MGMT Promoter Hypermethylation Research Articles

N2M2/NOA-20: Phase I/IIa umbrella trial of molecularly matched targeted therapies plus radiotherapy in patients with newly diagnosed glioblastoma without MGMT promoter hypermethylation.

2000 Background: Patients with glioblastoma without MGMT promoter hypermethylation are unlikely to benefit from temozolomide (TMZ). Trials aiming at replacing TMZ with targeted agents in not molecularly selected patient populations have failed. Methods: This phase I/IIa umbrella trial aimed at showing safety, feasibility, and preliminary efficacy of targeted compounds in addition to standard radiotherapy initiated within 42 days postoperatively. Molecular diagnostics and bioinformatic evaluation are performed within 28 days after surgery. Stratification for treatment takes place in five subtrials, including alectinib, idasanutlin, palbociclib, vismogedib and temsirolimus, according to the best matching molecular alteration. Patients without matching alterations are randomized between subtrials without strong biomarkers using atezolizumab and asunercept and TMZ as standard of care. Primary objective of the phase I parts of the trial was dose finding or dose validation. In the phase IIa trials, centrally determined progression-free survival at six months (PFS-6) is used as endpoint for efficacy with interim analyses for futility ( H0: p=0.231). Results: From May 2018 through July 2022, 301 patients were enrolled and 228 treated in 13 German NOA sites. The alectinib and vismodegib subtrials were closed since no molecularly matching patients were accrued; the idasanutlin subtrial was closed prior to the optimal dose at nine patients at discretion of the company providing the drug. The TMZ subtrial showed a PFS-6 of 18.52% (10/54 patients) (p=0.831) and a median overall survival (OS) of 12.1 months. Asunercept: PFS-6 of 15.4% (4/26) (p=0.8825) and OS of 12.8 months. Atezolizumab: PFS-6 of 21.4% (9/42) (p=0.660) and OS of 11.7 months. Palbociclib with patients demonstrating CDK4 amplification or CDKN2A/B codeletion: PFS-6 of 24.4% (10/41) (p=0.4823) and OS of 12.6 months. Temsirolimus with patients demonstrating mTOR activation: PFS-6 of 39.1% (18/46) (p=0.0109) and OS of 15.4 months. The regimen-limiting toxicity (RLT)-rate is 34.8%, which is insignificantly above the predefined unacceptable rate for RLTs of 30%. Most RLTs had severity grade 3, one RLT had severity grade 4. No RLTs resulted in death. Conclusions: N2M2 allows for elaborate molecular testing being integrated into the treatment decision and efficient determination of treatment activity for patients with newly diagnosed glioblastoma. There is clinical activity of temsirolimus in patients with tumors harboring an activated mTOR pathway although this is not positively prognostic without mTOR inhibition; there is no clinical activity for asunercept and atezolizumab in not molecularly selected patients and also palbociclib in molecularly selected patients. Clinical trial information: NCT03158389 .

‘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.

293 The Impact of Tumor Mutational Status on Protoporphyrin IX Accumulation in Tumor-Associated Macrophages in Glioblastoma Patients

INTRODUCTION: Immunotherapies provide promising new treatment options for lung cancer and melanoma, however success in glioblastoma (GBM) patients has been much rarer and uneven. Identifying candidates with GBM that are most likely to benefit from immunotherapy remains a challenge. The abundance of tumor-associated macrophages (TAMs) correlates response to immunotherapy in GBM patients. Here we leverage a new imaging technique for quantifying TAMs via tandem stimulated Raman histology (SRH) and two-photon excitation fluorescence microscopy (TPEF) to assess the impact of genetic classification on the abundance of TAMs. METHODS: We conducted tandem SRH and TPEF on specimens from 79 patients undergoing high-grade glioma resection with 5-ALA. We trained a MaskR-CNN deep neural network to identify TAMs and compute their density in each specimen. Corresponding clinical, demographic, genomic and methylomic data were collected for each patient. Independent sample t-tests were used to identify significant correlations between genetic markers, treatment, and TAM density. RESULTS: We analyzed 3,121,524 300 x 300 micron fields of view, from 151 fresh specimens, enabling segmentation of 611,789 TAMs. IDH-mutant tumors had significantly lower TAM density than IDH-wildtype tumors (p < 0.01). The absence of MGMT promoter hypermethylation was associated with significantly higher TAM density (p = 0.03). Tumors with EGFR amplification averaged significantly lower TAM density than EGFR wildtype specimens (p < 0.01). Recurrent tumors possessed 31.5% lower TAM density than primary resections. CONCLUSIONS: Tandem SRH and TPEF can estimate variations in TAM density in human GBMs. We found TAM density to be associated with IDH status, MGMT promoter methylation, and EGFR amplification. This imaging technique creates a new avenue for forecasting response to immunotherapy in GBM patients.

