($18,797 versus $68,753) due to the smaller survival gains for this population. PSA results indicate that TMZ is more likely to be cost-effective for methylated (89.3%) than unmethylated (77.4%) patients. Conclusions: Post-radiation clinical trial enrollment should be considered instead of TMZ for unmethylated glioblastoma patients. While the estimated ICER justifies the use of TMZ for unmethylated patients – in fact, does so more strongly than for methylated patients – the relatively small incremental health benefit is plausibly outweighed by the opportunity cost of not enrolling these patients in clinical trials that offer the possibility of more substantive gains to current and future glioblastoma patients.

The Potential Use of Radiomics with Pre-Radiation Therapy MR Imaging in Predicting Risk of Pseudoprogression in Glioblastoma Patients.

Glioblastoma (GBM) is the most common adult glioma. Differentiating post-treatment effects such as pseudoprogression from true progression is paramount for treatment. Radiomics has been shown to predict overall survival and MGMT (methylguanine-DNA methyltransferase) promoter status in those with GBM. A potential application of radiomics is predicting pseudoprogression on pre-radiotherapy (RT) scans for patients with GBM. A retrospective review was performed with radiomic data analyzed using pre-RT MRI scans. Pseudoprogression was defined as post-treatment findings on imaging that resolved with steroids or spontaneously on subsequent imaging. Of the 72 patients identified for the study, 35 were able to be assessed for pseudoprogression, and 8 (22.9%) had pseudoprogression. A total of 841 radiomic features were examined along with clinical features. Receiver operating characteristic (ROC) analyses were performed to determine the AUC (area under ROC curve) of models of clinical features, radiomic features, and combining clinical and radiomic features. Two radiomic features were identified to be the optimal model combination. The ROC analysis found that the predictive ability of this combination was higher than using clinical features alone (mean AUC: 0.82 vs. 0.62). Additionally, combining the radiomic features with clinical factors did not improve predictive ability. Our results indicate that radiomics is potentially capable of predicting future development of pseudoprogression in patients with GBM using pre-RT MRIs.

Automated machine learning to predict the co-occurrence of isocitrate dehydrogenase mutations and O6 -methylguanine-DNA methyltransferase promoter methylation in patients with gliomas.

Combining isocitrate dehydrogenase mutation (IDHmut) with O6 -methylguanine-DNA methyltransferase promoter methylation (MGMTmet) has been identified as a critical prognostic molecular marker for gliomas. The aim of this study was to determine the ability of glioma radiomics features from magnetic resonance imaging (MRI) to predict the co-occurrence of IDHmut and MGMTmet by applying the tree-based pipeline optimization tool (TPOT), an automated machine learning (autoML) approach. This was a retrospective study, in which 162 patients with gliomas were evaluated, including 58 patients with co-occurrence of IDHmut and MGMTmet and 104 patients with other status comprising: IDH wildtype and MGMT unmethylated (n=67), IDH wildtype and MGMTmet (n=36), and IDHmut and MGMT unmethylated (n=1). Three-dimensional (3D) T1-weighted images, gadolinium-enhanced 3D T1-weighted images (Gd-3DT1WI), T2-weighted images, and fluid-attenuated inversion recovery (FLAIR) images acquired at 3.0 T were used. Radiomics features were extracted from FLAIR and Gd-3DT1WI images. The TPOT was employed to generate the best machine learning pipeline, which contains both feature selector and classifier, based on input feature sets. A 4-fold cross-validation was used to evaluate the performance of automatically generated models. For each iteration, the training set included 121 subjects, while the test set included 41 subjects. Student's t-test or a chi-square test was applied on different clinical characteristics between two groups. Sensitivity, specificity, accuracy, kappa score, and AUC were used to evaluate the performance of TPOT-generated models. Finally, we compared the above metrics of TPOT-generated models to identify the best-performing model. Patients' ages and grades between two groups were significantly different (p=0.002 and p=0.000, respectively). The 4-fold cross-validation showed that gradient boosting classifier trained on shape and textual features from the Laplacian-of-Gaussian-filtered Gd-3DT1 achieved the best performance (average sensitivity=81.1%, average specificity=94%, average accuracy=89.4%, average kappa score=0.76, average AUC=0.951). Using autoML based on radiomics features from MRI, a high discriminatory accuracy was achieved for predicting co-occurrence of IDHmut and MGMTmet in gliomas. LEVEL OF EVIDENCE: 3 TECHNICAL EFFICACY STAGE: 3.

