Burns, Terence

Abstract BACKGROUND Progress for gliomas is slowed in part by the paucity of mechanistic feedback during treatment with experimental therapies. Access to extracellular tumor pharmacodynamic biomarkers could provide an avenue to accelerate progress. We have initiated a program of intra-operative microdialysis to accelerate biomarker discovery and to identify candidate outcome measures for translational therapies. METHODS Intraoperative microdialysis was performed with M-dialysis 100kDA catheters and 107 variable rate pumps under an IDE. Four IDH-mutant and two IDH-WT lesions were studied intraoperatively with 3 divergently placed catheters. Microperfusate (artifical CSF+ 3% dextran) was perfused at 2uL/min and collected in 20 min increments. Paired CSF was also obtained when accessible. A parallel cohort of nude mice bearing human IDH-mutant, IDH-WT, or sham intracranial xenografts (n=6-12) underwent intratumoral microdialysis. A pilot murine study of intracranial drug delivery was performed via concurrent microdialysis during convection-enhanced delivery (CED) of saline or the IDH-inhibitor AG120. RESULTS Microdialysate from IDH-mutant intracranial xenografts revealed >100 differentially abundant metabolites compared to sham or IDH-WT tumors, including D2-HG (21x) and MTA(18x), p< 10^-5. The most significantly abundant metabolite was DMA (4x, p< 10^-10). 15-1000uM D2HG was recovered from intra-operative human IDH-mutant tumors and 1-2uM from normal brain adjacent to IDH-WT gliomas and < 1uM in all IDH-WT samples. Forty metabolites differentiated enhancing tumor from adjacent brain in 3/3 paired human samples including upregulated Aminoacyl-tRNA biosynthesis and downregulated purine metabolism. Serial aliquots of microdialysate during saline CED yielded steady D2-HG levels whereas CED with AG120 yielded undetectable D2-HG within 6 hours. CONCLUSION The extracellular metabolic landscape of glioma is diverse, dynamic and reflects tumor biology and response to therapy. Collectively, these studies suggest that intra-tumoral drug testing should be feasible with realistic expectation of gaining metabolic feedback within a short timeframe. Leveraging this paradigm can provide opportunities to accelerate therapeutic translation for gliomas.

Abstract Medulloblastoma is a posterior fossa tumor rarely diagnosed in adults. Treatment includes craniospinal irradiation (CSI). Proton CSI is increasingly utilized to decrease radiation dose to normal tissues, despite the lack of randomized data demonstrating decreased toxicity compared to photon CSI. This single institution retrospective study of 39 adult medulloblastoma patients includes nineteen patients treated with photon CSI prior to 2015, and twenty treated with proton CSI thereafter. Median age was 28 years (range 18-66). Molecular subtype was most commonly sonic hedgehog (68%). The most common fractionation schedule was 36 Gy CSI in 20 fractions (85% of photon and 58% of proton patients) with a boost to 54-55.8 Gy (92%). Proton CSI delivered significantly lower mean doses to the cochleae (median 32 Gy vs. 44 Gy), lacrimal glands (8 vs. 36 Gy), lens (2 vs. 28 Gy), parotid glands (3 vs. 26 Gy), pharyngeal constrictors (6 vs. 15 Gy), esophagus (2 vs. 29 Gy), heart (0 vs. 14 Gy), lungs (1 vs. 7 Gy), liver (0.1 vs. 7 Gy), and skin (38 vs. 51 Gy) (all p< 0.001). Patients receiving proton CSI had significantly lower rates of acute dysphagia of any grade (5% vs. 35%, p= 0.044) and decreased median weight loss during radiation (+1.0 vs. -2.8 kg, p= 0.011). Acute hospitalization was associated with increased weight loss (p= 0.009). Median follow-up was 2.9 and 12.9 years for proton and photon patients, respectively, limiting late toxicity and outcome comparisons. At last follow-up five photon patients had died (two of progressive disease, three without recurrence ages 41-63) and 21% had experienced major cardiovascular events. At 10 years, 89% were alive and 82% were recurrence free. In conclusion, this study demonstrates dosimetric improvements with proton CSI, potentially leading to decreased acute toxicity including dysphagia and weight loss during treatment.

