Decker, Paul A.

Abstract Clinical management of IDH wildtype glioblastoma (GBM) and tumefactive multiple sclerosis (tMS) is drastically different. GBM requires maximal safe resection followed by chemoradiation, while tMS outcome is worsened by surgery and radiotherapy. Noninvasive methods are needed to help with accurate diagnosis of tumor and non-tumor etiologies. To develop an MRI-based classification model, tMS subjects diagnosed prior to January 1, 2020, were matched to tMS by age at diagnosis, sex, index MRI date, and 2D/3D acquisition. Inclusion criteria included one cm minimal lesion size and pre-operative post-contrast T1 and T2 images available for analysis. A 3D-DenseNet121 was used to develop a classification model using prespecified parameters: 650 epochs, batch size 16, learning rate 10-3, cross-entropy loss, and AdamW optimizer. The stopping rule was defined as three sequential differences in epoch cross-entropy loss <0.02. Models were developed using both T1gd and T2, as well as from only T1gd and only T2. Training included 220 subjects (110 GBM, 110 tMS). A 2-stage validation design was used, which included both retrospective and prospective cohorts. Stage 1 consisted of 272 retrospective GBM (diagnosed prior to January 1, 2020). Stage 2 consisted of 69 and 34 prospective (diagnosed after January 1, 2020) GBM and tMS, respectively. External validation on the 272 retrospective GBM demonstrated accuracy of 91%, 84%, and 78% for T1gd+T2, T1gd only, and T2 only, respectively. External validation on the 69 prospective GBM demonstrated an accuracy of 87%, 64%, and 67% for T1gd+T2, T1gd only, and T2 only, respectively. The 34 prospective tMS demonstrated accuracy of 76%, 76%, and 82% for T1gd+T2, T1gd only, and T2 only, respectively. This shows the feasibility of deep learning to aid in differential diagnosis of brain lesions. Future work entails the integration of germline variants into the classification model, including variants associated with the risk of glioma or MS.

Abstract Acquired tumor alterations are part of the WHO diagnosis of glioma. However, germline predisposition to these alterations is unknown. GWAS in European and Asian populations identified germline variants in 30 regions, many in or near genes that also acquire alterations, suggesting that interactions between inherited and acquired genetics are involved in glioma development. To evaluate such interactions, we used 3686 glioma cases and 1889 controls. A case-control design and multinomial logistic regression were used to identify germline variants associated with development of TERT-promoter mutant gliomas, generating two interesting observations. First, TERT germline variants were associated with predisposition to IDHwt glioma that have a TERT promoter mutation, but not with IDH-mutant codeleted gliomas that have a TERT promoter mutation. Second, two novel regions were observed in this more homogeneous subgroup of TERT mutated tumors: variants in TXNDC11 (OR=2.66, p=5.7x10-8) and RUVBL2 (OR=3.1, p=5.8x10-8). High tumor expression of TXNDC11 is associated with poor prognosis in glioma and RUVBL2 interacts with TERT to affect telomerase activity. Similar analyses are being performed on additional clinically relevant acquired alterations. In parallel, case-case GWAS analyses were performed to evaluate germline variants associated with other tumor alterations. Comparing IDH-mutant versus IDHwt stratified by rs55705857 genotype, the most significant variants were PHLDB1 (p=1.2x10-13) and D2HGDH (p=2.1x10-10), highlighting the importance of these two variants independent of CCDC26 for the development of IDH-mutant glioma. As a parallel approach, we utilized recursive partitioning and regression trees (rpart) to classify IDH mutation status using the 30 known variants. The top four branches of the regression tree included CCDC26, D2HGDH, PHLDB1, and MAML2, as well as two Asian variants (RAB27A, CYP4F12). This highlights the importance of utilizing variants discovered in non-European populations in analyses. Overall, understanding these interactions are necessary to develop therapeutics, stratify clinical trials, and to develop relevant tumor models.