CTNI-76. PHASE 2 CLINICAL TRIAL OF VAL-083 IN NEWLY DIAGNOSED MGMT-UNMETHYLATED GBM

Abstract Approximately 60% of glioblastoma multiforme (GBM) patients possess an unmethylated methylguanine DNA-methyltransferase (MGMT) promoter region, which confers a limited response to standard-of-care treatment with temozolomide (TMZ), resulting in shorter median survival when compared to patients with methylated MGMT promoter. VAL-083 is a novel bi-functional DNA targeting agent that induces inter-strand cross-links at N7-guanine, leading to DNA double-strand breaks and ultimately cell death. VAL-083 circumvents MGMT-mediated TMZ resistance in vitro and in vivo. A Phase 2 study has been initiated for VAL-083 in newly diagnosed MGMT unmethylated GBM. The study has 2 stages: Stage 1 is a dose-escalation safety and tolerability phase to confirm the phase 2 dose of VAL-083 when administered concurrently with radiation therapy (RT). Patients received VAL-083 at 20, 30, or 40 mg/m2/day x 3 days every 21 days along with standard radiation treatment (RT) (2 Gy/day, 5 days/week). The dose escalation stage is complete, and 30 mg/m2/day of VAL-083 in combination with RT was generally safe and well-tolerated. Stage 2 comprises an expansion phase to enroll up to 20 additional patients at the 30 mg/m2/day of VAL-083 in combination with RT. As of June 2, 2020, all patients have been enrolled, with a total of 29 patients in the study, and 25 patients receiving 30 mg/m2/day VAL-083. Of the 29 patients enrolled, 27 have completed their prospectively planned MRI scans and had their initial assessment for tumor response. Two additional patients died prior to their post-cycle 3 MRI. Consistent with our prior experience, myelosuppression was the most common adverse event. Three patients have experienced dose-limiting toxicities - one (1/3; 33%) at the 40 mg/m2/day and two (2/25; 8%) at the 30 mg/m2/day dose. Further safety and efficacy updates will be presented at the meeting. Clinicaltrials.gov identifier: NCT03050736.

TGF-β1 modulates temozolomide resistance in glioblastoma via altered microRNA processing and elevated MGMT.

BackgroundOur previous studies have indicated that miR-198 reduces cellular methylguanine DNA methyltransferase (MGMT) levels to enhance temozolomide sensitivity. Transforming growth factor beta 1 (TGF-β1) switches off miR-198 expression by repressing K-homology splicing regulatory protein (KSRP) expression in epidermal keratinocytes. However, the underlying role of TGF-β1 in temozolomide resistance has remained unknown.MethodsThe distribution of KSRP was detected by western blotting and immunofluorescence. Microarray analysis was used to compare the levels of long noncoding RNAs (lncRNAs) between TGF-β1–treated and untreated cells. RNA immunoprecipitation was performed to verify the relationship between RNAs and KSRP. Flow cytometry and orthotopic and subcutaneous xenograft tumor models were used to determine the function of TGF-β1 in temozolomide resistance.ResultsOverexpression of TGF-β1 contributed to temozolomide resistance in MGMT promoter hypomethylated glioblastoma cells in vitro and in vivo. TGF-β1 treatment reduced cellular MGMT levels through suppressing the expression of miR-198. However, TGF-β1 upregulation did not affect KSRP expression in glioma cells. We identified and characterized 2 lncRNAs (H19 and HOXD-AS2) that were upregulated by TGF-β1 through Smad signaling. H19 and HOXD-AS2 exhibited competitive binding to KSRP and prevented KSRP from binding to primary miR-198, thus decreasing miR-198 expression. HOXD-AS2 or H19 upregulation strongly promoted temozolomide resistance and MGMT expression. Moreover, KSRP depletion abrogated the effects of TGF-β1 and lncRNAs on miR-198 and MGMT. Finally, we found that patients with low levels of TGF-β1 or lncRNA expression benefited from temozolomide therapy.ConclusionsOur results reveal an underlying mechanism by which TGF-β1 confers temozolomide resistance. Furthermore, our findings suggest that a novel combination of temozolomide with a TGF-β inhibitor may serve as an effective therapy for glioblastomas.