Abstract 7123: Biomarker-targeted alkylator and DNA damage repair inhibitor combination for refractory and relapsed acute myeloid leukemia (AML)

Abstract Isocitrate dehydrogenase 1 and 2 (IDH1/2) convert isocitrate to α-ketoglutarate in the tricarboxylic acid (TCA) cycle. A missense mutation in IDH1/2 leads to accelerated conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG), an oncometabolite that competitively inhibits α-ketoglutarate-dependent dioxygenases including Ten-Eleven Translocation (TET) enzymes. TET enzymes are DNA demethylases that convert 5-methylcytosine to 5-carboxycytosine and regulate CpG island promoter methylation signatures. In IDH1/2 mutant glioma, inhibition of TET2 enzyme by 2-HG can lead to promoter hypermethylation of O6-methylguanine methyltransferase (MGMT) and reduce its expression. Approximately 10-15% of de novo or treatment-related AML harbor mutually exclusive mutations in IDH1/2 or TET2, and emerging resistance towards clinical IDH1/2 inhibitors, therefore warrants novel therapeutic options. We performed a pan-cancer TCGA analysis and observed MGMT promoter hypermethylation and reduced expression in IDH1/2 AML. Our independent study on 10 AML patients (5 IDH1/2 and 5 TET2 mutant) corroborated the TCGA data and showed a significantly reduced expression of MGMT in comparison to 7 wild-type AML samples. As MGMT resolves O6-alkylguanine lesions during DNA damage, we performed an exhaustive alkylator screen, where temozolomide (TMZ) and our novel compound KL-50 emerged as the top hits that imparted a maximum therapeutic index in multiple MGMT (MGMT+/–) isogenic AML cell line models. Both TMZ and KL-50 induced MGMT-dependent DNA strand breaks and replication stress and activated ATM and ATR DNA damage response pathways in a time-dependent manner. Inhibition of ATR activation with Ceralasertib, an ATR inhibitor (ATRi) currently in clinical trials for chronic myelomonocytic leukemia and myelodysplastic syndromes, imparted a profound MGMT-dependent synergy with TMZ and KL-50. Researchers have established in glioma that mismatch repair (MMR) loss confers resistance to TMZ, and reported studies also show evidence of MMR loss in refractory or relapsed AML patients. Thus, we developed numerous MMR-deficient isogenic AML cell lines in an MGMT+/– background and observed MMR loss-mediated resistance to TMZ and abrogation of its synergy with ATRi. Unlike TMZ, KL-50 retained a profound synergy with ATRi in an MGMT-dependent manner irrespective of MMR status. We are currently investigating our novel KL-50 and ATRi combination in MGMT/MMR isogenic cell line-based in vivo AML models and in AML patient-derived xenografts to establish it as a promising novel biomarker-targeted therapy for AML patients. Citation Format: Prateek Bhardwaj, Ranjini Sundaram, Amro Baassiri, Martin Matthews, Jennifer VanOudenhove, Susan Gueble, Stephanie Halene, Ranjit Bindra. Biomarker-targeted alkylator and DNA damage repair inhibitor combination for refractory and relapsed acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7123.

Improved Pathologic response to chemoradiation in MGMT methylated locally advanced rectal cancer.

With the growing interest in total neoadjuvant treatment for locally advanced rectal adenocarcinoma (LARC) there is an urgent unmet need to identify predictive markers of response to long-course neoadjuvant concurrent chemoradiotherapy (LCRT). O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) gene methylation has been associated in some malignancies with response to concurrent chemoradiotherapy. We attempted to find if pathologic response to LCRT was associated with MGMT promoter hypermethylation (MGMTh). Patients were identified with LARC, available pre-treatment biopsy specimens, and at least 1year of follow-up who received LCRT followed by surgical resection within 6months. Biopsies were tested for MGMTh using a Qiagen pyrosequencing kit (Catalog number 970061). The primary outcome of LCRT responsiveness was based on tumor regression grade (TRG), with grades of 0-1 considered to have excellent response and grades of 2-3 considered to be non-responders. Secondary outcomes included overall survival (OS) and recurrence free survival (RFS). Of 96 patients who met inclusion criteria, 76 had samples which produced reliable assay results. MGMTh corresponded with higher grade and age of the biopsy specimen. The percentage of responders to LCRT was higher amongst the MGMTh patients than the MGMTn patients (60.0% vs 27.5%, p value=0.0061). MGMTh was not significantly associated with improved OS (2-year OS of 96.0% vs 98.0%, p=0.8102) but there was a trend for improved RFS (2-year RFS of 87.6% vs 74.2%, p=0.0903). Significantly greater tumor regression following LCRT was seen in MGMTh LARC. Methylation status may help identify good candidates for close observation without surgery following LCRT.

What predicts survival in glioblastoma? A population-based study of changes in clinical management and outcome.

Glioblastoma is the most common and most aggressive primary brain tumor in adults. Despite multimodal treatment, the median survival time is 15-16 months and 5-year survival rate 5%-10%. The primary goal of this study was to identify prognostic factors for survival in an unselected population of patients operated for glioblastoma. The secondary goal was to explore changes in outcome and the clinical management of this patient group over time. We identified 222 consecutive adults operated for glioblastoma between November 2012 and June 2016 at the Department of Neurosurgery, Sahlgrenska University Hospital in Gothenburg, serving a health care region in the western part of Sweden with 1.900.000 inhabitants. Clinical variables were identified and tested as predictors for prognosis in extended Poisson regression models. The results were compared with a previously published cohort from 2004 to 2008, before current standard of care based on molecular tumor diagnosis was fully implemented. Median overall survival was 1.07 years, which was significantly longer than in the 2004-2008 cohort (1.07 vs. 0.73 y, age- and sex adjusted HR = 1.89, p < 0.0001). Variables associated with longer survival in the multivariable model were MGMT promoter hypermethylation, non-central tumor location, complete resection of enhancing tumor, WHO performance status 0-1, unilateral tumor location, fewer lobes involved, younger age and no comorbidities. The median survival for patients with glioblastoma treated according to current standard treatment has moderately but significantly increased, with MGMT promoter hypermethylation as the strongest predictor for survival.