Abstract Choosing effective chemotherapies for intravenous delivery to brain tumors is challenging, especially given the protective nature of the blood brain barrier (BBB). Connecting drug distribution to non-invasive, pre-surgical magnetic resonance imaging (MRI) could allow for predictive insight into drug distribution. In a previous study, we found that T2Gd images were predictive of a low BBB penetrant drug (Cefazolin), and FLAIR images were predictive of a high BBB penetrant drug (Levetiracetam). While these results are promising, we further seek to explore how advanced MRI sequences might inform image-based models of drug distribution. Prior to surgery, we acquired advanced dynamic contrast enhanced (DCE) and diffusion weighted imaging (DWI) MRI sequences for eight brain tumor patients (7 gliomas and 1 metastatic adenocarcinoma) in addition to the anatomic MRIs. All resulting quantitative maps and acquired images were co-registered. Prior to incision, patients received injections of cefazolin and levetiracetam. Next, multiple blood samples and biopsies were collected during surgery. Biopsies and plasma samples were analyzed for drug concentration using liquid chromatography mass spectrometry (LCMS), and biopsy drug levels were reported as Brain-Plasma Ratio (BPR). Mean image intensity was extracted from a 15x15 voxel window surrounding the biopsy location. We performed linear regression analyses to determine which combination of images were predictive of BPR. We found that considering quantitative imaging improved our initial ability to predict BPR for both drugs. For cefazolin, the third diffusion tensor eigenvalue (L3) map was significantly correlated with BPR (p< 0.001, R2= 0.36). For levetiracetam, the best model consisted of a combination of images and maps with the L3 map and the isotropic diffusion map (P) being the most influential (p= 0.001, R2= 0.63). Advanced MRI-based modeling is a promising tool for forecasting drug distribution in brain tumors and could be of great importance for understanding efficacy and selecting therapeutic strategies.

Abstract BACKGROUND Determination of the causation of germline single nucleotide polymorphisms (SNPs) located in non-coding regions of the genome is challenging. The genomic region of 8q24 has been identified as important in many kinds of cancer, linked to a topologically associated domain (TAD) encompassing MYC; this TAD contains a GWAS SNP (rs55705857) associated with IDH-mutant glioma. METHODS Germline genotyping data from 622 IDH-mutant glioma and 668 controls were used to fine map the rs55705857 locus by detailed haplotype analysis. Chromatin immunoprecipitation sequencing (ChIP-seq) of histone markers H3K4me1, H3K4me3, H3K27ac and H3K36me3 was performed on normal brain samples (n=8) and human glioma samples (n=11 IDH-wt and 52 IDH-mut). RNAseq from 9 normal and 83 brain tumors (n=26 IDH-wt and 55 IDH-mut) were used to assess differential gene expression. RESULTS Fine-mapping identified rs55705857 SNP as the most likely causative allele (OR=8.69; p<0.001) within 8q24 for the development of IDH-mutant glioma. At rs55705857, both H3K27ac and H3K4me1 in IDH-mutant vs IDH-wt tumors were increased 3.05- and 1.58-fold, respectively (DiffBind; p=5.81×10-7 and p=2.31×10-3). ChromHMM analysis of the marks indicated that promoter and enhancer functions were significantly increased, and the activity broadened at rs55705857 in IDH-mut gliomas compared to IDH-wt tumors and normal brain samples. This enhancement correlated with significant increased MYC expression in IDH-mut gliomas (p=3.1×10-13), as well as alterations of Myc signaling targets. Publicly available ATACseq, ChIPseq and long-range DNA interaction data demonstrated that the rs55705857 locus is open and interacts with the MYC promoter. CONCLUSIONS Fine-mapping of the 8q24 locus provided strong evidence that rs55705857 is the causative 8q24 locus associated with IDH-mut glioma. Functional experiments suggest that IDH mutation facilitates rs55705857 interaction with MYC to alter downstream MYC targets.

