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Purpose Autologous tumor lysate-loaded dendritic cell vaccine (DCVax-L) is a promising treatment modality for glioblastomas. The purpose of this study was to investigate the potential utility of multiparametric MRI-based prediction model in evaluating treatment response in glioblastoma patients treated with DCVax-L. Methods Seventeen glioblastoma patients treated with standard-of-care therapy + DCVax-L were included. When tumor progression was suspected and repeat surgery was being contemplated, we sought to ascertain the number of cases correctly classified as true progression (TP) + mixed response or pseudoprogression (PsP) from multiparametric MRI-based prediction model using histopathology/mRANO criteria as ground truth. Multiparametric MRI model consisted of predictive probabilities (PP) of tumor progression computed from diffusion and perfusion MRI-derived parameters. A comparison of overall survival (OS) was performed between patients treated with standard-of-care therapy + DCVax-L and standard-of-care therapy alone (external controls). Additionally, Kaplan-Meier analyses were performed to compare OS between two groups of patients using PsP, Ki-67, and MGMT methylation status as stratification variables. Results Multiparametric MRI model correctly predicted TP + mixed response in 72.7% of cases (8/11) and PsP in 83.3% (5/6) with an overall concordance rate of 76.5% with final diagnosis as determined by histopathology/mRANO criteria. There was a significant concordant correlation coefficient between PP values and histopathology/mRANO criteria (r = 0.54; p = 0.026). DCVax-L-treated patients had significantly prolonged OS than those treated with standard-of-care therapy (22.38 ± 12.8 vs. 13.8 ± 9.5months, p = 0.040). Additionally, glioblastomas with PsP, MGMT methylation status, and Ki-67 values below median had longer OS than their counterparts. Conclusion Multiparametric MRI-based prediction model can assess treatment response to DCVax-L in patients with glioblastoma.

Background Recurrent gliomas are therapeutically challenging diseases with few treatment options available. One area of potential therapeutic vulnerability is the presence of targetable oncogenic fusion proteins. Methods To better understand the clinical benefit of routinely testing for fusion proteins in adult glioma patients, we performed a retrospective review of 647 adult patients with glioma who underwent surgical resection at our center between August 2017 and May 2021 and whose tumors were analyzed with an in-house fusion transcript panel. Results Fifty-two patients (8%) were found to harbor a potentially targetable fusion with 11 (21%) of these patients receiving treatment with a fusion-targeted inhibitor. The targetable genes found to be involved in a fusion included FGFR3, MET, EGFR, NTRK1, NTRK2, BRAF, ROS1, and PIK3CA. Conclusions This analysis demonstrates that routine clinical testing for gene fusions identifies a diverse repertoire of potential therapeutic targets in adult patients with glioma and can offer rational therapeutic options for patients with recurrent disease.

Re-irradiation is therapeutically considered for select rGBM patients and may induce immunogenic cell death to stimulate anti-tumor immune responses. Our phase 2 study of re-irradiation with pembrolizumab among rGBM patients evaluated the efficacy and safety of this regimen. METHODS Adult rGBM patients with KPS ≥ 70, a maximum supratentorial tumor diameter of 6 cm who were on ≤ 2mg dexamethasone/day and were ≥ 6 months from initial conventional radiation therapy were eligible. Cohort A (bevacizumab [BEV]-naïve) had ≤ 2 prior progressions while cohort B (BEV-refractory) allowed unlimited progressions but only one on prior BEV. Re-irradiation included 35 Gy over 10 fractions to residual enhancing (cohort A) as well as enhancing + non-enhancing (cohort B) disease. Pembrolizumab was administered at 200 mg every three weeks beginning within one week of re-irradiation start. BEV was administered at 15 mg/kg every three weeks for cohort B only. RESULTS Sixty patients enrolled (n = 30 per cohort) with a median age of 61 years (range 20-76), 47% were female and 53% enrolled after 2 or more progressions. Grade 3 events deemed at least possibly related to study therapy in > one patient for cohort A included headache (n = 2) and for cohort B included elevated ALT (n = 2) and hypertension (n = 5). No grade 4 events occurred in more than single patients per cohort and no grade 5 events occurred. Median PFS and PFS-6 were 4.9 months (95% CI: 3.5, 5.6) and 26.0% (95% CI: 12.3%, 43.0%) for cohort A and 4.14 months (95% CI: 3.45, 5.42) and 16.9% (95% CI: 5.4, 33.7) for cohort B. Median survival for cohorts A and B were 11.5 months (95% CI: 9.6, 14.1) and 7.6 months (95% CI 5.5, 9.3), respectively. CONCLUSIONS Re-irradiation with pembrolizumab was overall well tolerated and achieved comparable efficacy to historical salvage therapy established with lomustine.