The success of volumetric means ADC in predicting MGMT promoter hypermethylation in glioblastomas

Aims: This study aimed to investigate the relationship between volumetric mean ADC values and MGMT promoter hypermethylation status in glioblastoma (GB) patients segmented into perilesional edema area, solid tumor area, and necrosis area.&#x0D; Methods: The 212 GB patients in the University of California San Francisco Preoperative Diffuse Glioma MRI (UCSF-PDGM) dataset were retrieved from the Cancer Imaging Archive (TCIA). The mean volumetric ADC value was calculated in patients with shared segmentation data in the UCSF-PDGM dataset. The difference in mean volumetric ADC value was investigated in patients divided into groups based on MGMT promoter hypermethylation (MGMT+/ MGMT-).&#x0D; Results: Of the patients in our study, 125 (59.0%) were male. The median age of the patients was 62 years (26-94). MGMT promoter hypermethylation was observed in 152 (71.7%) patients. Mean Survival was calculated as 574.14±345.57 days in the MGMT+ group and 484.68±301.71 days in MGMT- group. According to volumetric mean ADC values, a difference was observed in the solid tumor and perilesional edema areas according to MGMT promoter hypermethylation (p1.14 in ADC measurements from the edema area with 72% sensitivity and 90% specificity. MGMT+ group could be determined with a sensitivity of 88% and specificity of 69% with a cut-off value of &gt;1.01 in ADC measurements from the solid tumor area.&#x0D; Conclusion: Volumetric ADC measurements from the perilesional edema and solid tumor areas revealed higher ADC values in the MGMT+ group.

Quantitative DNA Repair Biomarkers and Immune Profiling for Temozolomide and Olaparib in Metastatic Colorectal Cancer.

O6-methylguanine DNA methyltransferase (MGMT)-silenced tumors reveal sensitivity to temozolomide (TMZ), which may be enhanced by PARP inhibitors. Approximately 40% of colorectal cancer has MGMT silencing and we aimed to measure antitumoral and immunomodulatory effects from TMZ and olaparib in colorectal cancer. Patients with advanced colorectal cancer were screened for MGMT promoter hypermethylation using methylation-specific PCR of archival tumor. Eligible patients received TMZ 75 mg/m2 days 1-7 with olaparib 150 mg twice daily every 21 days. Pretreatment tumor biopsies were collected for whole-exome sequencing (WES), and multiplex quantitative immunofluorescence (QIF) of MGMT protein expression and immune markers. MGMT promoter hypermethylation was detected in 18/51 (35%) patients, 9 received study treatment with no objective responses, 5/9 had stable disease (SD) and 4/9 had progressive disease as best response. Three patients had clinical benefit: carcinoembryonic antigen reduction, radiographic tumor regression, and prolonged SD. MGMT expression by multiplex QIF revealed prominent tumor MGMT protein from 6/9 patients without benefit, while MGMT protein was lower in 3/9 with benefit. Moreover, benefitting patients had higher baseline CD8+ tumor-infiltrating lymphocytes. WES revealed 8/9 patients with MAP kinase variants (7 KRAS and 1 ERBB2). Flow cytometry identified peripheral expansion of effector T cells. Our results indicate discordance between MGMT promoter hypermethylation and MGMT protein expression. Antitumor activity seen in patients with low MGMT protein expression, supports MGMT protein as a predictor of alkylator sensitivity. Increased CD8+ TILs and peripheral activated T cells, suggest a role for immunostimulatory combinations. TMZ and PARP inhibitors synergize in vitro and in vivo in tumors with MGMT silencing. Up to 40% of colorectal cancer is MGMT promoter hypermethylated, and we investigated whether TMZ and olaparib are effective in this population. We also measured MGMT by QIF and observed efficacy only in patients with low MGMT, suggesting quantitative MGMT biomarkers more accurately predict benefit to alkylator combinations.

Is sarcopenia a predictor of overall survival in primary IDH-wildtype GBM patients with and without MGMT promoter hypermethylation?