Abstract Histologic features, presence of CDKN2A/B homozygous deletion and methylation subtype are strong determinants of outcome and of therapy in IDH-mutant gliomas. We were interested in learning if there are additional markers of outcome in these tumors. To address this question, we assembled a cohort of 963 IDH-mutant gliomas with overall survival data (median follow-up alive 2.7 years, median follow-up deceased 3.9 years). Array-based copy number data, targeted NGS, germline genotyping data and Illumina EPIC array methylation data were available on 872, 226, 542, and 401 patients, respectively. Morphologic type (oligodendroglioma versus astrocytoma, grade (2/3 vs 4) and methylation subtype (A_IDH_LG vs A_IDH_HG) were all significantly associated with survival (p <0.001). There was no significant difference in survival between grade 2 vs 3 IDH-mutant astrocytomas (p=0.53 adjusting for age and sex). However, subjects with the risk allele (G) for rs55705857 had a better overall survival than subjects with the non-risk allele (A) in IDH-mutant oligodendroglioma (AG/GG vs AA, median overall survival 21.6 vs 15.7 years, p=0.048 after adjusting for grade, age, and sex). Conversely, rs55705857 was associated with a markedly worse survival in grade 2 IDH-mutant astrocytomas (AG/GG vs AA, median overall survival 8.6 vs 19.6 years, p=0.007 after adjusting for age and sex). Despite similar sample sizes and number of deaths, there was no significant difference in overall survival by rs55705857 genotype in grade 3 or 4 IDH-mutant astrocytomas. Upon further functional analyses, TLX1 and GALNT17 were found to be differentially methylated in IDH1-mutant astrocytomas and oligodendrogliomas, respectively, when stratified by rs55705857 risk allele. This is the first observation of a germline characteristic (e.g. rs55705857) predicting survival in low-grade IDH-mutant astrocytoma. While this observation requires validation, it suggests that germline genotype may be practice changing and inform results of trials such as CATNON and CODEL.

Abstract Glioblastoma (GBM) remains one of the most therapy-resistant malignancies with frequent local failures despite aggressive surgery, chemotherapy, and ionizing radiation (IR). Small molecule inhibitors of DNA-dependent protein kinase (DNA-PKi) are potent radiosensitizers currently in clinical trials. Determining which patients may benefit from radiosensitization with DNA-PKi is critical to avoid unnecessary increased risk of normal tissue toxicity. In this study, we used GBM patient-derived xenografts (PDX) in orthotopic murine models to study the relationship between molecular features, pharmacokinetics, and the radiosensitizing potential of the DNA-PKi peposertib. We show that peposertib radiosensitizes established and PDX GBM lines in vitro at 300 nmol/L and above, with a significant increase in radiosensitization by maintaining post-IR exposure for >12 hours. Radiosensitization by peposertib is mediated by catalytic inhibition of DNA-PK, and knockdown of DNA-PK by short hairpin RNA (shRNA) largely abolished the radiosensitizing effect. Peposertib decreased auto-phosphorylation of DNA-PKcs after IR in a dose-dependent manner with a delay in resolution of γH2AX foci at 24 hours. The addition of peposertib to IR significantly increased survival in GBM120 orthotopic xenografts, but not in GBM10. There was no difference in plasma or average tumor concentrations of peposertib in the two cohorts. Although the mechanism underpinning this discordant effect in vitro versus in vivo is not clear, there was an association for greater sensitization in TP53 mutant lines. Transfection of a dominant-negative TP53 mutant in baseline TP53 wild-type GBM lines significantly delayed growth and decreased nonhomologous end joining efficiency (but not homologous recombination), after peposertib exposure. See related commentary by Buchsbaum, p. 840

