Methylated Tumors Research Articles

Immunophenotypic Profile of Adult Glioblastoma IDH-Wildtype Microenvironment: A Cohort Study.

Background: Glioblastoma IDH-wildtype (GBM IDH-wt) is the most aggressive brain tumor in adults and is characterized by an immunosuppressive microenvironment. Different factors shaping its tumor microenvironment (TME) regulate tumor progression and treatment response. The aim of this study was to characterize the main immunosuppressive elements of the GBM IDH-wt TME. Methods: Immunohistochemistry for CD3, CD4, CD8, CD163, programmed death ligand 1 (PD-L1) and programmed death 1 (PD1) was performed on surgical tumor specimens from patients diagnosed with GBM IDH-wt, according to the CNS WHO 2021 criteria. The impact of categorical variables on time-dependent outcomes such as overall survival (OS) and progression-free survival (PFS) has been estimated through the Kaplan-Meier method. Results: We included 30 patients (19 males and 11 females), median age of 59.8 years (range 40.2-69.1 years). All patients underwent surgery followed by temozolomide concurrent with and adjuvant to radiotherapy. MGMT was methylated in 14 patients (47%) and unmethylated in 16 patients (53%). The overall absolute percentages of CD4+ lymphocytes, both intratumoral and perivascular, were significantly more represented than CD8+ lymphocytes in the TME (p = 0.02). A low density of CD4+ lymphocytes (≤10%) was found to be a favorable prognostic factor for GBM outcome (p = 0.02). Patients with MGMT methylated and unmethylated tumors exhibited a distinct TME composition, with a significant higher number of perivascular CD8+ lymphocytes (p = 0.002), intratumoral CD8+ lymphocytes (p = 0.0024) and perivascular CD4+ lymphocytes (p = 0.014) in MGMT unmethylated tumors. PD-L1 expression in tumor cell surface was observed in four tumors (13.3%), and PD1 expression in infiltrating T lymphocytes was observed in nine (30%) tumors, with predominantly perivascular distribution. Conclusions: MGMT methylated and unmethylated tumors exhibit different immune profiles, likely reflecting the different biology of these tumors. The expression of PD-L1 in GBM IDH-wt patients is confined to a small subpopulation. While we found a significant association between low CD4+ lymphocyte density (≤10%) and survival, given the small numbers of our cohort, the prognostic value of CD4+ lymphocyte density will need to be validated in large-scale studies.

INNV-27. TEMOZOLOMIDE FOR PATIENTS WITH AGGRESSIVE PITUITARY TUMORS: A CASE SERIES

Abstract BACKGROUND Pituitary adenomas are often effectively managed with medical therapy, surgery, and radiation therapy. However, up to 15% of pituitary neoplasms exhibit rapid growth, invasive behavior, or, rarely, metastasize despite optimal standard treatments. Temozolomide is associated with a survival benefit for patients with these aggressive pituitary tumors. METHODS We present a case series of 8 patients with an aggressive pituitary tumor (APT) or pituitary carcinoma (PC) treated with temozolomide. RESULTS Eight patients (3 female, median age at diagnosis 44) with APT (n=6) or PC (n=2) were treated. A variety of pituitary tumor types were represented (1 prolactinoma, 5 corticotroph adenomas, 1 gonadotroph adenoma, 1 thyrotropinoma). Prior to temozolomide initiation, patients underwent a median 5 treatments including medical management (n=4), surgery (n=7), radiation therapy (n=7). Temozolomide was started at median 3.5 recurrence. Median treatment duration was 6 cycles (n=6, 150-200 mg/m2 days 1-5, 28-day cycles; n=1, 75 mg/m2, days 1-21, 28-day cycles). Seven patients experienced progression after temozolomide treatment, median progression-free survival (PFS) was 4 months. One patient remains on treatment, progression-free at 5 months. MGMT methylation status was assessed for 3 tumors. Among 2 patients with MGMT promoter methylated tumors, PFS was 4 months for one patient, the other remains on treatment. For one patient with MGMT promoter unmethylated corticotroph adenoma, PFS was 9 months. No ≥ grade 4 toxicities were noted. Most common toxicities were nausea (n=5), thrombocytopenia (n=3), and fatigue (n=2). CONCLUSION In this group of heavily pretreated patients, temozolomide monotherapy was well-tolerated and associated with median PFS of 4 months. Interestingly, PC patients had longer PFS at 9 months and 24 months. Our findings support use of single-agent temozolomide chemotherapy for patients with APT/PC, but further prospective analysis is needed to identify patients more likely to benefit from treatment and optimal timing of chemotherapy initiation.

BRCA promoter methylation in triple-negative breast cancer is preserved in xenograft models and represents a potential therapeutic marker for PARP inhibitors.

Most triple-negative breast cancers (TNBC) are sporadic in nature and often associated with dysfunction of the BRCA1 or BRCA2 genes. Since somatic BRCA mutations are rare in breast cancer (BC), this dysfunction frequently is the result of BRCA promoter methylation. Despite the phenotypic similarities of these tumors to those with germline or somatic BRCA mutation, the evidence of response to PARP inhibitors is unclear. We analyzed the prevalence of BRCA promoter methylation in 29 BC metastases through the well-established Illumina Infinium EPIC Human Methylation Bead Chip. In cases with BRCA methylation, the xenograft of the same tumor was tested. Additionally, we compared BC xenografts with an identified BRCA methylation to their matched primary tumors and subsequently investigated the efficacy of PARP inhibitors on tumor organoids from a BRCA2 promoter-methylated BC. BRCA2 promotor hypermethylation was identified in one pleural metastasis of a young patient as well as in the xenograft tissue. We also identified five more xenograft models with BRCA2 promotor hypermethylation. Analysis of one matched primary tumor confirmed the same BRCA2 methylation. PARP inhibitor treatment of tumor organoids derived from the BRCA2 methylated xenograft tumor tissue of the young patient showed a significant decline in cell viability, similar to organoids with somatic BRCA1 mutation, while having no effect on organoids with BRCA1 wildtype. BRCA promotor hypermethylation seems to be a rare event in metastatic BC but is preserved in subsequent xenograft models and might represent an attractive therapeutic marker for PARP inhibitors.