Background: In this study, we aimed to examine the success of temporal muscle thickness (TMT) and masseter muscle thickness (MMT) in predicting overall survival (OS) in primary IDH-wild glioblastoma (GBM) patients with and without MGMT promoter hypermethylation through publicly available datasets. Methods: We included 345 primary IDH-wild GBM patients with known MGMT promoter hypermethylation status who underwent gross-total resection and standard treatment, whose data were obtained from the open datasets. TMT was evaluated on axial thin section postcontrast T1-weighted images, and MMT was evaluated on axial T2-weighted images. The median TMT and MMT were used to determine the cut-off point. Results: The findings showed that median TMT 9.5 mm and median MMT 12.7 mm determined the cut-off value in predicting survival. Both TMT and MMT values less than the median muscle thickness were negatively associated with OS (TMT&lt;9.5: HR 3.63 CI 2.34–4.23, p &lt;0.001, MMT&lt;12.7: HR 3.53 CI 2.27–4.07, p &lt;0.001). When patients were classified according to MGMT positivity, the findings showed MGMT-negative patients (TMT&lt;9.5: HR 2.54 CI 1.89–3.56, p &lt;0.001, MMT&lt;12.7: HR 2.65 CI 2.07–3.62, p &lt;0.001) and MGMT-positive patients (TMT&lt;9.5: HR 3.84 CI 2.48–4.28, p &lt;0.001, MMT&lt;12.7: HR 3.73 CI 2.98–4.71, p &lt;0.001). Conclusion: Both TMT and MMT successfully predict survival in primary GBM patients. In addition, it can successfully predict survival in patients with and without MGMT promoter hypermethylation.

Potential predictive biomarker for response to radiotherapy and CXCL12 inhibition in glioblastoma in the phase I/II GLORIA trial.

2048 Background: Standard of care (SOC) treatment achieves poor clinical outcomes in patients with glioblastoma (GBM), particularly in the absence of MGMT promoter hypermethylation. Preclinical models suggest that GBM recurrence is facilitated by CXCL12-mediated recruitment of bone marrow-derived cells capable of vasculogenesis after radiotherapy (RT). We have recently reported favorable safety and feasibility data of the phase I/II GLORIA trial, which combines RT and the CXCL12-neutralizing L-RNA aptamer olaptesed pegol (NOX-A12) in patients with newly diagnosed, incompletely resected or unresected GBM lacking MGMT promoter hypermethylation (NCT04121455). Here we report on clinical outcomes and their correlation with potential biomarkers. Methods: 10 patients with newly diagnosed incompletely resected (n=8) or biopsied (n=2) GBM with ECOG≤2, age≥18 and without MGMT promoter hypermethylation were enrolled. All patients received standard RT (60 Gy in 30 fractions or 40.05 Gy in 15 fractions) and escalating dose levels of continuous (24/7) i.v. infusions of NOX-A12 for 26 weeks. While the previously reported primary endpoint was safety, secondary efficacy endpoints included progression-free survival (PFS) and overall survival (OS). Biomarker assessment was performed with a six-plex immunofluorescence staining (CODEX) of neuropathologically confirmed tumor areas of pretreatment tissues for CXCL12, CD31 (endothelial cells), GFAP (glioma cells), CD68 (macrophages, microglia), aSMA (pericytes), Ki-67 (proliferating cells) and a nuclear marker. An independent patient cohort treated with SOC (n=15) matched by clinical and histopathological features was used for comparisons of efficacy and biomarker assessment. Results: Median PFS of the GLORIA cohort was 5.7 (range 1.9–8.5) months and the median OS was 12.7 (4.7–18.4) months. Biomarker analyses revealed that a higher frequency of CXCL12 expressing endothelial and glioma cells (EG12 score) significantly correlated with PFS (r=0.87; p=0.005) in patients treated with RT and NOX-A12, but not with SOC (r=-0.10; p=0.724). GLORIA patients with a high EG12 score (median classifier) had a significantly longer PFS than those with lower scores (3.0 vs. 6.0 months; p=0.031) and a trend towards prolonged OS (11.1 vs. 15.8 months; p=0.075). These correlations were not seen in the reference cohort treated with SOC (PFS: 6.0 vs. 4.6 months; p=0.502; OS: 10.0 vs. 9.6 months; p=0.243). Conclusions: We show superior clinical efficacy of RT and NOX-A12 in patients with high frequency of CXCL12 expressing endothelial and glioma cells, suggesting the use of the EG12 score as a novel predictive biomarker for CXCL12-directed therapies in GBM. Clinical trial information: NCT04121455 .

Abstract PR005: Features and susceptibility to dietary-induced alkylating damage in colorectal cancer