The Impact of Surgery in IDH 1 Wild Type Glioblastoma in Relation With the MGMT Deregulation.

Object: The treatment of choice in glioblastoma (GBM) is the maximal surgical extent of resection (EOR) followed by adjuvant chemo-radiotherapy. Furthermore, methylguanine-DNA methyltransferase (MGMT) promoter methylation is associated with prolonged overall survival (OS) and progression free survival (PFS). The objective of the present study is correlate the biomolecular aspects in relation with EOR.Materials and methods: We analyzed a series of 116 patients with IDH-1 wild type GBM and different EOR (Gross Total Resection—GTR-, Partial Resection—PR- and Biopsy), treated with adjuvant chemo-radiotherapy. The MGMT status was analyzed in terms of promoter methylation and protein expression.Results: When GTR was possible, OS and PFS were significantly better compared to the other two groups (p = 0.001 and p = 0.035, respectively). MGMT methylation was significantly associated with better OS in the biopsy group (p = 0.022) and better OS and PFS in PR (p = 0.02 and p = 0.012, respectively), but not in the GTR group (p = 0.252 for OS, p = 0.256 for PFS) nor the PFS in the biopsy group (p = 0.259). MGMT protein expression levels do not show any association with OS and PFS, regardless of the type of surgery.Conclusions: Our study confirms the positive association of a safe maximal EOR with better OS and PFS, and indicates a positive prognostic value of MGMT methylation status only in case of the presence of residual tumor tissue. MGMT protein expression seems not to play a clinical role in relation with the type of surgery.

Risk Factors for Cognitive Impairment in High-Grade Glioma Patients Treated with Postoperative Radiochemotherapy

PurposeFractionated radiotherapy as well as concomitant and adjuvant chemotherapy such as temozolomide for postoperative high-grade glioma (HGG) patients improves progression-free survival and overall survival. Multiple factors such as chemotherapy, radiotherapy, tumor grade, residual tumor volume, and genetic modifications might play a role in the formation of cognitive impairment. The risk factors of cognitive impairment in postoperative patients with HGG receiving radiotherapy and chemotherapy remains a concern in this population. The purpose of this study was to identify risk factors for cognitive impairment in patients of postoperative HGG.Materials and MethodsA total of 229 patients with HGG who underwent surgery were analyzed. Cognitive impairment was defined as a decrease of Cognitive Assessment Montreal (MoCA)’s score in at least two cognitive domains or any MoCA’s score of less than 26 points at the time of study compared with baseline level. Multiple potential risk factors including methylated status of the O6-methylguanine-DNA methyltransferase (MGMT) promoter, glioma World Health Organization (WHO) grade, residual tumor volume, education, and sex were analyzed. Cox univariate and multivariate regression analysis was used to detect the significant risk factors for cognitive impairment.ResultsAt the end of follow-up among the 229 patients, 147 patients (67%) developed cognitive impairment. 82 patients (36%) remained in normal cognitive condition. In multivariate analysis, unmethylated MGMT promoter (hazard ratio [HR], 1.679; 95% confidence interval [CI], 1.212 to 2.326; p=0.002), glioblastoma (HR, 1.550; 95% CI, 1.117 to 2.149; p=0.009), and residual tumor volume > 5.58 cm3 (HR, 1.454; 95% CI, 1.047 to 2.020; p=0.026) were independent risk factors for cognitive impairment.ConclusionMethylated status of the MGMT promoter, glioma WHO grade, and residual tumor volume might be risk factors for the cognitive impairment in postoperative patients with HGG.