Abstract Background Antibody drug conjugates (ADCs) targeting the epidermal growth factor receptor (EGFR), such as depatuxizumab mafodotin (Depatux-M), is a promising therapeutic strategy for glioblastoma (GBM) but recent clinical trials did not demonstrate a survival benefit. Understanding the mechanisms of failure for this promising strategy is critically important. Methods PDX models were employed to study efficacy of systemic vs intracranial delivery of Depatux-M. Immunofluorescence and MALDI-MSI were performed to detect drug levels in the brain. EGFR levels and compensatory pathways were studied using quantitative flow cytometry, Western blots, RNAseq, FISH, and phosphoproteomics. Results Systemic delivery of Depatux-M was highly effective in nine of 10 EGFR-amplified heterotopic PDXs with survival extending beyond one year in eight PDXs. Acquired resistance in two PDXs (GBM12 and GBM46) was driven by suppression of EGFR expression or emergence of a novel short-variant of EGFR lacking the epitope for the Depatux-M antibody. In contrast to the profound benefit observed in heterotopic tumors, only two of seven intrinsically sensitive PDXs were responsive to Depatux-M as intracranial tumors. Poor efficacy in orthotopic PDXs was associated with limited and heterogeneous distribution of Depatux-M into tumor tissues, and artificial disruption of the BBB or bypass of the BBB by direct intracranial injection of Depatux-M into orthotopic tumors markedly enhanced the efficacy of drug treatment. Conclusions Despite profound intrinsic sensitivity to Depatux-M, limited drug delivery into brain tumor may have been a key contributor to lack of efficacy in recently failed clinical trials.

Abstract Background: Genome-wide association studies (GWAS) revealed that 25 regions in 24 genes are associated with adult diffuse glioma development. These regions were identified by performing GWAS of glioma overall and by pathology (GBM and nonGBM). The 2016 WHO Classification of Tumors of the Central Nervous System utilizes two somatic alterations to molecularly-classify adult diffuse glioma: IDH mutation and 1p/19q codeletion. We hypothesized that germline variants may increase susceptibility to, or interact with, these somatic alterations to accelerate the development of specific molecular subtypes of glioma. We further hypothesize that germline variants associated with IDH-mutated glioma might be associated with other IDH-mutated tumors, namely, cholangiocarcinoma, acute myeloid leukemia (AML) and melanoma. Methods: We performed a GWAS by glioma molecular subtype - as defined by presence or absence of IDH somatic mutation and 1p/19q codeletion. A total of 1320 glioma cases and 1889 controls were used in the discovery set, and 799 glioma cases and 808 controls in the validation set. A meta-analysis was performed with a genome-wide p-value threshold of 5 × 10−8. GTEx data were used to perform an expression quantitative trait loci (eQTL) analysis. For germline variants that were significantly associated with IDH-mutated glioma, we evaluated pleiotropy with cholangiocarcinoma, AML and melanoma using TCGA and Mayo Biobank controls. Results: Variants in or near D2HGDH on chromosome 2 were genome-wide significant in IDH-mutated glioma (meta p-value = 2.82 × 10−10). TCGA reported that the D2HGDH region was commonly deleted in IDH-mutated gliomas that do not have 1p/19q codeletion. In TCGA data for IDH-mutated, non-codeleted glioma, we observed that the D2HGDH variant was inversely associated with tumor deletions of D2HGDH (odds ratio=0.57, p-value=0.015). The eQTL analyses demonstrated significant associations between D2HGDH germline variant and expression of D2HGDH (p=2.2 × 10−11). Further stratifying IDH-mutated glioma by 1p/19q codeletion status, one variant near FAM20C on chromosome 7 was genome-wide significant in gliomas that have IDH mutation and 1p/19q codeletion (meta p-value=9.56 × 10−9). Analyses are currently underway to evaluate pleiotropy of these IDH-mutated glioma germline variants with other IDH-mutated tumors including cholangiocarcinoma, AML and melanoma. Variants in or near GMEB2 on chromosome 20 were genome-wide significant in IDH wild-type glioma (meta p-value=2.60 × 10−10). The most significant variant in the GMEB2 region remained significant after adjustment for the known RTEL1 glioma risk variant nearby on chromosome 20 (p=0.029). Conclusions: We identified and validated novel germline variants in two genes that are associated with etiology of IDH-mutated and one gene that is associated with IDH wild-type adult diffuse glioma. Citation Format: Jeanette E. Eckel-Passow, Kristen L. Drucker, Thomas M. Kollmeyer, Matthew L. Kosel, Paul A. Decker, Annette M. Molinaro, Terri Rice, Corrine E. Praska, Lauren E. Clark, Alissa A. Caron, Alexej Abyzov, Anthony Batzler, Jun S. Song, Melike Pekmezci, Helen M. Hansen, Lucie S. McCoy, Paige M. Bracci, Joseph Wiemels, John K. Wiencke, Stephen Francis, Terence C. Burns, Caterina Giannini, Daniel H. Lachance, Margaret Wrensch, Robert B. Jenkins. Adult diffuse glioma GWAS by molecular subtype identifies variants in D2HGDH, FAM20C and GMEB2 [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1193.