Abstract Introduction: Colorectal cancer (CRC) has several established risk factors, including diet. However, their mutagenic effect has not been observed directly in patients’ tumors and the individuals or ethnic groups who are most susceptible to diet-induced carcinogenesis are yet to be identified. We hypothesized that mutational signature analyses in CRC, coupled with epidemiologic, tumor molecular and patient germline data, can be linked to pre-diagnosis diet and specific germline alterations which can further inform cancer prevention efforts. Methods: We analyzed 900 CRCs with whole-exome sequencing (WES) and prospectively collected pre-diagnosis dietary data from the Nurses’ Health Studies I and II (NHS) and the Health Professionals Follow-up Study (HPFS), as well as 540 The Cancer Genome Atlas (TCGA) tumors and 295 non-Western CRCs from the Pan-Cancer Analysis of Whole Genomes. We additionally examined a sequencing dataset of normal colonic crypts. Following variant calling, mutational signature analyses were performed on cancer specimens using Non-negative Matrix Factorization. We leveraged the WES results to identify all patients that harbored the O-6-Methylguanine-DNA Methyltransferase (MGMT) germline rs16906252 single-nucleotide polymorphism (SNP), which is associated with tumor MGMT promoter hypermethylation and impaired alkylating damage repair. Results: We identified a novel alkylating mutational signature in CRC, which was associated with pre-diagnosis intake of red meats (overall red meat P = 0.017, unprocessed red meat P = 7.8×10-3, processed red meat P = 7.1×10-3, Mann-Whitney test), distal tumor location (P = 1.4×10-4, Mann-Whitney test) and worse CRC-specific survival (P trend = 0.036, multivariable Cox regression). We found a similar mutational signature in normal colonic crypts, suggesting an early oncogenic process. We showed that the alkylating signature’s oncogenicity is mainly mediated by KRAS p.G12D and KRAS p.G13D hotspot mutations. In addition, individuals harboring the MGMT rs16906252 germline SNP had a significant enrichment in tumors with alkylating damage (P = 0.015, Mann-Whitney test) and the effect of the SNP was synergistic with pre-diagnosis red-meat intake (P = 0.0099, Wald Test). Notably, we did not observe the alkylating mutational signature in CRCs from African and East-Asian patients, populations in which the MGMT rs16906252 SNP is largely absent. Conclusions: Through the integration of molecular and epidemiologic data, we identified evidence of red meat-associated alkylating damage in CRCs. This alkylating mutational signature was associated with distal colon location, KRAS oncogenic drivers, and poor CRC-specific survival. In addition, tumor alkylating damage was enriched among individuals harboring the MGMT germline rs16906252 polymorphism, absent in East Asian and African CRC patients and was synergistic with pre-diagnosis red-meat intake. Together, these results have significant implications for dietary-induced carcinogenesis and precision prevention in CRC. Citation Format: Carino Gurjao, Rong Zhong, Koichiro Haruki, Chichun Tan, Yvonne Li, Henry Lee-Six, Brendan Reardon, Tomotaka Ugai, Mingyang Song, Eliezer M. Van Allen, Charles S. Fuchs, Jonathan A. Nowak, Kana Wu, Jeffrey A. Meyerhardt, Edward L. Giovannucci, Jeffrey P. Townsend, Shuji Ogino, Marios Giannakis. Features and susceptibility to dietary-induced alkylating damage in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference: Precision Prevention, Early Detection, and Interception of Cancer; 2022 Nov 17-19; Austin, TX. Philadelphia (PA): AACR; Can Prev Res 2023;16(1 Suppl): Abstract nr PR005.

CTNI-67. DUAL INHIBITION OF POST-RADIOGENIC ANGIO-VASCULOGENESIS BY OLAPTESED PEGOL (NOX-A12) AND BEVACIZUMAB IN GLIOBLASTOMA – INTERIM DATA FROM THE FIRST EXPANSION ARM OF THE GERMAN PHASE 1/2 GLORIA TRIAL

Abstract BACKGROUND We recently reported favorable safety, promising clinical efficacy and immunohistochemical indicators of response after radiotherapy (RT) plus escalating doses of the CXCL12-neutralizing RNA-Spiegelmer olaptesed pegol (NOX-A12) for glioblastoma in the German multicenter phase 1/2 GLORIA trial (NCT04121455). Here, we report outcomes after RT plus dual inhibition of vasculogenesis (NOX-A12) and angiogenesis (bevacizumab). METHODS After establishing safety in the monotherapy arm, we enrolled six patients with incompletely resected GBM, ECOG ≤ 2, age ≥ 18 and without MGMT promoter hypermethylation into a pre-planned expansion arm. Patients received standard RT (60 Gy in 30 fractions), continuous i.v. infusions of NOX-A12 (600 mg/week) and i.v. infusions of bevacizumab (10 mg/kg q2w). The primary endpoint was safety. Secondary endpoints included radiographic response, perfusion/diffusion imaging and neurologic performance. RESULTS Dual treatment was well-tolerated and safe. Of all G ≥ 2 AEs (n = 37), two G2 events (5.4%) were deemed related to NOX-A12. There were no dose-limiting toxicities and no treatment-related deaths. Longitudinal NANO assessment revealed stable neurologic functioning in all patients. Five out of six patients achieved partial responses (PRs) as per mRANO in week 9. All PRs remained durable at a median follow up of 5.6 months (range 3.6 to 9.3 months). No progression occurred. The mean best response was -65.9% (-13.3% to -99.9%) for target lesion sums and -92.1% (-76.2% to -100%) for non-target lesion (NTL) sums. In all three patients with NTL at least one lesion disappeared. The mean best change from baseline of the highly perfused-tumor fraction was -84.5% (-51.9% to -100%) and the mean best change of the apparent diffusion coefficient was 20.1% (-24.5% to 59.1%). CONCLUSION Interim data of the ongoing trial confirm the previously established safety profile of NOX-A12 and suggest improved efficacy of dual inhibition of post-radiogenic angio- and vasculogenesis by the addition of bevacizumab.

