e14016 Background: Glioblastoma is a lethal brain cancer. Clinical patterns of disease suggest that androgens influence glioblastoma risk and prognosis, and androgen receptor (AR) transcript levels and protein expression are upregulated in glioblastoma compared to normal brain. Thus, potent brain-penetrant anti-androgen therapies may be an effective treatment strategy in glioblastoma. Methods: Clinical samples and patient-derived glioma xenografts (PDXs) were assessed for AR expression by immunohistochemistry. Glioblastoma cell lines and patient-derived models maintained under stem cell conditions were tested in vitro for their response to anti-androgen therapies (abiraterone, enzalutamide and seviteronel). Change in plasticity marker ZEB1 was measured using immunofluorescence. Stem cell function was assessed using a tumoursphere assay. In an intracranial AR positive glioblastoma PDX model (RN1) in an immunocompromised mouse, the most effective drug in vitro, seviteronel, was tested alone and in combination with temozolomide or radiation. Tumor growth was monitored with weekly bioluminescent imaging, with survival as the primary endpoint. Results: Cytoplasmic AR staining was present in ~55% of glioblastoma samples using immunohistochemistry; this biomarker has shown to be predictive in triple-negative breast cancer. AR positive model RN1 was inhibited by low concentrations of anti-androgen agents, with seviteronel having the lowest half maximal inhibitory concentration (IC 50 ) after 96 h (enzalutamide 52 µM, abiraterone 12 µM, seviteronel 7 µM). AR negative model WK1 was inhibited though with higher IC 50 values after 96 h (enzalutamide 63 µM, seviteronel 21 µM). Cell lines U87 and U251 were also inhibited by anti-androgen monotherapy. Immunofluorescence analysis showed downregulation of plasticity marker ZEB1 with anti-androgens (normalized mean fluorescent intensity: abiraterone 0.84, enzalutamide 0.91, seviteronel 0.74). Tumoursphere assays demonstrated that anti-androgens inhibit the tumor-forming ability of cells, with seviteronel and abiraterone showing more inhibition than enzalutamide. In mice intracranially implanted with RN1 cells, seviteronel improved overall survival compared to vehicle alone (36 vs 27.5 days, p=0.03). There was no observed benefit when seviteronel was added to radiotherapy (33 vs 39 days, p=0.19). Adding seviteronel to temozolomide limited tumor growth by IVIS but did not significantly improve overall survival compared to temozolomide alone (63.5 vs 56.5 days, p=0.37). Conclusions: Targeting AR with brain penetrant anti-androgen drugs may be a promising biomarker-directed therapeutic strategy for glioblastoma. To address the limitations of our study, further experiments to optimize translational treatment protocols, address the role of biological sex and examine the role of the immune microenvironment are underway.

(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.

Abstract BACKGROUND Glioblastoma is a lethal brain cancer. Androgen receptor (AR) transcript levels and protein expression are upregulated in glioblastoma compared to normal brain. AR signaling has a role in cancer stem cell function in various cancers. Stem cell activity and cancer cell plasticity represent a key resistance mechanism in glioblastoma. Thus, potent brain-penetrant anti-androgen therapies have the potential to improve treatment outcomes in glioblastoma. METHODS Clinical samples and patient derived xenografts were assessed for AR expression by immunohistochemistry. Cytoplasmic AR+ and AR- glioblastoma patient-derived xenografts maintained under stem cell conditions were tested in vitro for their response to anti-androgen therapies (abiraterone, enzalutamide and seviteronel). Stem cell function was assessed using a tumoursphere assay. Changes in plasticity markers, including ZEB1, were measured with immunofluorescence. RESULTS ~55% of people with glioblastoma have detectable cytoplasmic AR based on immunohistochemistry. AR-positive patient-derived glioma stem cell line RN1 was inhibited by low concentrations of anti-androgen agents, with seviteronel having the lowest half maximal inhibitory concentration (IC50) (at 96 hours: enzalutamide 52µM, abiraterone 12µM, seviteronel 7µM). AR-negative line WK1 was also inhibited though with higher IC50s (at 96 hours: enzalutamide 63µM, seviteronel 21µM). Conventional cell lines U87 and U251 were also inhibited by anti-androgen monotherapy. Tumoursphere assays demonstrated that anti-androgen agents inhibit the tumour-forming ability of cells, with seviteronel and abiraterone showing more inhibition than enzalutamide. Immunofluorescence analysis showed downregulation of plasticity marker ZEB1 with anti-androgen treatment (normalised mean fluorescent intensity: abiraterone 0.84, enzalutamide 0.91, seviteronel 0.74). CONCLUSIONS Glioblastoma is a devastating disease, lacking effective or targeted treatments. Targeting AR with repurposed anti-androgen drugs may be a promising therapeutic strategy, with the potential to abrogate treatment resistance. We are now evaluating these anti-androgen regimens in animal experiments, which will provide a more replicative microenvironment and confirm blood-brain barrier penetrance.