CBMT-22. REVERSING THE WARBURG EFFECT BY TARGETING NICOTINAMIDE PHOSPHORIBOSYLTRANSFERASE (NAMPT), THE RATE-LIMITING ENZYME OF NAD+ SALVAGE PATHWAY INCREASES, CHEMOSENSITIVITY TO TEMOZOLOMIDE IN GLIOMA CELLS

Abstract BACKGROUND Glioma cells metabolize glucose primarily through incomplete, non-oxidative glycolysis even in the presence of oxygen (the Warburg effect) which provides a constant supply of substrate for proliferation and generates an acidic environment that favors invasion and immunosuppression. NAMPT, the rate-limiting enzyme of the NAD+ salvage pathway, regulates key metabolic processes preferentially used in glioma energy metabolism. Inhibition of NAMPT could have global impact on glioma cell metabolism and could be a promising strategy for glioma therapy. This study examined the effect of NAMPT inhibition using KPT-9274 and FK866 (NAMPTi) on glycolysis and glioma cell metabolism. METHODS Metabolic changes related to NAMPT inhibition were examined in glioma cell lines and patient derived glioma stem-like cells (GSC). Mass spectrometry was used for metabolic profiles and the generated data was annotated by comparison against an extensive metabolite library to identify relevant metabolites. The Agilent Seahorse assay was utilized to determine treatment-related changes in glycolysis. RESULTS NAMPT inhibition caused NAD, NADH, NADP, NADPH, and ATP depletion in glioma cells. Global metabolomics results showed an accumulation of GMP, IMP, AMP and myo-inositol indicating reprogramming of glioma cell metabolism for de novo purine biosynthesis. Additionally, an accumulation of glucose- and fructose-1,6 bisphosphate in NAMPTi treated cells indicated dysfunctional glycolysis. This was confirmed by the Agilent glycolysis stress test by which NAMPTi-treated glioma cells showed a reduction in glycolysis, glycolytic capacity and reserves. NAMPTi treated cells showed downregulation of LDHA due to NAD depletion. Further, downregulation of LDHA enhanced the chemosensitivity of glioma cells to temozolomide. CONCLUSIONS NAMPTi cause profound disruption of glycolysis, reduced glycolytic capacity and glycolytic reserve in gliomas and GSCs irrespective of O[6]-methylguanine-DNA methyltransferase promoter methylation or IDH1 status. Targeting NAMPT to disrupt the Warburg effect is a novel strategy for developing effective therapies against glioma to overcome heterogeneity.

Is a reduction of radiation dose feasible in patients affected by glioblastoma undergoing radio-chemotherapy according to MGMT promoter methylation status without jeopardizing survival?

ObjectiveTo explore therapeutic results of different radiotherapy (RT) dose schedules combined to Temozolomide (TMZ)-RT treatment in newly diagnosed glioblastoma (GB), according to the O (6)-methylguanine-DNA methyltransferase (MGMT) methylation status. Patients and methodsPatients with newly diagnosed GB received either standard (60-59.4 Gy) or reduced (54-52 Gy) dose radiation therapy (RT) with concurrent and adjuvant TMZ between June 2010 and October 2016. We retrospectively evaluated the therapeutic effectiveness of the RT ranges schedules in terms of overall survival (OS) with univariate and multivariate analysis, after analyzing the MGMT methylation status. ResultsOne hundred and seventeen patients were selected for the present analysis out of 146 total treated patients accrued. Seventy-two out of the selected cases received the standard RT-TMZ course (SDRT-TMZ) whereas the remaining 45 underwent the reduced dose schedule (RDRT-TMZ). The analysis according to the MGMT promoter methylation status showed that, in methylated-MGMT GB patients, SDRT-TMZ and RDRT-TMZ groups did not show different median OS (p = ns) according to the two RT schedules, independently by the extent of surgical resection. Instead, a difference in survival outcomes was confirmed in unmethylated-MGMT GB patients with better survival for patients undergoing to SDRT, particularly in sub-total resection. ConclusionIn our experience, a reduction of radiation dose schedule does not seem to jeopardize survival in methylated-MGMT patients independently by the extent of resection.A therapeutic approach to a standard reduction of RT dose for the methylated subset of patients may be feasible and could deserve prospective trials for validation.