Targeted genetic and epigenetic profiling of esophageal adenocarcinomas and non-dysplastic Barrett’s esophagus

BackgroundDespite the efforts to describe the molecular landscape of esophageal adenocarcinoma (EAC) and its precursor lesion Barrett’s esophagus (BE), discrepant findings are reported. Here, we investigated the prevalence of selected genetic (TP53 mutations and microsatellite instability (MSI) status) and epigenetic (DNA promoter hypermethylation of APC, CDKN2A, MGMT, TIMP3 and MLH1) modifications in a series of 19 non-dysplastic BE and 145 EAC samples. Additional biopsies from adjacent normal tissue were also evaluated. State-of-the-art methodologies and well-defined scoring criteria were applied in all molecular analyses.ResultsOverall, we confirmed frequent TP53 mutations among EAC (28%) in contrast to BE, which harbored no mutations. We demonstrated that MSI and MLH1 promoter hypermethylation are rare events, both in EAC and in BE. Our findings further support that APC, CDKN2A, MGMT and TIMP3 promoter hypermethylation is frequently seen in both lesions (21–89%), as well as in a subset of adjacent normal samples (up to 12%).ConclusionsOur study further enlightens the molecular background of BE and EAC. To the best of our knowledge, this is one of the largest studies addressing a targeted analysis of genetic and epigenetic modifications simultaneously across a combined series of non-dysplastic BE and EAC samples.

MGMT inactivation as a new biomarker in patients with advanced biliary tract cancers.

Biliary tract cancers (BTCs) have poor prognosis and limited therapeutic options. The impact of O 6‐methylguanine‐DNA methyltransferase (MGMT) inactivation in advanced BTC patients is not established. We investigated the prevalence, prognostic, and predictive impact of MGMT inactivation in two multicenter cohorts. MGMT inactivation was assessed through PCR and immunohistochemistry (IHC) in an Italian cohort; the results were then externally validated using RNA sequencing (RNA‐seq) data from the BTC subcohort of the Molecularly Aided Stratification for Tumor Eradication Research (MASTER) precision oncology program of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. Among 164 Italian cases, 18% presented MGMT promoter hypermethylation (> 14%) and 73% had negative MGMT protein expression. Both were associated with worse overall survival (OS; HR 2.31; P < 0.001 and HR 1.99, P = 0.012, respectively). In the MASTER cohort, patients with lower MGMT mRNA expression showed significantly poorer OS (median OS [mOS] 20.4 vs 31.7 months, unadjusted HR 1.89; P = 0.043). Our results suggest that MGMT inactivation is a frequent epigenetic alteration in BTC, with a significant prognostic impact, and provide the rationale to explore DNA‐damaging agents in MGMT‐inactivated BTCs.

EPEN-06. Comprehensive profiling of myxopapillary ependymomas identifies a distinct molecular subtype with relapsing disease

Abstract Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients’ clinical course are unknown. We assembled a cohort of 185 tumors classified as MPE based on DNA methylation from pediatric, adolescent, and adult patients. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPEB, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. Tumors in the methylation class MPE were histologically diagnosed as WHO grade I (59%), WHO grade II (37%), or WHO grade III tumors (4%). 75/77 analyzed tumors expressed HOXB13, which is a diagnostic feature not detected in other spinal ependymal tumors. Based on DNA methylation, our series split into two subtypes. MPE-A occurred in younger patients (median age 27 vs. 45 years, p=7.3e-05). They were enriched with WHO grade I tumors and associated with papillary morphology and MGMT promoter hypermethylation (all p&amp;lt;0.001). MPE-B included most tumors initially diagnosed as WHO grade II and cases with tanycytic morphology. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. 15/30 MPE-A could not be totally resected compared to 1/58 MPE-B (p=6.3e-08), and progression-free survival was significantly better for MPE-B (p=3.4e-06, 10-year relapse rate 33% vs. 85%). We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.

Randomized phase II/III trial of veliparib or placebo in combination with adjuvant temozolomide in newly diagnosed glioblastoma (GBM) patients with MGMT promoter hypermethylation (Alliance A071102).

2001 Background: PolyADP-ribose polymerase (PARP) is an important modulator of DNA repair following temozolomide (TMZ) therapy. Pre-clinical testing demonstrated significant survival benefit for the combination of TMZ and PARP inhibitor veliparib in a subset of GBM pt-derived xenografts with MGMT promoter hypermethylation. Methods: After central pathology review and MGMT testing, patients (pts) with newly diagnosed, MGMT promoter hypermethylated GBM who had completed concurrent radiation and TMZ were randomized to adjuvant therapy with TMZ (Days 1-5 q28 days) combined with either placebo or veliparib (Days 1-7 q28 days). Veliparib/placebo+TMZ treatment was continued for up to 6 cycles. Pts accrued on the phase II and III portions of the trial were included in the primary endpoint analysis of overall survival (OS), with 90% power to detect a hazard ratio of 0.71 using a one-sided log-rank test with type I error rate of 0.05. The planned phase III sample size was 400 pts with data maturity after 302 deaths. Results: The phase II and III portions of the trial were open to accrual from 12/15/2014 to 2/6/2017 and 11/8/2017 to 10/15/2018, respectively; 447 pts were accrued to the trial and used in this intention to treat analysis. The two treatment groups were well balanced for prognostic factors, 421 pts initiated treatment, median follow-up was 57.8 months (mos), 380 pts had disease progression and 335 pts have died. There was no difference in OS (p = 0.15; HR 0.89 (0.71-1.11), median OS 28.1 vs. 24.8 mo. for TMZ+veliparib vs. TMZ+placebo, respectively) and no difference in secondary endpoint progression free survival (PFS, p = 0.31; HR 1.05 (0.86-1.30), median 13.2 vs. 12.1 mo, respectively). There was a notable trend for extended OS with TMZ+veliparib treatment at intermediate time-points between 24 and 42 mos (3-year OS 36.6% vs. 28.9% with TMZ+placebo, p = 0.09). In an unplanned exploratory analysis, treatment with TMZ at the time of first recurrence was associated with extended post-recurrence OS (p = 0.03) for pts treated on the experimental arm; median post-recurrence OS with TMZ salvage was 17.0 mo in the TMZ+veliparib arm and 12.6 mo in the TMZ+placebo arm, as compared to 9.6 mo in either arm if TMZ salvage was not used. These data are consistent with a possible effect of veliparib limiting the emergence of TMZ resistance in a subset of GBM pts. Conclusions: Veliparib combined with adjuvant TMZ therapy was not associated with significant extension in OS or PFS in newly diagnosed, MGMT hypermethylated GBM pts. However, a subset of pts treated with TMZ+veliparib may have an extended survival following re-treatment with TMZ at first recurrence. Clinical trial information: NCT02152982.