Circulating methylated HOXA9 tumor DNA as a biomarker for mortality in recurrent breast cancer: An exploratory study.

Methylated homeobox A9 (meth-HOXA9) circulating tumor DNA may be a relevant biomarker in breast cancer, although its clinical significance remains unknown. The present exploratory study aimed to investigate the association between meth-HOXA9 and mortality in patients with recurrent breast cancer. The cohort study enrolled 51 patients with breast cancer recurrence from the Department of Oncology, Vejle Hospital, University Hospital of Southern Denmark (Vejle, Denmark). Tissue samples from primary surgery and biopsies, and plasma samples obtained at the time of recurrence were analyzed for meth-HOXA9 using a methylation-specific droplet digital polymerase chain reaction. Using Cox regression, hazard ratios (HRs) for mortality with 95% confidence intervals (CIs) comparing patients with detectable and undetectable meth-HOXA9 in both tumor tissue and plasma were estimated. Among the 50 patients with data on tumor tissue meth-HOXA9, there was no association between meth-HOXA9 in the primary tumor and mortality (HR 1.09, 95% CI 0.47-2.52). A total of 34 patients had data on plasma meth-HOXA9 at the time of recurrence. Detectable plasma meth-HOXA9 was associated with higher mortality (HR 3.95, 95% CI 1.50-10.37). Among the 20 patients with data on both plasma and metastatic tissue meth-HOXA9, meth-HOXA9 was detectable in 90% of metastases and 65% of plasma samples. In conclusion, detectable plasma meth-HOXA9 was significantly associated with higher mortality in recurrent breast cancer; therefore, plasma meth-HOXA9 may prove useful as a prognostic marker in patients with breast cancer.

Promoter Hypermethylation of the BRCA1 Gene as a Novel Biomarker for Prostate Cancer.

Prostate cancer (PCa) is recognized as one of the most common malignancies that greatly affects the male population globally. Breast cancer gene 1 (BRCA1) is an important tumor suppressor gene that plays a central role in the maintenance of genomic integrity by promoting the repair of double-strand breaks of DNA. Here, we present a pilot study to examine the promoter methylation and gene expression of the BRCA1 gene in patients with PCa in Erbil governorate, Iraq. The collection of samples took place in Erbil City, Iraq, specifically at Rizgary Hospital, PAR Hospital, and Al-Mufti's private laboratory. A total of 40 tissue samples were collected from age-matched individuals, comprising 30 pathologically confirmed PCa cases and 10 normal prostatic tissue taken from individuals who, during diagnosis, were found to be negative for PCa. Data on demographic and clinical information, such as pathological stage, age, and prostate-specific antigen (PSA) level, were gathered from the medical records. The impact of the promoter methylation was forecasted using the DNA bisulfite conversion technique and methyl-specific PCR (MSP) with specific primers for the BRCA1 promoter region. The assessment of BRCA1 expression was conducted using quantitative real-time PCR (qPCR). Among the 30 patients examined, 76.6% (23 cases) were found to have BRCA1 promoter methylation, and none of the normal tissues appeared to have DNA methylation. BRCA1 promoter methylation was positively associated with the advanced stage of disease (p=0.01) and Gleason score (p=0.007). The analysis revealed a significant downregulation of the BRCA1 gene expression in methylated tumor samples as compared to non-methylated tumors and normal tissues, suggesting the role of epigenetic silencing. To the best of our knowledge, this is the first study investigating methylation status and level of BRCA1 mRNA transcripts among PCa patients in Iraq. Our findings suggest that promoter hypermethylation of the BRCA1 gene could serve as a viable biomarker for PCa, marking a significant discovery.

Detection of dual-methylated BRCA1/BRCA2 cell-free DNA for ovarian cancer on an aptamer-based integrated microfluidic system

A new screening strategy has been developed to select nucleic acid aptamer probes with an “in vivo-like” systematic evolution of ligands by exponential enrichment (SELEX) process featuring positive, negative selection and in vivo-like selection steps. One methylation-specific aptamer with high affinity and specificity was selected and it could capture methylated cell-free DNA (cfDNA) from unfiltered blood of ovarian cancer (OvCa) patients. This aptamer was conjugated onto magnetic beads and integrated into a new microfluidic chip for rapid and automatic detection and quantification of two prevalent methylated tumor suppressor genes, breast cancer type 1 and 2 susceptibility protein (BRCA 1/2), in the molecular profile of OvCa. The assay was conducted under physiological conditions that mimic the microenvironment of cancer patients at 25–37 °C, with a low pH value (5.5) and/or high blood ion levels, without requiring additional sample preparation steps. The results of this study could be extrapolated to the direct detection of methylated cfDNA at levels as low as 1 pM in blood or plasma. Not only was DNA methylation observed in OvCa patients, but it was also detected in many other types of cancer; it is therefore considered an important factor in carcinogenesis. Hence, it is inferred that this screened aptamer should not only be used for OvCa diagnosis but also for multiple cancer epigenetic detection and drug development research in the future.

Genetic Prognostic Factors in Adult Diffuse Gliomas: A 10-Year Experience at a Single Institution.