The Prognostic Value of the Pretreatment Neutrophil/Lymphocyte Ratio in Patients with Glioblastoma Multiforme Brain Tumors: A Retrospective Cohort Study of Patients Treated with Combined Modality Surgery, Radiation Therapy, and Temozolomide Chemotherapy

Background: Recent studies have shown that the peripheral blood pretreatment neutrophil/lymphocyte ratio (NLR) is a prognostic measure in various cancers. The few studies evaluating NLR in glioblastoma multiforme (GBM) patients yielded inconsistent results. Objectives: The primary objective of our study was to test the ability of pretreatment NLR to predict the overall survival (OS) and progression-free survival (PFS) of patients with GBM treated by combined modality therapy (surgery, radiation, and temozolomide chemotherapy). A secondary objective was to evaluate the toxicity of the combined modality protocol in a consecutive series of patients treated in our center, in the context of a real-world universal health-care setting. Methods: We analyzed 89 patients with GBM in a retrospective cohort analysis who were treated in Soroka University Medical Center’s Oncology Department between the years 2005–2016. We analyzed NLR as a dichotomous variable at 3 cut-off points, 2.5, 3 and 4, as a predictor of OS and PFS. Methylation status of the O⁶-methylguanine-DNA methyltransferase (MGMT) promoter was not determined. Results: No significant correlation was found between NLR and either OS or PFS. Factors that predicted a shorter OS were age and extent of surgery. Patients over 70 years of age had a statistically significant shorter OS, 12.5 months (95% CI: 10.4–14.5 months) versus 17.6 months (95% CI: 14.2–21.1 months) in those 70 years of age and younger (p = 0.004). The OS of patients undergoing partial resection (12.7 months 95% CI: 8.3–17.1 months) or biopsy only (9.3 months 95% CI: 7.8–24.6 months), was significantly shorter than that of patients undergoing total resection (18.9 months, 95% CI: 11.8–26.0 months; p = 0.035). There were no treatment-related deaths. The most common grade III–IV toxicities were thrombocytopenia, 12.4%, and fatigue, 13.5%. Conclusions: In our cohort of GBM patients treated with combined modality therapy, pretreatment NLR was not prognostic. Toxicity of treatment was acceptable. Investigation of the NLR with larger groups of patients selected by MGMT status is warranted.

Predictive MGMT status in a homogeneous cohort of IDH wildtype glioblastoma patients

Methylation of the O(6)-Methylguanine-DNA methyltransferase (MGMT) promoter is predictive for treatment response in glioblastoma patients. However, precise predictive cutoff values to distinguish “MGMT methylated” from “MGMT unmethylated” patients remain highly debated in terms of pyrosequencing (PSQ) analysis. We retrospectively analyzed a clinically and molecularly very well-characterized cohort of 111 IDH wildtype glioblastoma patients, who underwent gross total tumor resection and received standard Stupp treatment. Detailed clinical parameters were obtained. Predictive cutoff values for MGMT promoter methylation were determined using ROC curve analysis and survival curve comparison using Log-rank (Mantel-Cox) test. MGMT status was analyzed using pyrosequencing (PSQ), semi-quantitative methylation specific PCR (sqMSP) and direct bisulfite sequencing (dBiSeq). Highly methylated (> 20%) MGMT correlated with significantly improved progression-free survival (PFS) and overall survival (OS) in our cohort. Median PFS was 7.2 months in the unmethylated group (UM, < 10% mean methylation), 10.4 months in the low methylated group (LM, 10-20% mean methylation) and 19.83 months in the highly methylated group (HM, > 20% mean methylation). Median OS was 13.4 months for UM, 17.9 months for LM and 29.93 months for HM. Within the LM group, correlation of PSQ and sqMSP or dBiSeq was only conclusive in 51.5% of our cases. ROC curve analysis revealed superior test precision for survival if additional sqMSP results were considered (AUC = 0.76) compared to PSQ (cutoff 10%) alone (AUC = 0.67). We therefore challenge the widely used, strict PSQ cutoff at 10% which might not fully reflect the clinical response to alkylating agents and suggest applying a second method for MGMT testing (e.g. MSP) to confirm PSQ results for patients with LM MGMT levels if therapeutically relevant.