Radiotherapy and olaptesed pegol (NOX-A12) in partially resected or biopsy-only MGMT-unmethylated glioblastoma: Interim data from the German multicenter phase 1/2 GLORIA trial.

2050 Background: Pre-clinical studies consistently demonstrate that inhibition of the CXCL12/CXCR4/CXCR7 axis abrogates recruitment of pro-vasculogenic bone marrow-derived cells after radiotherapy (RT) of glioblastoma (GBM) and promotes T cell exclusion from the tumor microenvironment (TME). The German multicenter phase 1/2 trial GLORIA (NCT04121455) assesses safety of RT plus escalating dose levels (DL) of the CXCL12-neutralizing RNA-Spiegelmer Olaptesed pegol (OLA; NOX-A12) in patients with chemotherapy-resistant GBM. Methods: Until now, GLORIA enrolled 10 patients newly diagnosed with incompletely resected (n = 8) or biopsied (n = 2) GBM with ECOG≤2, age ≥18 and without MGMT promoter hypermethylation. All patients receive standard RT (60 Gy in 30 fractions or 40.05 Gy in 15 fractions) and continuous (24/7) i.v. infusions of either 200 mg (DL1; n = 3), 400 mg (DL2; n = 3) or 600 mg (DL3; n = 4) per week of OLA for 26 weeks. The primary endpoint (EP) is safety as per incidence of treatment-related adverse events (AE). Secondary EPs include radiographic response as per mRANO criteria, dynamic susceptibility contrast perfusion (DSC) and the fraction of highly-perfused tumor (FTBhigh) as well as the apparent diffusion coefficient (ADC). Target lesions (TL) and non-target lesions (NTL, i.e. in-field satellite lesions) are analyzed separately. Tumor tissue is assessed by high-plex immunofluorescence imaging (co-detection by indexing; CODEX). Matched reference cohorts serve as controls for MRI (n = 14) and CODEX (n = 8) data. Results: Combination of RT and OLA was well-tolerated and safe. Of all G ≥ 2 AEs (n = 77), 3 (4%) were deemed to be solely OLA-related, including 1 grade 3 AE at DL3. There were no dose limiting toxicities and no treatment-related deaths. In total, eight of the nine patients (89%) with TLs at baseline showed a TL response during OLA therapy, with four (40%) reaching partial remission (PR) as per radiologic mRANO criteria (n = 2 at DL1 and n = 2 at DL3). All three patients treated at DL1 and all four of DL3 reached PR of one or more NTLs. In three cases (n = 2 at DL1; n = 1 at DL3), at least one NTL completely disappeared. Under OLA, radiographic responses of NTL were best at the highest DL (DL1 +49.5/DL2 +488.3/DL3 -59%), as was the increase in diffusion (mean ADC increase +46.4/+28.2/+56.7%) and the decrease in FTBhigh (mean -33.5/-32.8/-47.7%). Matched pre-/post-surgery CODEX of a confirmed pseudoprogression revealed intralesional clusters of proliferating cytotoxic T cells. Analysis of tissue from a non-responding patient showed T-cell encapsulation by M2-polarized macrophages in an immune-cell enriched TME. Additional follow-up is ongoing. Conclusions: Interim data from the ongoing GLORIA trial demonstrates safety of RT plus OLA and suggests promising clinical efficacy of a new class of drugs targeting CXCL12 in GBM. Clinical trial information: NCT04121455.

Effects of Long-Term Temozolomide Treatment on Glioblastoma and Astrocytoma WHO Grade 4 Stem-like Cells.