Gliomas are primary brain lesions involving cerebral structures without well-defined boundaries and constitute the most prevalent central nervous system (CNS) neoplasms. Among gliomas, glioblastoma (GB) is a glioma of the highest grade and is associated with a grim prognosis. We examined how clinical variables and molecular profiles may have affected overall survival (OS) over the past ten years. A retrospective study was conducted at Sina Hospital in Tehran, Iran and examined patients with confirmed glioma diagnoses between 2012 and 2020. We evaluated the correlation between OS in GB patients and sociodemographic as well as clinical factors and molecular profiling based on IDH1, O-6-Methylguanine-DNA Methyltransferase (MGMT), TERTp, and epidermal growth factor receptor (EGFR) amplification (EGFR-amp) status. Kaplan-Meier and multivariate Cox regression models were used to assess patient survival. A total of 178 patients were enrolled in the study. The median OS was 20 months, with a 2-year survival rate of 61.0%. Among the 127 patients with available IDH measurements, 100 (78.7%) exhibited mutated IDH1 (IDH1-mut) tumors. Of the 127 patients with assessed MGMT promoter methylation (MGMTp-met), 89 (70.1%) had MGMT methylated tumors. Mutant TERTp (TERTp-mut) was detected in 20 out of 127 cases (15.7%), while wildtype TERTp (wildtype TERTp-wt) was observed in 107 cases (84.3%). Analyses using multivariable models revealed that age at histological grade (p < 0.0001), adjuvant radiotherapy (p < 0.018), IDH1 status (p < 0.043), and TERT-p status (p < 0.014) were independently associated with OS. Our study demonstrates that patients with higher tumor histological grades who had received adjuvant radiotherapy exhibited IDH1-mut or presented with TERTp-wt experienced improved OS. Besides, an interesting finding showed an association between methylation of MGMTp and TERTp status with tumor location.

Molecular profiling of serial liquid biopsy specimens utilizing cell free DNA (cfDNA) and circulating tumor cells (CTCs) in TBCRC041: A phase II study of alisertib in endocrine resistant metastatic breast cancer (MBC).

1037 Background: TBCRC041, a randomized phase 2 trial of alisertib (A) ± fulvestrant (F) in women with HR+/HER2- MBC demonstrated promising clinical activity for alisertib (1). Interrogation of baseline (PreC1) and end of cycle 1 (EOC1) cfDNA and CTCs may identify biomarkers associated with response to A±F. Methods: Plasma cfDNA was sequenced using the Guardant INFINITY platform, which includes genomic and methylated tumor fraction (mTF) analysis. Pathogenic variants were determined using publicly available databases and Mayo Clinic annotation pipelines. CTCs were identified as nucleated, EpCAM+/ cytokeratin+/ CD45- cells and assessed for ER/HER2 staining (RareCyte, Seattle). PFS was defined as the time from randomization to progression or death. EOC1-PFS differs from PFS as time starts at C2D1. Stratified log-rank tests and conditional Cox modeling were used to assess whether PFS or EOC1-PFS differed by mutations in ESR1 or PIK3CA, pre-CTC ≥ 5, or mTF&gt;1%. Results: Of the 91 eligible pts, 84 progressed, and 73 were deceased. In the 86 pts with PreC1-cfDNA results, variants were identified in ESR1 (42; 48.8%), PIK3CA (36; 41.9%), AKT1 (7; 8.1%), PTEN(7; 8.1%), BRCA1 (4; 4.7%), and BRCA2 (7; 8.1%). Pts with a PIK3CAmut had significantly decreased PFS (HR 1.8; 95%CI: 1.1 -2.9, p=0.014) but not by ESR1mut status (p=0.583). PreC1 and EOC1-mTF were determined for 78 and 76 pts, respectively. PreC1-mTF was 0-1% in 21; 1.1-10.0% in 27; and 10.1-100% in 30. mTF levels remained between 0-1% in 17 pts fell to 0-1% in 11 pts, and was 0-1% in 5 pts without a preC1-mTF determination. PFS did not differ with respect to PreC1-mTF (p=0.210), but EOC1-PFS was worse in pts with mTF &gt;1% (HR 3.0; 95%CI: 1.6 -5.2, p&lt;0.001). PreC1 and EOC1-CTC were determined for 78 and 76 pts. 36 (46.2%) pts had a PreC1 CTC count ≥ 5. EOC1-CTC levels remained at (n=17) or increased to (n=1) ≥ 5 and was ≥ 5 in 2 pts without a preC1-CTC determination. EOC1-PFS was not significantly different for EOC1-CTC ≥ 5 (p=0.065), but was significantly decreased in pts with PreC1-CTC≥5 (HR=1.8; 95%CI: 1.1-3.0; p=0.018). Broader cfDNA NGS panel and CTC-derived ER/HER2 staining results will be presented at the meeting. Conclusions: Among patients receiving A±F, PFS varied based on the PIK3CAmutation status. cfDNA and CTC assessment PreC1 and EOC1 provided complementary information. Further validation in larger datasets is needed. 1. JAMA Oncol 2023;9:815. Clinical trial information: NCT02860000 .

Recent Advancements in Research on DNA Methylation and Testicular Germ Cell Tumors: Unveiling the Intricate Relationship.

Testicular germ cell tumors (TGCTs) are the most common type of testicular cancer, with a particularly high incidence in the 15-45-year age category. Although highly treatable, resistance to therapy sometimes occurs, with devastating consequences for the patients. Additionally, the young age at diagnosis and the treatment itself pose a great threat to patients' fertility. Despite extensive research concerning genetic and environmental risk factors, little is known about TGCT etiology. However, epigenetics has recently come into the spotlight as a major factor in TGCT initiation, progression, and even resistance to treatment. As such, recent studies have been focusing on epigenetic mechanisms, which have revealed their potential in the development of novel, non-invasive biomarkers. As the most studied epigenetic mechanism, DNA methylation was the first revelation in this particular field, and it continues to be a main target of investigations as research into its association with TGCT has contributed to a better understanding of this type of cancer and constantly reveals novel aspects that can be exploited through clinical applications. In addition to biomarker development, DNA methylation holds potential for developing novel treatments based on DNA methyltransferase inhibitors (DNMTis) and may even be of interest for fertility management in cancer survivors. This manuscript is structured as a literature review, which comprehensively explores the pivotal role of DNA methylation in the pathogenesis, progression, and treatment resistance of TGCTs.