2071 Background: Brigimadlin (BI 907828) is a potent small molecule inhibitor of the p53-MDM2 pathway currently in a phase 0/1 clinical trial in combination with radiotherapy in newly diagnosed MGMTunmethylated glioblastoma (GBM). To inform development of brigimadlin for GBM, we developed a pharmacokinetic (PK)/ pharmacodynamic (PD)/ efficacy model using GBM patient derived xenografts (PDXs). Methods: Brigimadlin PK parameters were evaluated in FVB wild-type and knockout (Mdr1a/b-/-Bcrp1-/-) mice and in athymic nude mice. Drug binding was measured by rapid equilibrium dialysis, and drug levels quantified by LC/MS-MS. Dose-response relationships were evaluated in subcutaneous and orthotopic PDXs across a range of doses. Response to brigimadlin (2 mg/kg weekly) +/- RT (2 Gy x 10 fractions) was evaluated in orthotopic PDXs. Results: Brigimadlin significantly delayed median time to regrowth of GBM108 ( MDM2amplified) subcutaneous tumors (vs. placebo; p<0.05) at dose levels of 0.25 mg/kg (1.3-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (2.6-fold), and 2 mg/kg (5.3-fold). A single dose of brigimadlin resulted in corresponding intra-tumoral drug levels 24 hours post-dose: 84 nM (0.25 mg/kg), 175 nM (0.5 mg/kg), 398 nM (1 mg/kg) and 924 nM (2 mg/kg) and dose-dependent upregulation of p53 target genes (p21 and PUMA). In contrast, GBM14 ( MDM2non-amplified) showed regrowth delay (1.6-fold) only at 2 mg/kg (p=0.008). Brigimadlin was highly bound (fraction unbound = 0.0006 in brain; 0.0017 in GBM tumor tissue), and CNS distribution was limited by efflux; in wild-type mice Kp,uu_brain = 0.002 ± 0.003; in Mdr1a/b-/-Bcrp1-/- mice Kp,uu = 0.043 ± 0.025. Efficacy of brigimadlin was compared in orthotopic GBM108 PDXs grown in athymic nude mice vs. immuno/efflux-deficient (Rag2-/-Mdr1a-/-Bcrp1-/-) mice. In nude mice, median survival was extended at 2 mg/kg brigimadlin (1.4-fold, p=0.009), with corresponding intra-tumoral drug levels of 29 nM. In efflux deficient mice, median survival was extended (p<0.05) at 0.25 mg/kg (1.5-fold), 0.5 mg/kg (1.5-fold), 1 mg/kg (1.8-fold), and 2 mg/kg (>4-fold). Measurement of intra-tumoral drug levels is in progress. In combination with RT in GBM108, brigimadlin (2 mg/kg x 2 weeks) extended median survival vs. vehicle (66 vs. 50 days; p=0.012) and enhanced median survival following RT (128.5 vs. 82 days; p=0.009). In GBM14, 2 mg/kg brigimadlin (2 mg/kg x 12 weeks) extended median survival (39.5 vs. 32.5 days; p=0.0004) and enhanced survival following RT (97 vs. 81 days; p<0.0001). Conclusions: Efficacy of brigimadlin is dependent on adequate CNS delivery. Studies in subcutaneous PDX demonstrated intra-tumoral total drug levels in the mid-nanomolar range provide meaningful tumor effects in a highly sensitive, MDM2 amplified PDX. In orthotopic PDXs with and without MDM2 amplification, brigimadlin provided further benefit in combination with RT. These data support ongoing clinical evaluation of brigimadlin in GBM.

2012 Background: Navtemadlin (nvtm) is a potent, selective, orally available small molecule inhibitor of murine double minute 2 homologue (MDM2), which has completed a Ph 0 surgical window of opportunity study in glioblastoma, IDH-wildtype (GBM; Lee SNO 2022). To optimally interpret the clinical data, an extensive evaluation of nvtm PK, PD, and efficacy was performed in GBM patient derived xenografts (PDXs). Methods: Response to nvtm +/- radiotherapy (RT) was characterized in vitro and in vivo across a panel of GBM PDXs with various TP53 and MDM2 alterations. Efficacy, PK, and PD were evaluated in flank or orthotopic PDX models. p53 pathway activation was evaluated by qPCR, Western blot, and IHC. Nvtm brain penetration was evaluated in immunodeficient abcb1a/b-/- (P-glycoprotein; P-gp), abcg2-/- (BCRP), or double-knockout mice. Binding was assessed by rapid equilibrium dialysis, and nvtm concentrations by LC/MS-MS and MALDI-MSI. Results: Nvtm decreased in vitro viability of TP53WT GBM PDX explant cultures with IC50s <100 nM, but was ineffective in TP53-mutant GBM. In flank PDX models, nvtm (100 mg/kg daily for 4 weeks) delayed tumor regrowth by 1.9 to 4.8-fold in TP53 WT PDXs without MDM2amplification (n=9 models), and more markedly by 20.1 to 29.4-fold in MDM2amplified models (n=4; 2 PDXs did not recur). In a flank PDX dose-response study, nvtm delayed the growth of MDM2amplified GBM108 at 25 mg/kg vs. placebo (61 vs. 11 days; p=0.014; mean intra-tumoral nvtm = 590 nM). In contrast, growth of MDM2 non-amplified GBM14 was delayed only at 100 mg/kg (37 vs. 15 days; p=0.082; mean intra-tumoral nvtm = 2990 nM). In both flank models, p53 target gene upregulation was similar between 25 and 100 mg/kg and did not correlate with regrowth. Nvtm was highly bound (fraction unbound = 0.090 in culture media and 0.014 in GBM tissue homogenate). In mice after oral dosing, distribution to CNS was limited by P-gp, with a brain to plasma ratio of 0.009 and unbound ratio of 0.005, whereas BCRP deficiency did not affect CNS distribution. With orthotopic GBM108, nvtm efficacy was evaluated in nude vs. efflux-deficient mice. In nude mice, nvtm was ineffective at doses up to 100 mg/kg; however, in efflux deficient mice, the mean intra-tumoral nvtm level at 25 mg/kg was similar to flank tumors (690 nM) and survival was significantly extended (134 vs. 62 days, p=0.0002) with additional benefit at 100 mg/kg (>300 vs. 62 days, p=0.002). Conclusions: Nvtm showed significant efficacy in TP53 WT GBM flank PDX models but efficacy in orthotopic PDX was highly dependent on CNS penetration. Targeting nvtm levels based on the in vitroIC50 may significantly underestimate the in vivo effective concentration . In vivo studies showed lower intra-tumoral nvtm levels are required to significantly inhibit growth of MDM2amplified compared with non-amplified tumors.