Prognostic value of test(s) for O6 -methylguanine-DNA methyltransferase (MGMT) promoter methylation for predicting overall survival in people with glioblastoma treated with temozolomide

Background Glioblastoma is an aggressive form of brain cancer. Approximately five in 100 people with glioblastoma survive for five years past diagnosis. Glioblastomas that have a particular modification to their DNA (called methylation) in a particular region (the O6-methylguanine-DNA methyltransferase (MGMT) promoter) respond better to treatment with chemotherapy using a drug called temozolomide. Objectives To determine which method for assessing MGMT methylation status best predicts overall survival in people diagnosed with glioblastoma who are treated with temozolomide. Search methods We searched MEDLINE, Embase, BIOSIS, Web of Science Conference Proceedings Citation Index to December 2018, and examined reference lists. For economic evaluation studies, we additionally searched NHS Economic Evaluation Database (EED) up to December 2014. Selection criteria Eligible studies were longitudinal (cohort) studies of adults with diagnosed glioblastoma treated with temozolomide with/without radiotherapy/surgery. Studies had to have related MGMT status in tumour tissue (assessed by one or more method) with overall survival and presented results as hazard ratios or with sufficient information (e.g. Kaplan-Meier curves) for us to estimate hazard ratios. We focused mainly on studies comparing two or more methods, and listed brief details of articles that examined a single method of measuring MGMT promoter methylation. We also sought economic evaluations conducted alongside trials, modelling studies and cost analysis. Data collection and analysis Two review authors independently undertook all steps of the identification and data extraction process for multiple-method studies. We assessed risk of bias and applicability using our own modified and extended version of the QUality In Prognosis Studies (QUIPS) tool. We compared different techniques, exact promoter regions (5'-cytosine-phosphate-guanine-3' (CpG) sites) and thresholds for interpretation within studies by examining hazard ratios. We performed meta-analyses for comparisons of the three most commonly examined methods (immunohistochemistry (IHC), methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ)), with ratios of hazard ratios (RHR), using an imputed value of the correlation between results based on the same individuals. Main results We included 32 independent cohorts involving 3474 people that compared two or more methods. We found evidence that MSP (CpG sites 76 to 80 and 84 to 87) is more prognostic than IHC for MGMT protein at varying thresholds (RHR 1.31, 95% confidence interval (CI) 1.01 to 1.71). We also found evidence that PSQ is more prognostic than IHC for MGMT protein at various thresholds (RHR 1.36, 95% CI 1.01 to 1.84). The data suggest that PSQ (mainly at CpG sites 74 to 78, using various thresholds) is slightly more prognostic than MSP at sites 76 to 80 and 84 to 87 (RHR 1.14, 95% CI 0.87 to 1.48). Many variants of PSQ have been compared, although we did not see any strong and consistent messages from the results. Targeting multiple CpG sites is likely to be more prognostic than targeting just one. In addition, we identified and summarised 190 articles describing a single method for measuring MGMT promoter methylation status. Authors' conclusions PSQ and MSP appear more prognostic for overall survival than IHC. Strong evidence is not available to draw conclusions with confidence about the best CpG sites or thresholds for quantitative methods. MSP has been studied mainly for CpG sites 76 to 80 and 84 to 87 and PSQ at CpG sites ranging from 72 to 95. A threshold of 9% for CpG sites 74 to 78 performed better than higher thresholds of 28% or 29% in two of three good-quality studies making such comparisons.