Methylated Cell-Free Tumor DNA in Sputum as a Tool for Diagnosing Lung Cancer—A Systematic Review and Meta-Analysis

Lung cancer is the leading cause of cancer-related mortality worldwide. Early diagnosis is pivotal for the prognosis. There is a notable overlap between lung cancer and chronic bronchitis, and the potential use of methylated tumor DNA in sputum as a biomarker for lung cancer detection is appealing. This systematic review and meta-analysis followed the PRISMA 2020 statement. A comprehensive search was conducted in Embase, Medline, Web of Science, and the Cochrane Library, using these search strings: Lung cancer, sputum, and methylated tumor DNA. A total of 15 studies met the eligibility criteria. Studies predominantly utilized a case–control design, with sensitivity ranging from 10 to 93% and specificity from 8 to 100%. A meta-analysis of all genes across studies resulted in a summary sensitivity of 54.3% (95% CI 49.4–59.2%) and specificity of 79.7% (95% CI 75.0–83.7%). Notably, two less explored genes (TAC1, SOX17) demonstrated sensitivity levels surpassing 85%. The study’s findings highlight substantial variations in the sensitivity and specificity of methylated tumor DNA in sputum for lung cancer detection. Challenges in reproducibility could stem from differences in tumor site, sample acquisition, extraction methods, and methylation measurement techniques. This meta-analysis provides a foundation for prioritizing high-performing genes, calling for a standardization and refinement of methodologies before potential application in clinical trials.

Clinical validation of Northstar Response, a novel methylated ctDNA therapy response monitoring assay in patients with advanced GI cancer undergoing active treatment.

756 Background: ctDNA is a non-invasive way to holistically assess the tumor. Several assays developed for therapy response monitoring rely on variant allele frequencies to detect scarce somatic variants in the blood. In contrast, methylated ctDNA measurement has shown promise as a treatment monitoring biomarker without requiring a tumor biopsy, yet accuracy is limited in ability to precisely quantify the amount of methylation present in the ctDNA. The CLIA-approved Northstar Response assay (BillionToOne, Inc.) quantifies on average 90 methylated loci, a 10-fold increase in signal over the average tissue-informed assays. Methods: This prospective, observational study assesses the clinical validity of Northstar Response, a quantitative methylated ctDNA therapeutic response monitoring assay that does not require prior tumor tissue. A cohort of 100 advanced cancer patients with a variety of GI malignancies submit serial ctDNA measurements in a blinded fashion. Eligibility includes advanced GI cancer patients starting a new line of systemic therapy (IO, targeted, chemo or combination) who are not pregnant or s/p organ or bone marrow transplantation. Blood samples are collected at baseline, at the time of imaging, and at least two intermediate time points (~30 days and 60 days post tx initiation). The primary endpoint is to measure the concordance and temporal relationship between disease progression measured by ctDNA compared to RECISTv1.1, clinical confirmation, or death. A Cox proportional hazards model will be used to quantify the association between the change in tumor methylation score (TMS; total methylated tumor molecules per 1000 assayed genomic equivalents) and clinical progression. The cohort will be split equally between a training and validation set. Results: Enrollment began in June 2023 and continues. To date, 28 patients with a variety of advanced GI cancers (table) have been enrolled with 37 samples processed. All samples have passed established QC metrics with average turn-around-time from receipt of sample to final report of 9.1 days (95% were &lt;15 days). TMS ranged from &lt;120 to &gt;160,000 across individual samples, indicating that the assay can detect a broad range of tumor load in blood. We detected a median of 69 methylated loci per sample (range 4-451). Out of 21 pts, 17 had at least one sample with TMS measured above the noise floor of 120. Conclusions: The Northstar Response assay can consistently detect ctDNA across a variety of advanced GI cancers. Enrollment continues with formal comparison to clinical progression planned. Additional data on the complete training set will be available at the meeting.[Table: see text]

Abstract IA006: Multifaceted effects of DNA damage response inhibitors on radiation responses of glioblastoma and the normal brain

Abstract PARP inhibitors (PARPi) and ATM inhibitors (ATMi) enhance radiation sensitivity in multiple cancer models, both in vitro and in vivo. Our observation that the radiosensitizing properties of PARPi are most pronounced in rapidly proliferating cells is reflected in early phase clinical trial data showing exacerbation of acute radiation toxicity in rapidly proliferating tissues such as oropharyngeal and esophageal mucosa. Lack of radiosensitization in late responding, slowly proliferating normal tissues indicates that PARPi may be more effectively combined with radiation therapy (RT) in patients with brain tumors. ATMi are much more potent radiosensitizers than PARPi but less is known about their impact on normal tissue toxicity since they have only recently progressed to the clinic, although preclinical in vivo data are encouraging. We are evaluating the oral PARPi olaparib and the oral ATMi AZD1390 in combination with RT +/- temozolomide (TMZ) in the treatment of glioblastoma (GBM), the most prevalent and most aggressive primary brain tumor. Patients with GBM experience very poor outcomes in terms of both median survival (c.1 year) and neurocognitive decline, which is caused primarily by RT. Early phase testing of the olaparib-RT combination is underway in three populations of patients with newly diagnosed GBM. Patients aged &amp;gt;65 with MGMT unmethylated GBM are being recruited to a randomized, placebo-controlled phase II study (PARADIGM, ISRCTN52658296) after a phase I dose escalation study showed that olaparib (200 mg twice daily) was extremely well tolerated when combined with brain irradiation (40 Gray in 15#). Good performance status patients aged under 70 are being recruited to PARADIGM-2 (ISRCTN51253312) which comprises two parallel phase I dose escalation studies: patients with MGMT unmethylated tumors are receiving daily olaparib with RT (60 Gy in 30#) without TMZ, while patients with MGMT methylated tumors are receiving intermittent olaparib with standard chemoradiation. AZD1390 is being evaluated in a first-in-human phase I dose escalation study (NCT03423628) in combination with re-irradiation (35 Gy in 10#) in patients with recurrent GBM, and with first-line radical radiotherapy (60 Gy in 30#) in patients with newly diagnosed, MGMT unmethylated GBM. In parallel, we are undertaking preclinical studies to investigate the impact of PARPi and ATMi on RT induced neurotoxicity. In vivo imaging studies support the emerging concept that RT induced neuroinflammation is important in the pathogenesis of neurotoxicity, and we have generated preliminary behavioral data indicating that both PARPi and ATMi can alleviate the neurotoxic effects of RT. Ongoing experiments are defining the roles of microglia, astrocytes and neurogenesis in this phenomenon. We will present these diverse datasets in the context of our hypothesis that combining PARPi or ATMi with RT has potential to improve outcomes for GBM patients by enhancing tumor control while simultaneously suppressing neuroinflammation and alleviating RT related neurocognitive decline. Citation Format: Anthony J. Chalmers. Multifaceted effects of DNA damage response inhibitors on radiation responses of glioblastoma and the normal brain [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr IA006.