OBJECTIVE With the revised WHO 2016 classification of brain tumors, there has been increasing interest in imaging biomarkers to predict molecular status and improve the yield of genetic testing for diffuse low-grade gliomas (LGGs). The T2-FLAIR–mismatch sign has been suggested to be a highly specific radiographic marker of isocitrate dehydrogenase ( IDH ) gene mutation and 1p/19q codeletion status in diffuse LGGs. The presence of T2-FLAIR mismatch indicates a T2-hyperintense lesion that is hypointense on FLAIR with the exception of a hyperintense rim. METHODS In accordance with PRISMA guidelines, we performed a systematic review of the Ovid Medline, Embase, Scopus, and Cochrane databases for reports of studies evaluating the diagnostic performance of T2-FLAIR mismatch in predicting the IDH and 1p/19q codeletion status in diffuse LGGs. Results were combined into a 2 × 2 format, and the following diagnostic performance parameters were calculated: sensitivity, specificity, positive predictive value, negative predictive value, and positive (LR+) and negative (LR−) likelihood ratios. In addition, we utilized Bayes theorem to calculate posttest probabilities as a function of known pretest probabilities from previous genome-wide association studies and the calculated LRs. Calculations were performed for 1) IDH mutation with 1p/19q codeletion ( IDHmut-Codel ), 2) IDH mutation without 1p/19q codeletion ( IDHmut-Noncodel ), 3) IDH mutation overall, and 4) 1p/19q codeletion overall. The QUADAS-2 (revised Quality Assessment of Diagnostic Accuracy Studies) tool was utilized for critical appraisal of included studies. RESULTS A total of 4 studies were included, with inclusion of 2 separate cohorts from a study reporting testing and validation (n = 746). From pooled analysis of all cohorts, the following values were obtained for each molecular profile— IDHmut-Codel : sensitivity 30%, specificity 73%, LR+ 1.1, LR− 1.0; IDHmut-Noncodel : sensitivity 33.7%, specificity 98.5%, LR+ 22.5, LR− 0.7; IDH : sensitivity 32%, specificity 100%, LR+ 32.1, LR− 0.7; 1p/19q codeletion: sensitivity 0%, specificity 54%, LR+ 0.01, LR− 1.9. Bayes theorem was used to calculate the following posttest probabilities after a positive and negative result, respectively— IDHmut-Codel : 32.2% and 29.4%; IDHmut-Noncodel : 95% and 40%; IDH : 99.2% and 73.5%; 1p/19q codeletion: 0.4% and 35.1%. CONCLUSIONS The T2-FLAIR–mismatch sign was an insensitive but highly specific marker of IDH mutation and I DHmut-Noncodel profile, although significant exceptions may exist to this finding. Tumors with a positive sign may still be IDHwt or 1p/19q codeleted. These findings support the utility of T2-FLAIR mismatch as an imaging-based biomarker for positive selection of patients with IDH -mutant gliomas.