Abstract B082: PD-1 to CD8 ratio on tumor-infiltrating lymphocytes and peripheral blood mononuclear cells as a predictor for determining response of glioblastoma patients to radiation-induced ICD-based DC vaccine therapy

Abstract Glioblastoma multiforme (GBM) is the most common and aggressive glioma within the central nervous system in adults. Radiation-induced ICD-based DC vaccine (RICD-DC vaccine) therapy plus conventional multi-modal regimen for GBM has been demonstrated with promising outcomes in clinical trials. However, some GBM patients received RICD-DC vaccine therapy did not increased survival rates than other GBM patients who only received conventional therapy. To investigate this issue, we conducted a retrospective study to analyze clinical and laboratory data to evaluate the factors which is critical for affecting the response rate of RICD-DC vaccine treatment. Patients with de novo GBM were enrolled (n=47) and divided into two subgroups: the first subgroup received post-surgical adjuvant immunotherapy with autologous RICD-DC vaccine (n=27) and the second received conventional treatment without immunotherapy (control, n=20). Quantitative immunohistochemistry for CD45, CD4, CD8, programmed death-1 (PD-1) and programmed death ligand 1 (PD-L1) was performed on patient tumor samples and peripheral blood mononuclear cells (PBMCs) at initial resection/biopsy before treatment. Pearson’s correlation, Cox proportional hazard model, and Kaplan-Meier analyses were performed to examine the correlations between these biomarkers expression and survival rates. In the RICD-DC vaccine group, patients with a lower PD-1+/CD8+ ratio (≤0.21) on tumor infiltrating lymphocytes (TILs) had longer overall survival (OS) (median 60.97 months, P &amp;lt; 0.001) than patients with a higher PD-1+/CD8+ ratio (median 20.07 months, , P &amp;lt; 0.001); 80% of the patients with a lower PD-1+/CD8+ ratio survived longer than 2 years whereas only 20% of patients with a higher PD-1+/CD8+ ratio. Similar results were observed in progression-free survival (PFS). Consistently, lymphocyte counts with lower PD-1+/CD8+ ratio (≤0.197) had much longer OS and PFS in patients’ PBMCs. In addition, there was a significant correlation of PD-1+/CD8+ ratio between TILs and PBMCs (Pearson’s correlation R2 = 0.6002, P &amp;lt; 0.001). By contrast, OS and PFS did not be impacted by CD4−, CD8−, but PD-1+, CD45+ TILs (P = 0.073 and P = 0.249, respectively). Moreover, the significant prognostic factors of OS and PFS also contained younger age (&amp;lt;57 years), gross total resection, and conventional treatment of concomitant chemoradiotherapy (CCRT) and PD-1+ lymphocyte counts in the RICD-DC vaccine group. In contrast to other factors, gender, counts of CD45+, CD4+ or CD8+ lymphocytes, tumoral PD-L1 expression, isocitrate dehydrogenase 1 mutation, and methylation status of O6 methylguanine-DNA methyltransferase promoter have no impact in both groups. Taken together, we found that TILs or PBMCs with lower PD-1+/CD8+ ratio in GBM patients with younger age, with gross tumor resection may be the desirable candidates who can be benefited from conventional multimodal regimen plus RICD-DC vaccine immunotherapy. Citation Format: Fang-Yu Lin, Chia-Ing Jan, Wan-Chen Tsai, Horng-Jyh Harn, Hsin-Man Lu, Ming-Chao Liu, Shao-Chih Chiu, Der-Yang Cho. PD-1 to CD8 ratio on tumor-infiltrating lymphocytes and peripheral blood mononuclear cells as a predictor for determining response of glioblastoma patients to radiation-induced ICD-based DC vaccine therapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B082.