Abstract A002: The utility of pre-clinical trials in glioblastoma patient-derived xenografts (PDXs) models for informing clinical trial development of therapeutic strategies

Abstract Large PDX collections provide broad representation of tumor genetics and are commonly used to evaluate the clinical potential of novel therapeutic strategies. In many solid malignancies, profound benefit in PDX preclinical studies has foreshadowed eventual FDA approval of clinically impactful therapeutics. With a lack of corresponding progress in glioblastoma (GBM), the value of PDX testing has been questioned. Mayo Clinic has developed 111 GBM PDXs from newly diagnosed (72 PDX) and recurrent (39 PDX) patients, and over the past 23 years, we have used this collection for extensive in vivo preclinical testing of 145 drugs in both orthotopic and heterotopic settings. As part of this work, the impact of standard of care therapy with temozolomide (TMZ) and bevacizumab was evaluated in orthotopic models using clinically relevant dosing regimens. For each animal study, the ratio of median survival for treated vs. control animals were used to define a ‘survival ratio’ as a normalized metric of benefit. TMZ efficacy was evaluated in 26 newly diagnosed GBM PDXs, with equal representation of MGMT methylated and unmethylated tumors. As expected, MGMT methylated PDXs were much more sensitive to TMZ as compared to unmethylated PDXs (median survival ratio 4.47 vs 1.48, respectively), and similar to clinical experience, 2 MGMT unmethylated PDXs were markedly sensitive to TMZ, while 1 MGMT methylated PDX was relatively resistant. In a more limited study, dose-dense vs. standard TMZ dosing was compared in 7 PDXs (4 MGMT methylated, 3 MGMT unmethylated). In a pooled analysis of all lines, both standard (HR=0.09; 95% CI: 0.06–0.14) and dose-dense TMZ therapy (HR=0.09; 95% CI: 0.06–0.14) were superior to vehicle controls (p&amp;lt;0.001). Similar to the clinical results in the Phase III randomized RTOG 0525 trial, survival with the dose-dense TMZ was no different than standard dosing (p=0.88, HR=0.98; 95% CI: 0.72-1.32). In an analysis of bevacizumab across 32 PDXs, mice were dosed once a week until moribund, and a minimum overall median survival benefit of 20% was used to define ‘responders’. Thirteen PDXs met this criterion, although the overall survival ratio among this subset was limited (range 1.21-1.65). Only 2 models had a 50% or greater survival benefit. Stratification by disease state (24 newly diagnosed vs 8 recurrent) showed equivalent performance (median survival benefit 1.13 for both). These results are consistent with the limited survival benefit observed with bevacizumab and the RTOG 0825 clinical trial. In summary, the results from these relatively inexpensive PDX studies demonstrate the potential of pre-clinical PDX trials to evaluate the extent of benefit and fraction of responsive PDXs to a novel therapeutic. If analyzed across an ‘adequate’ number of PDXs, these results potentially can be used to identify predictive biomarkers or to compare various treatment regimens prior to clinical testing. Citation Format: Danielle M Burgenske, Ann C Mladek, Katrina K Bakken, Zeng Hu, Brett L Carlson, Paul A Decker, Jeanette E Eckel-Passow, Jann N Sarkaria. The utility of pre-clinical trials in glioblastoma patient-derived xenografts (PDXs) models for informing clinical trial development of therapeutic strategies [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A002.