Abstract Purpose: Antibody-drug conjugates (ADC) are targeted therapies with robust efficacy in solid cancers, and there is intense interest in using EGFR-specific ADCs to target EGFR-amplified glioblastoma (GBM). Given GBM’s molecular heterogeneity, the bystander activity of ADCs may be important for determining treatment efficacy. In this study, the activity and toxicity of two EGFR-targeted ADCs with similar auristatin toxins, Losatuxizumab vedotin (ABBV-221) and Depatuxizumab mafodotin (Depatux-M), were compared in GBM patient-derived xenografts (PDX) and normal murine brain following direct infusion by convection-enhanced delivery (CED). Experimental Design: EGFRviii-amplified and non-amplified GBM PDXs were used to determine in vitro cytotoxicity, in vivo efficacy, and bystander activities of ABBV-221 and Depatux-M. Nontumor-bearing mice were used to evaluate the pharmacokinetics (PK) and toxicity of ADCs using LC-MS/MS and immunohistochemistry. Results: CED improved intracranial efficacy of Depatux-M and ABBV-221 in three EGFRviii-amplified GBM PDX models (Median survival: 125 to &gt;300 days vs. 20–49 days with isotype control AB095). Both ADCs had comparable in vitro and in vivo efficacy. However, neuronal toxicity and CD68+ microglia/macrophage infiltration were significantly higher in brains infused with ABBV-221 with the cell-permeable monomethyl auristatin E (MMAE), compared with Depatux-M with the cell-impermeant monomethyl auristatin F. CED infusion of ABBV-221 into the brain or incubation of ABBV-221 with normal brain homogenate resulted in a significant release of MMAE, consistent with linker instability in the brain microenvironment. Conclusions: EGFR-targeting ADCs are promising therapeutic options for GBM when delivered intratumorally by CED. However, the linker and payload for the ADC must be carefully considered to maximize the therapeutic window.

Abstract Introduction Alaska Native and American Indian (ANAI) peoples in Alaska currently experience a disproportionate burden of morbidity and mortality from tobacco cigarette use. Financial incentives for smoking cessation are evidence-based, but a family-level incentive structure has not been evaluated. We used a community-based participatory research and qualitative approach to culturally adapt a smoking cessation intervention with ANAI families. Aims and Methods We conducted individual, semistructured telephone interviews with 12 ANAI adults who smoke, 12 adult family members, and 13 Alaska Tribal Health System stakeholders statewide between November 2022 and March 2023. Through content analysis, we explored intervention receptivity, incentive preferences, culturally aligned recruitment and intervention messaging, and future implementation needs. Results Participants were receptive to the intervention. Involving a family member was viewed as novel and aligned with ANAI cultural values of commitment to community and familial interdependence. Major themes included choosing a family member who is supportive and understanding, keeping materials positive and encouraging, and offering cash and noncash incentives for family members to choose (eg, fuel, groceries, activities). Participants indicated that messaging should emphasize family collaboration and that cessation resources and support tips should be provided. Stakeholders also reinforced that program materials should encourage the use of other existing evidence-based cessation therapies (eg, nicotine replacement, counseling). Conclusions Adaptations, grounded in ANAI cultural strengths, were made to the intervention and recruitment materials based on participant feedback. Next steps include a beta-test for feasibility and a randomized controlled trial for efficacy. Implications This is the first study to design and adapt a financial incentives intervention promoting smoking cessation among ANAI peoples and the first to involve the family system. Feedback from this formative work was used to develop a meaningful family-level incentive structure with ANAI people who smoke and family members and ensure intervention messaging is supportive and culturally aligned. The results provide qualitative knowledge that can inform future family-based interventions with ANAI communities, including our planned randomized controlled trial of the intervention.