Glioblastoma leads to a fatal course within two years in more than two thirds of patients. An essential cornerstone of therapy is chemotherapy with temozolomide (TMZ). The effect of TMZ is counteracted by the cellular repair enzyme O6-methylguanine-DNA methyltransferase (MGMT). The MGMT promoter methylation, the main regulator of MGMT expression, can change from primary tumor to recurrence, and TMZ may play a significant role in this process. To identify the potential mechanisms involved, three primary stem-like cell lines (one astrocytoma with the mutation of the isocitrate dehydrogenase (IDH), CNS WHO grade 4 (HGA)), and two glioblastoma (IDH-wildtype, CNS WHO grade 4) were treated with TMZ. The MGMT promoter methylation, migration, proliferation, and TMZ-response of the tumor cells were examined at different time points. The strong effects of TMZ treatment on the MGMT methylated cells were observed. Furthermore, TMZ led to a loss of the MGMT promoter hypermethylation and induced migratory rather than proliferative behavior. Cells with the unmethylated MGMT promoter showed more aggressive behavior after treatment, while HGA cells reacted heterogenously. Our study provides further evidence to consider the potential adverse effects of TMZ chemotherapy and a rationale for investigating potential relationships between TMZ treatment and change in the MGMT promoter methylation during relapse.

Comprehensive profiling of myxopapillary ependymomas identifies a distinct molecular subtype with relapsing disease.

Myxopapillary ependymoma (MPE) is a heterogeneous disease regarding histopathology and outcome. The underlying molecular biology is poorly understood, and markers that reliably predict the patients' clinical course are unknown. We assembled a cohort of 185 tumors classified as MPE based on DNA methylation. Methylation patterns, copy number profiles, and MGMT promoter methylation were analyzed for all tumors, 106 tumors were evaluated histomorphologically, and RNA sequencing was performed for 37 cases. Based on methylation profiling, we defined two subtypes MPE-A and MPE-B, and explored associations with epidemiological, clinical, pathological, and molecular characteristics of these tumors. MPE-A occurred at a median age of 27 years and were enriched with tumors demonstrating papillary morphology and MGMT promoter hypermethylation. Half of these tumors could not be totally resected, and 85% relapsed within 10 years. Copy number alterations were more common in MPE-A. RNA sequencing revealed an enrichment for extracellular matrix and immune system-related signatures in MPE-A. MPE-B occurred at a median age of 45 years and included many tumors with a histological diagnosis of WHO grade II and tanycytic morphology. Patients within this subtype had a significantly better outcome with a relapse rate of 33% in 10 years (P = 3.4e-06). We unraveled the morphological and clinical heterogeneity of MPE by identifying two molecularly distinct subtypes. These subtypes significantly differed in progression-free survival and will likely need different protocols for surveillance and treatment.

Clinical significance and biomarker potential of MGMT protein measurement in colorectal cancer.

136 Background: O6-methylguanine DNA methyltransferase (MGMT) is the principal repair mechanism of DNA damage created by alkylating agents. Approximately 40% of colorectal cancers (CRC) exhibit MGMT promoter hypermethylation, expected to result in gene silencing. The role of MGMT as a biomarker is poorly understood, and quantitative measurement of MGMT protein has not been performed in CRC. We hypothesized that altered tumor MGMT would affect DNA damage response, modulate adaptive anti-tumor immune responses, and influence survival in CRC. Methods: We used multiplexed quantitative immunofluorescence (QIF) to study 4 clinically annotated retrospective patient cohorts treated from 2000 to 2017 at Yale, consisting of 400 formalin-fixed paraffin-embedded CRC cases represented in tissue microarray format. These included paired tumors and adjacent non-tumor colonic mucosa (n = 112 pairs in Cohort 1), paired primary CRC and lymph node metastases (n = 31 pairs in Cohort 2), and 2 cohorts with primary stage I-IV tumors (n = 250 in Cohort 3; n = 150 in Cohort 4). We established a QIF panel for simultaneous, localized measurement of DAPI (all cells), cytokeratin (CK; tumor epithelial cells), MGMT, γH2AX (DNA damage response), and CD8. The measurement of fluorescent signals in CK-positive tumor cells or CK-negative stromal cells was achieved using co-localization strategies and the AQUA QIF platform. Results: We identified lower MGMT protein levels in CRC cells than in non-tumor colonic epithelium (p &lt; 0.0001), and in lymph node metastases compared to paired primary CRC tumors (p = 0.0411). Using the visual detection threshold, tumor selective MGMT protein downregulation was identified in 20% of cases in Cohorts 3 and 4. Microsatellite instability-high (MSI-H) cases showed decreased tumoral MGMT protein compared to microsatellite stable (MSS) cases (p = 0.002). In Cohorts 3 and 4, low tumoral MGMT was consistently associated with increased CD8+ tumor infiltrating lymphocytes (p = 0.0472; p = 0.0002). The association between MGMT and γH2AX was inconsistent. The association between MGMT and CD8 was significant only in MSS cases (p = 0.0001) but not in MSI-H cases (p = 0.0979), supporting that the effect is not driven by MSI-H status. Tumor-selective MGMT downregulation evidenced by a low tumor-to-stroma ratio was associated with improved progression-free survival and overall survival in both Cohorts 3 and 4, but statistical significance was only seen in Cohort 3. Conclusions: MGMT protein downregulation occurs in 20% of CRCs and is associated with increased adaptive anti-tumor immune responses, mismatch repair (MMR) deficiency and better prognosis. MGMT deficiency may alter DNA repair in tumor cells and mediate the accumulation of antigenic mutations or neopeptides, independent from MMR status. Our results support a biomarker role of MGMT protein and suggest a role for immunotherapy combinations in MGMT deficient tumors.