DNAR-10. HETEROGENEOUS MGMT PROMOTER METHYLATION IN A MULTIFOCAL GLIOBLASTOMA: A CASE REPORT

Abstract MGMT promoter methylation status is an important predictive biomarker for treatment response in glioblastoma. MGMT unmethylated glioblastoma has elevated levels of MGMT which ultimately leads to chemoresistance. Clinicians consider the MGMT methylation status when treatment recommendations are given, and clinical trials use this marker for the selection of a homogenous group of glioblastoma patients based on prognosis. Here, we present a case of multifocal glioblastoma with heterogeneous MGMT methylation status. A 56-year-old male presented with left leg weakness for 2 months and left arm weakness for 1 month. Imaging revealed a calcified, rim enhancing right temporoparietal cystic lesion and a heterogeneously enhancing right parietal mass. Perilesional edema surrounded and connected the two lesions. The patient underwent gross total resection of the parietal mass and pathology revealed a WHO grade 4 glioblastoma, IDH wildtype, unmethylated tumor. Postoperatively, the patient was neurologically intact. He completed a 6-week long course of concomitant chemoradiation without any significant side effects. Two weeks later the patient developed rapidly worsening headaches, nausea, vomiting, dizziness, and left side weakness. Imaging revealed significant enlargement of the previously unresected temporoparietal cystic lesion with marked increase in surrounding edema and a 2-3 mm right to left midline shift. Patient underwent craniotomy with cyst drainage and tumor resection of the right cystic temporoparietal lesion, demonstrating a WHO grade 4 glioblastoma, IDH wildtype, methylated tumor. Post-operatively the patient was again neurologically intact and started adjuvant temozolomide. MGMT methylation status of glioblastoma can alter recommendations for chemotherapy with temozolomide. MGMT methylation status might vary in distinct parts of the tumor as molecular heterogeneity is a hallmark of glioblastoma. This heterogeneity should be considered by clinicians when treatments are recommended and by clinical trials for glioblastoma patients, especially when multifocal lesions have a distinct radiographic appearance.

CNSC-20. MGMT PROMOTER METHYLATED HIGH GRADE GLIOMA WITH DISTANT RECURRENCE AND STABLE ORIGINAL TUMOR SITE: CASE SERIES

Abstract Recurrence pattern in high grade glioma is classified as local and distant recurrence based on distance from original resection cavity and radiation field. We report the first series of three cases of O6-methylguanine-DNA methyltransferase (MGMT) promoter methylated high grade glioma with distant recurrence without local recurrence throughout the disease course. Patient 1: 65 year-old female with WHO grade 4 glioblastoma, isocitrate dehydrogenase enzyme (IDH) wild type (WT), MGMT methylated. She underwent gross total resection followed by Stupp protocol. Postoperative ischemic volume was &amp;lt; 1 ml. Distant recurrence occurred 4 months after completion of Stupp protocol. Patient 2: 55 year-old male with WHO grade 4 glioblastoma, IDH wild type, MGMT methylated. He underwent gross total resection followed by Stupp protocol. Postoperative ischemic volume was 18 ml. Distant recurrence occurred 5 months after completion of Stupp protocol. Patient 3: 31 year-old male with WHO grade 4 astrocytoma, IDH mutant, MGMT promoter methylated. He underwent gross total resection followed by Stupp protocol. Postoperative ischemic volume was 2 ml. Distant recurrence occurred 10 months after completion of Stupp protocol. Distant recurrence is more common in MGMT promoter methylated tumors. All 3 patients in our case series had poor outcomes after distant recurrence without local recurrence at the original tumor site. This observation indicates good local control in MGMT promoter methylated high grade glioma with current standard treatment. For one patient, next generation sequencing (NGS) was available for both original and recurrent tumor and did not reveal any major differences in the genetics of original tumor and distant recurrent tumor other than temozolomide induced high tumor mutational burden in the distant recurrent tumor. Understanding risk factors for distant recurrence in MGMT promoter methylated high grade gliomas and investigating correlations between recurrences will help plan therapeutic strategies to prevent distant recurrence and improve outcomes.

A Combined Phase 0/2 "Trigger" Trial of Niraparib in Combination with Radiation in Patients with Newly-Diagnosed Glioblastoma

Poly ADP-ribose (PAR) polymerase (PARP) mediates DNA damage response. Niraparib is an investigational PARP1/2-selective inhibitor. We conducted a combined phase 0/2 study to evaluate niraparib pharmacokinetics (PK) and pharmacodynamics (PD) in patients with newly-diagnosed glioblastoma (GBM), graduating patients to a phase 2 study evaluating a therapeutic regimen of niraparib with concurrent conventionally-fractionated radiotherapy (RT) in O6-methylguanine methyltransferase (MGMT) unmethylated tumors exceeding a prespecified PK threshold in non-enhancing tumor. Patients with presumed newly-diagnosed GBM were enrolled in a phase 0 study receiving 4 days of niraparib (300 or 200 mg QD) prior to planned resection 3-5 or 8-12 hours following the last dose. Tumor tissue (enhancing and non-enhancing regions), cerebrospinal fluid (CSF), and plasma were collected. Total and unbound niraparib concentrations were measured using validated LC-MS/MS methods. PARP inhibition was assessed by quantification of PAR induction after 10 Gy ex vivo irradiation in surgical tissue compared to non-irradiated control tissue. A PK 'trigger' determined eligibility for the therapeutic phase 2 expansion portion of the study. This was defined as unbound [niraparib] > 5-fold biochemical IC50 (i.e., 19 nM) in non-enhancing tumor. Patients with MGMT unmethylated tumors exceeding this PK threshold were eligible for expansion phase dosing of niraparib with concurrent RT followed by a maintenance phase of niraparib. Patients with MGMT methylated tumors were not eligible for the expansion phase and proceeded with temozolomide (TMZ) plus RT followed by maintenance TMZ. RT dose was 60 Gy in 30 fractions using volumetric-modulated arc therapy (VMAT). All 29 patients enrolled in the phase 0 portion of the study met the PK threshold. In non-enhancing regions, the mean unbound concentration of niraparib was 258.2 nM. The suppression of PAR levels after ex vivo RT was observed in 79% of the patients (17/22). Sixteen patients had unmethylated tumors, and of those, 11 patients enrolled in phase 2. Five of the 6 initial patients enrolled in phase 2 experienced thrombocytopenia related to niraparib, and 3/5 cases were deemed serious and life-threatening. Consequently, starting dose in both phases was lowered to 200 mg, and no serious AEs were observed thereafter. At a median follow-up of 8.1 months [range: 6.0-12.9 months], 6-month PFS was 64% with 4 patients remaining on treatment and 5 patients ongoing survival follow-up. Niraparib achieves pharmacologically-relevant concentrations in non-enhancing, newly-diagnosed GBM tissue in excess of any other studied PARP inhibitor. When delivered with concurrent RT, niraparib was well-tolerated, with low rates of grade 3+ toxicity. Initial clinical efficacy data are encouraging.