In early triple-negative breast cancer (eTNBC), platinum-based chemotherapy has been shown to improve pathological complete response, but recommendations to include platinum chemotherapy are not consistent in international guidelines. We performed a systematic review of published randomized control trials to assess to assess survival outcomes and quality of life for people with early TNBC. The results presented in this abstract have not yet been peer-reviewed by Cochrane. If the final version of the review meets the necessary standards, the review is expected to be published in the Cochrane Database of Systematic Reviews. Methods: Randomized controlled trials examining neoadjuvant or adjuvant platinum chemotherapy for eTNBC were included. We searched for published and unpublished data using standard Cochrane search strategies. The primary outcomes assessed were disease free survival (DFS) and overall survival (OS). Secondary outcomes included rate of pathological complete response (pCR), dose intensity and completion of regimens, grade III or IV toxicity related to chemotherapy, and quality of life. Prespecified subgroups included BRCA mutation status, HRD status, lymph node status, frequency of chemotherapy, type of platinum agent used, and the presence or absence of anthracycline chemotherapy. Results: From 3972 records, 19 published studies were eligible. Twenty six ongoing studies were identified. Risk of bias was judged low for most trials. There were 14 neoadjuvant chemotherapy trials, 4 adjuvant chemotherapy trials, and one trial of neoadjuvant and adjuvant therapy. Most trials used carboplatin (16 trials) followed by cisplatin (2), and lobaplatin (1). Eight trials had an anthracycline free intervention arm, 5 of which had a carboplatin-taxane intervention compared to an anthracycline-taxane control. All studies reporting DFS and OS used carboplatin. Twelve of 19 studies with a total of 3347 participants reported DFS data. Inclusion of platinum chemotherapy improved DFS in neoadjuvant and adjuvant setting (HR 0.63, 95% confidence interval (CI) 0.53-0.75; HR 0.69, 95% CI 0.54-0.88 respectively)). Eleven studies collected OS data, with a total of 3229 participants and 460 deaths reported. Inclusion of platinum chemotherapy in the regimen improved OS (neoadjuvant: HR 0.69, 95% CI 0.69, 0.55-0.86; adjuvant: 0.70, 95% CI 0.50 to 0.96). Median follow up for survival outcomes ranged from 36 – 97.6 months. Our analysis confirmed platinum chemotherapy increased pCR rates (RR 1.46 [1.33-1.61], p< 0.00001). Subgroup analysis revealed that survival outcome benefits were seen regardless of BRCA mutation status, HRD status, lymph node status, or whether the intervention arm contained anthracycline chemotherapy or not. Platinum chemotherapy was associated with reduced dose intensity, with participants more likely to require chemotherapy delays (RR 2.23 [1.70-2.94], 4 studies), dose reductions (RR 1.77 [1.56-2.02], 6 studies) and early cessation of treatment (RR 1.20 [1.04-1.38], 16 studies). Increased hematological toxicity occurred in the platinum group who were more likely to experience grade III/IV neutropenia (RR 1.55 [1.45-1.66]), anemia (RR 10.12 [6.61-15.50]) and thrombocytopenia (RR 7.59 [5.10-11.29]). There was no increase in febrile neutropenia (RR 1.16 [0.89-1.49]). Treatment-related death was very rare (7 events in 3094 patients) and similar across treatment groups (RR 0.58 [0.14-2.33]). Five studies collected quality of life data but did not report it. Conclusion: Platinum-based chemotherapy using carboplatin in the adjuvant or neoadjuvant setting improves long-term outcomes in eTNBC, regardless of the examined subgroups. This was at the cost of more frequent chemotherapy delays and dose reductions, and greater haematological toxicity. Benefit from platinum was seen both when platinum agents were added to anthracycline containing regimens, as well as in anthracycline-free regimens. Citation Format: Sofia Mason, Melina Willson, Annabel Goodwin, Jane Beith, Sam J. Egger, Rachel F. Dear. Platinum-based chemotherapy for early triple-negative breast cancer: A Cochrane systematic review and meta-analysis [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-06-01.

PURPOSE:Glioblastoma is a universally fatal cancer of the central nervous system which responds poorly to treatment. MGMT has potential as a predictive biomarker in glioblastoma patients to determine treatment response. However, methods of measuring MGMT are currently unsatisfactory, and as such, use of this marker has not translated well into the clinic. This paper aims to review current methodology of MGMT measurement, with a focus on immunohistochemistry as a potential way forward. TOPICS AND METHODS: Studies of glioma patients where MGMT immunohistochemistry was undertaken, as well as the literature surrounding methylation analyses and the regulation of MGMT, were reviewed. RESULTS:All methods of measuring MGMT were disputed in some way in the literature. A trend of discordance between methylation analyses and protein analyses was present. There is a lack of standardisation in the measurement of MGMT, and as a result, it seems that there are highly variable results. CONCLUSIONS:No single method of MGMT analysis has emerged as a clear choice for routine clinical testing of MGMT in glioma patients. Although methylation analyses are favoured, their expense and inaccessibility are barriers to their use in routine clinical practice. More research into immunohistochemistry is needed to determine whether it can serve as a reliable and cost-effective alternative to methylation analyses.