Longitudinal Multilevel Omic Analysis of Pediatric T-ALL Reveals Distinct Mechanisms for Disease Progression in Type 1 and in Type 2 Relapses

We aimed at understanding the relapse-driving processes in pediatric T-ALL and performed an integrated longitudinal multi-level omics analysis of 13 T-ALL patients at initial diagnosis (INI) and relapse (REL). We compared the mutation (SNV/InDels) and copy number alteration (CNA) patterns as well as gene expression, methylation levels and chromatin accessibility by ATAC-Seq.Aberrant expression of T-ALL transcription factors (TAL1, TAL2,LMO2, TLX1, TLX3, NKX2.4 and NKX2.5) was preserved from initial presentation to relapse in all patients. These leukemia-driving events defined the expression patterns, methylation profiles and the chromatin accessibility landscapes.A global differential analysis of the RNA-Seq data (DESeq2, padj<.05) revealed only 0.3% of the genes to be either up- or down-regulated at relapse when compared to the matched initial sample. Likewise, we detected methylation changes in only 3% of the promoters and differential chromatin accessibility in only 0.26% of the analyzed ATAC-peaks (DESeq2, padj<.05).We then focused our analysis on the 2 types of relapse in pediatric T-ALL, which we have previously defined on the basis of subclonal mutation profiles (Kunz et al., 2015). These types of relapse are characterized by either clonal evolution of cells derived from the major clone at initial presentation (type 1) or emergence and evolution of a minor initial clone showing a molecular profile that is distinct from the predominant initial clone (type 2). When considering type 1 and type 2 relapses separately we identified a strong trend for type 2 relapses to acquire more mutations (p=0.0879, ttest) than type 1 relapses. Further to the known activating mutations in NT5C2 acquired at relapse by 8/13 patients no other mutations or CNAs were recurrently acquired in the relapses of this group of patients. However, mutations in proto-oncogenes or genes involved in DNA surveillance were acquired by 7/8 type 2 relapse patients in our series. Changes of CNAs also occurred more frequently in type 2 than in type 1 relapses (pval= 0.0267, ttest). This increased complexity on the genetic level was also apparent on the epigenetic level, with an increase of changes in the methylation pattern (mean difference in β value between INI and REL: type 1 - 0.00034; type2 - 0.002 (pval< 0.0001, chi2)), chromatin accessibility (number of differentially accessible ATAC-peaks: type 1 - 4 (0.006%) ; type 2 - 1018 (1.3%); (pval< 0.0001, chi2)) and on the expression level (number of differentially expressed genes: type 1 - 11; type 2 - 111, pval<0.0001, chi2).When considering differences between leukemias at the time of initial diagnosis, which later develop either type 1 or type 2 relapses we found 1.016 genes to be differentially expressed (524: up; 492: down in type 1; DE-Seq2: padj<0.05). Differential expression analysis revealed that genes involved in early T-cell differentiation were upregulated at initial diagnosis of type 1 in comparison to initial diagnosis of type 2, which was also reflected by more accessible chromatin surrounding their cis-regulatory regions as analyzed by ATAC-seq suggesting an early arrest of these samples during differentiation process. Altogether 1.4% of all ATAC-peaks were more accessible in type 1, whereas 0.7% of peaks were more accessible in type 2 leukemia (DE-Seq2: p<0.05). Remarkably, the chromatin surrounding DNA-repair genes was more accessible in initial leukemias of type 1, which was reflected by up-regulated mRNA expression level of such genes. These data suggest that an increased propensity of DNA repair may represent an important mechanism of T-ALL to develop a type 1 relapse, an interpretation that is consistent with previous reports linking the epigenetic silencing of the methylguanine-DNA methyltransferase promoter with compromised DNA repair and longer survival in patients with glioblastoma receiving alkylating agents (Esteller et. al., 2000).In sum, the multilevel omic comparison of pediatric T-ALL that develop either a type 1 or a type 2 relapse show remarkably more complex changes of the genetic and epigenetic profiles during the transition from initial to relapsing disease. Notably, pediatric T-ALLs, who later develop a type 1 relapse display an epigenetic and transcriptomic landscape predicting an upregulation of DNA repair functions, which we suggest to potentially play a role in developing resistance to DNA damaging agents in this type of relapse. DisclosuresMuckenthaler:Novartis: Research Funding. Bourquin:Amgen: Other: Travel Support. Kulozik:bluebird bio: Consultancy, Honoraria.