OS09.3.A FINAL RESULTS OF THE MGMT UNMETHYLATED ARM OF PARADIGM-2: A PHASE I STUDY OF OLAPARIB AND RADIOTHERAPY IN PATIENTS WITH NEWLY DIAGNOSED GLIOBLASTOMA (GBM)

Abstract BACKGROUND The poly(ADP-ribose) polymerase (PARP) inhibitor olaparib has radio and chemosensitizing effects in preclinical models and penetrates GBM in patients at radiosensitizing concentrations. PARP inhibitors sensitize proliferating cells to temozolomide (TMZ) but exacerbate haematological toxicity. Patients with MGMT unmethylated tumours do not benefit from TMZ so it can be withheld, enabling continuous olaparib dosing with radiotherapy (RT). Patients with MGMT methylated tumours benefit from TMZ so olaparib must be combined with chemoradiation (CRT) in an intermittent, low-dose manner. PARADIGM-2 is a clinical trial comprising two parallel phase I studies of olaparib/RT or olaparib/RT/TMZ in newly diagnosed GBM patients stratified by MGMT status. MATERIAL AND METHODS GBM patients aged &amp;lt;70 with WHO performance status 0-1 were eligible. Tumour tissue underwent central pyrosequencing testing for MGMT promoter methylation. MGMT unmethylated patients received daily oral olaparib throughout RT (60 Gray in 30#) and for four weeks afterwards. MGMT methylated patients received intermittent olaparib during CRT (60 Gray plus TMZ 75mg/m2 daily) and for four weeks after. In both arms, olaparib dose was escalated in a 3 + 3 cohort design. ISRCTN 51253312. RESULTS By January 2023, 24 MGMT methylated patients and 43 MGMT unmethylated patients had been recruited. Dose escalation in MGMT unmethylated patients was completed with no dose-limiting toxicities and the recommended phase II dose (RP2D) of olaparib was established at 300 mg twice daily (the full single agent dose). Maximum tolerated dose was not reached. The RP2D cohort was expanded to 30 patients of whom one was excluded because the diagnosis was revised to anaplastic oligodendroglioma. Median overall survival (OS) of the 42 eligible patients was 14.1 months (80% confidence intervals (CI) 12.2 - 15.9), with 12- and 24-month OS estimates of 64.3% (54.0-72.9) and 16.7% (10.1-24.7) respectively. In the dose expansion cohort, 6 of 29 patients are alive with median follow-up time of 22.9 months. Median OS is 14.2 months (12.2-16.9); 12- and 24-month OS estimates are 69.0% (56.5-78.5) and 24.1% (14.8-34.8) respectively. Updated survival data will be presented at the conference. Dose escalation in MGMT methylated patients continues with the current cohort receiving olaparib 150 mg daily 4 days per week. CONCLUSION Pre-treatment stratification by MGMT status in a phase I study in GBM is feasible and enables optimisation of olaparib dosing and scheduling with RT and TMZ. In MGMT unmethylated patients, olaparib can be combined with RT at the full single agent dose (300 mg twice daily) if TMZ is withheld. Of 29 patients receiving this dose, a quarter have survived more than 2 years. In MGMT methylated patients, olaparib can be safely combined with radical CRT when given at lower doses on an intermittent basis (currently 150 mg four days per week).

Olaparib in HR-deficient (HRD), metastatic triple-negative breast cancer (TNBC) and relapsed ovarian cancer (ROC) without germline mutations in BRCA1 or BRCA2: Phase 2 EMBRACE trial.

e15079 Background: Homologous recombination deficiency (HRD) caused by germline BRCA1/2 mutations (gBRCAm) sensitize high grade serous ovarian cancers (HGSOC) and TNBC to PARP inhibitors (PARPi) such as olaparib. Loss of function of BRCA1 or RAD51C, due to promoter methylation or mutation of non-BRCA HR genes may also confer sensitivity to PARPi. This investigator-initiated, proof-of-concept trial evaluated olaparib in platinum(Pt)-sensitive, relapsed HGSOC and metastatic TNBC, with HRD due to mechanisms other than gBRCAm. EMBRACE is the first trial evaluating PARPi in loss of function of BRCA1/ RAD51C by promoter methylation. Methods: Single-arm, phase 2 trial of olaparib 300 mg orally twice daily in adults with metastatic TNBC or Pt-sensitive relapsed HGSOC and HRD due to mechanisms other than gBRCAm. 1 line of prior Pt-chemotherapy was allowed (HGSOC); additional lines of non-Pt therapy were allowed (TNBC). Tumor BRCA1/RAD51C promoter methylation was determined by methylation-sensitive high-resolution melting PCR and next generation sequencing (NGS, Pillar Biosciences BRCA1 &amp; RAD51C Methylation test, high methylation threshold 70%). Unmethylated cases underwent NGS HR gene mutation testing (Pillar Biosciences HRD test, paired tumor/normal). The primary outcome was objective tumor response rate (OTRR) at 6 months(m). Secondary objectives: progression-free survival (PFS), OTRR according to HR gene mutation type, safety. Tertiary: associations between biomarkers and clinical outcomes. Results: 22 participants (15 HGSOC, 7 TNBC) enrolled from 208 screened (Sep 2018 - Mar 2022). Methylation was detected in 8/15 HGSOC (all BRCA1) and 5/7 TNBC (4 BRCA1, 1 RAD51C). Both cohorts included mutations in gBRIP1 (1), gPALB2 (1), gRAD51C (3), sBRCA1 (2), with 1 COSMIC mutational signature 3, and 1 positive HRD score in HGSOC. OTRR at 6m was 40% in HGSOC (6/15: 5 partial, 1 complete), and 0% in TNBC (0/7). OTRR was 38% (3/8) in methylated tumors vs 43% (4/7) with other HRD. Stable disease at 6m was 47% (7) in HGSOC and 43% (3) in TNBC durations were 7m in HGSOC and 3m in TNBC. PFS was 53% at 6m and 25% at 12m in HGSOC, vs 17% at 6m and NE at 12m in TNBC. TNBC 2-3 prior lines of therapy. Conclusions: Olaparib demonstrated clinically relevant activity in HGSOC without gBRCAm. Objective responses were seen in HGSOC with methylated BRCA1. Olaparib had limited activity in heavily pre-treated TNBC. Further research is needed into the effects of non-Pt chemotherapy on methylation of BRCA1/RAD51C to improve selection of HGSOC and TNBC for PARPi. (ANZCTRN12617000855325) Acknowledgements: Funding from Cancer Australia and the NBCF(PS-15-048), provision of study drug plus an untied educational grant from AstraZeneca and its group of companies. EMBRACE was led by the NHMRC CTC, University of Sydney in collaboration with ANZGOG, BCT-ANZ, and GCCTI. Clinical trial information: ACTRN12617000855325 .