Abstract INTRODUCTION Neuroimaging dogma for glioblastoma asserts that hyperintensity on T1Gd MRI reveals the bulk of the tumor, while T2/FLAIR signal indicates edema. However, it is unclear whether this edema results from immune response or increased tumor cells. Further, one significant driver of the known sex differences in glioblastoma may be differences in immune response, due to the X-linkage of many immune genes. Based on this, we hypothesized that assumptions regarding tumor cellularity in T2/FLAIR images should be tailored to the biological sex of the patient. METHODS Using a retrospective cohort of 18 primary glioblastoma patients receiving multiple image-localized biopsies (82 total) and standard MRI, we assessed: distance of biopsy from T1Gd and T2 areas; a pathologist’s score of percent tumor cell density; and an imaging-based invasion metric, D/ρ. This metric is derived from the biomathematical Proliferation-Invasion model of glioma growth, which features two parameters, net growth rate (ρ) and net invasion rate (D). Their ratio D/ρ is related to degree of invasion, and is estimated from volumetric measurements of MRI abnormalities. Additionally, 25 patient-derived xenograft models implanted in females were grown until moribund, at which point brains were excised and stained for DAPI (to show all cells) and Lamin (to highlight tumor cells). Image processing of lamin-stained sections defines contours of intensity correlating with cell density. RESULTS Outside both the T1Gd and T2 region, male patient biopsies had higher tumor cell densities than females. Males also tended to have higher invasion metrics. Although each set derived from different patients, preclinical metrics of invasion were positively correlated with clinical invasion in females but negatively correlated in males. CONCLUSION Our preliminary finding that cell distribution patterns correlate with imaging metrics differently between the sexes supports the hypothesis that the degree of tumor cell density represented on certain MRI sequences may be sex-specific.

Abstract OBJECTIVES Dogma suggests that for brain tumors, regions of enhancement on T1-weighted gadolinium contrast enhanced (T1Gd) magnetic resonance imaging (MRI) correlate with intravenously delivered drug distribution as enhancement indicates a compromised blood-brain barrier (BBB). However, poor response to intravenous therapies highlights the importance of the diffuse disease beyond enhancing regions. This study investigated whether imaging features can provide an accurate prediction of drug distribution. METHODS Eight brain tumor patients (7 gliomas and 1 metastatic adenocarcinoma) were included in this Phase 0 trial. Presurgery T1-weighted, T1Gd, T2-weighted gadolinium contrast enhanced (T2Gd), and T2-weighted Fluid Attenuated Inversion Recovery (T2FLAIR) MRIs were acquired. All images underwent bias correction using the N4 algorithm, standardization of intensities, and registration. Prior to incision, patients received both an antibiotic cefazolin (6% BBB penetrance) and levetiracetam (80% BBB penetrance), an anti-seizure drug. Subsequently, multiple blood samples and image-guided biopsies were taken and analyzed for drug concentration using liquid chromatography mass spectrometry. Biopsy drug levels are reported as Brain-Plasma Ratio (BPR), the ratio of biopsy concentration relative to plasma concentration. Mean image intensity was extracted from an 8x8 mm window surrounding each biopsy location. Regression analysis was performed to determine which combination of image types were linearly predictive of BPR for both drugs. Correlations were also analyzed according to the biopsy location radiographic appearance. RESULTS Regression analysis revealed that T2Gd intensity was linearly predictive of cefazolin BPR and FLAIR intensity was linearly predictive of levetiracetam BPR (p=0.009 and 0.041, respectively). Grouping samples according the the radiographic appearance revealed that levetiracetam BPR had a similar pattern of values to that of FLAIR intensities and cefazolin BPR had a similar pattern to T2, further supporting the regression analysis results. CONCLUSIONS Local concentrations of drug may be related to T2-weighted signals (T2Gd and T2FLAIR) rather than the gadolinium distribution on T1Gd images.