High-risk constitutional MLH1 methylation as a cause of early-onset colorectal and endometrial cancers displaying mismatch repair deficiency and MLH1 methylation.

10518 Background: Mismatch repair deficiency (MMRd) occurs in ~15% of colorectal (CRC) and ~30% of endometrial (EC) cancers, of which ~20% are caused by Lynch syndrome (LS). Universal screening of all incident CRC and EC for MMRd uses MLH1 methylation (in tumors with MLH1-loss by immunohistochemistry) to omit common sporadic cases from follow-up germline testing for LS. However, this overlooks unusual cases with constitutional MLH1 methylation (epimutation), a poorly-recognized high-risk mechanism for LS-type cancers that are MLH1 methylated. We aimed to determine the role and prevalence of constitutional MLH1 methylation among CRC and EC cases with tumor MLH1 methylation from clinical and population-based series and if age limits could be used to triage cases warranting additional germline methylation testing. Methods: We screened blood DNA for constitutional MLH1 methylation by pyrosequencing and methylation-specific qPCR in patients with MLH1-loss, MLH1 methylated CRC and EC ascertained from: i) cancer clinics, referred by oncologists and genetic counsellors; ii) Ohio population-based “Columbus” and “OCCPI” cohorts. Bisulfite sequencing confirmed constitutional MLH1 methylation. Results: In clinical cases, constitutional MLH1 methylation was identified in 3/7 EC and 3/8 CRC cases &lt; 60 years. In population-based cohorts, constitutional MLH1 methylation was identified in 4/95 (4.2%) and 4/281 (1.4%) CRC cases of all ages from Columbus and OCCPI, respectively. Highest rates of detection with the majority of cases detected were at age ≤55 years, with 3/4 (75%) in Columbus and 4/17 (23.5%) in OCCPI ≤55 years. For EC, constitutional MLH1 methylation was identified in 0/68 Columbus cases of all ages and 1/24 OCCPI cases &lt; 60 years (age-limited testing). She was one of six (~17%) cases &lt; 50 years in the combined cohorts. EC was the first/dual-first presenting “sentinel” cancer in three female patients with constitutional MLH1 methylation. Of the 14 cases with constitutional MLH1 methylation, nine had hemiallelic methylation (~50% alleles methylated, affecting a single parental allele) and five had mosaicism across various normal tissues. Somatic “second-hits” affecting the unmethylated allele were found in the tumors of mosaic cases, demonstrating causation. Conclusions: A correct diagnosis of constitutional MLH1 methylation at first cancer presentation is important as it will significantly alter clinical management of these high-risk patients. Although rare overall, high rates of detection were found ≤55 years for CRC and ≤50 years for EC cases with a MMRd, MLH1 methylated tumor, entailing minimal extra screening. Testing for constitutional MLH1 methylation is warranted in patients with young-onset CRC or EC, or syn/metachronous cancers at any age, with MMRd and MLH1 methylated tumor(s).

DISTRIBUTION AND IMAGING CHARACTERISTICS OF HIGH-GRADE GLIOMAS ACCORDING TO METHYLATION STATUS OF MGMT GENE PROMOTER

Background: Many molecular investigations have identified that the methylation status of the O-6-methylguanine-DNA methyl transferase (MGMT) gene promoter in patients with high grade glioma is associated with a improved prognosis and survival when treated with temozolamide (TMZ). Aims and Objective: The aim of the study was to find out the distribution of methylation status and imaging characteristics in patients with High Grade Gliomas. Method: The study included forty consecutive patients who presented with neurological symptoms and were subsequently diagnosed as having a primary brain tumour on conventional MRI scan. MR-perfusion imaging was performed to determine the grade of the tumor which was subsequently compared with histopathological grade of the tumor after brain biopsy. The biopsy or surgical specimens were further analysed for determining the MGMT promoter methylation status using Methylation-specific PCR (MSP). Results: The mean age of patients was 50.3 years in the methylated group and 49.1 years in the unmethylated group (p value = 0.671). Out of 40 patients, 18 had methylated MGMT promoters while 22 had unmethylated MGMT promoters. We found that ring-like enhancement was seen more frequently in the unmethylated tumours (72.7%) than in methylated ones (61.1%). The presence of T2 heterogeneous signal intensity was seen more in the methylated group (77.8%) than unmethylated group (68.2%). Severe necrosis (affecting &gt;50% of the tumour) was seen more in the methylated tumours (61.1%) than in the unmethylated tumours (40.09%). However, these differences were found to be statistically insignificant. Conclusion: The distribution and imaging characteristics may provide some information about methylation of high-grade gliomas but a multicentric large data maybe required to reach a consensus about its reliability when biopsy of the methylation status of the tumour is not available.