We have previously demonstrated an independent association between high levels of plasma circulating cell-free DNA (ccfDNA) concentration and poor survival outcomes in patients with newly diagnosed glioblastoma. Given that somatic mutations are rarely detected in glioblastoma patient plasma, we reasoned that DNA shed by tumor (circulating tumor DNA, ctDNA) was not likely to be a driver of prognostic increases in ccfDNA. To investigate the tissue of origin for this prognostic ccfDNA, we analyzed the plasma ccfDNA methylation signatures of 23 patients with newly diagnosed glioblastoma using the Illumina Infinium EPIC array, a genome-wide DNA methylation technique covering over 850,000 CpG sites. Deconvolution of the ccfDNA methylation signatures based on published reference methylomes revealed an increased proportion of ccfDNA derived from granulocytes in glioblastoma specimens compared to healthy controls (p < 0.0001). Further, this granulocyte proportion was increased in patients with high ccfDNA levels (above median value) compared to patients with low ccfDNA (p = 0.0001). Granulocyte proportion correlated with ccfDNA concentration (Spearman r = 0.64, p = 0.001). The top two gene sets identified by differential methylation analysis followed by gene set enrichment analysis (methylGSA) between high- and low-ccfDNA specimens were “neutrophil activation involved in immune response” (GO:0002283, p = 3.4E-23) and “neutrophil degranulation” (GO:0043312, p = 3.4E-23). Additional analysis of ccfDNA fragment size identified larger fragments ( > 700 bp) as the major source of the prognostic signal (log-rank p = 0.002 for progression-free survival) compared to traditionally sized ccfDNA fragments (50-700 bp, log-rank p = 0.12). Taken together, these data suggest that granulocytes are the primary contributing source of prognostic ccfDNA in glioblastoma. Future studies are needed to determine the mechanism through which granulocyte-derived ccfDNA is associated with clinical outcomes in glioblastoma and to explore related therapeutic opportunities.

Differentiation of true tumor progression (TP) from pseudoprogression (PsP) is a major unmet need in patients with glioblastoma. [18F]Fluciclovine is a synthetic amino acid PET radiotracer that is FDA-approved in biochemical recurrence in prostate cancer. The study aim was to assess the value of [18F]Fluciclovine PET in differentiation of histologically confirmed (“true”) TP and PsP in post-treatment of glioblastoma. METHODS: 23 patients with glioblastoma with new contrast-enhancing lesions or lesions showing increased enhancement ( > 25% increase) on standard MRI after completion of radiation underwent 60-minutes dynamic [18F]Fluciclovine PET imaging. Patients subsequently underwent resection of enhancing lesion and tumor percentage vs. treatment-related changes were quantified on histopathology. Patients were considered "true” TP if tumor represented ≥ 50% of the resected specimen, mixed TP-PsP if < 50% and > 10%, and PsP if tumor represented ≤ 10%. Summed 30- to 40-minute post-injection PET images were used to measure SUVpeak and SUVmax (g/mL units). RESULTS: 15 patients with “true” TP, 3 with mixed TP-PsP, and 5 with PsP were included. There was a positive correlation between SUVpeak by PET and tumor percentage by histology (Rho= 0.56, p= 0.006). Patients who demonstrated “true” TP had significantly higher SUVpeak compared to patients with histological PsP (4.8±1.6 vs 2.9± 1.0, p= 0.02, AUC= 0.91, n=20). SUVpeak cut-off of 3.3 provided 93% sensitivity, 80% specificity, and 90% accuracy for differentiation of “true” TP from PsP. Patients with “true” TP/mixed TP-PsP also had significantly higher SUVpeak than patients with PsP (4.6±1.5 vs 2.9± 1.0, p= 0.03, AUC= 0.88, n=23). SUVmax and partial volume-corrected SUVpeak and SUVmax exhibited similar performance. CONCLUSION: Our results indicated that [18F]Fluciclovine PET imaging can accurately differentiate “true” TP from PsP. Further studies are required to confirm these promising early results and determine optimal criteria for interpreting [18F]Fluciclovine PET to distinguish PsP from TP.

BACKGROUND Tumor-associated macrophages (TAMs) are a key component of glioblastoma (GBM) tumor microenvironment. Considering the differential role of different TAM phenotypes in iron metabolism with the M1 phenotype storing intracellular iron, and M2 phenotype releasing iron in the tumor microenvironment, here we investigated non-invasive quantitative susceptibility mapping (QSM) and T2* MRI relaxometry to quantify iron as imaging biomarkers for TAMs in adult patients with GBM. METHODS In this prospective study, 21 adult patients with GBM were enrolled between 2016 to 2019. Patients underwent a 3D single echo gradient echo sequence in addition to standard anatomical sequences on a 3 Tesla MRI. QSM images were reconstructed using the morphology-enabled dipole inversion (MEDI) algorithm. In 3 subjects, ex vivo imaging of surgical specimens was performed on a Bruker 9.4 Tesla 8.9 cm vertical bore MR using 3D multi-echo GRE scans, and R2* (1/T2*) maps were generated by a pixel-wise monoexponential fitting. Each specimen was stained with hematoxylin and eosin, as well as with CD68, CD86, CD206, and L-Ferritin. RESULTS Significant positive correlation was observed between mean susceptibility for the tumor enhancing zone and the L-ferritin positivity percent (r =0.56, p=0.018) and the combination of tumor’s enhancing zone and necrotic core and the L-Ferritin positivity percent (r=0.72; p=0.001). Moreover, the mean susceptibility significantly correlated with positivity percent for CD68 (ρ=0.52, p=0.034) and CD86 (r=0.7 p=0.001), but not for CD206 (ρ=0.09; p=0.7). There was a positive correlation between mean R2* values and CD68 positive cell counts (r =0.6, p=0.016). Similarly, mean R2* values significantly correlated with CD86 (r=0.54, p=0.03) but not with CD206 (r=0.15, p=0.5). High mean susceptibility in the necrotic core was associated with inferior PFS (hazard ratio,4.5, p=0.016). CONCLUSION MR Susceptibility Imaging can quantify the iron content of GBM and provide a non-invasive method for TAM quantification and phenotyping.

Purpose Tumor-associated macrophages (TAMs) are a key component of glioblastoma (GBM) microenvironment. Considering the differential role of different TAM phenotypes in iron metabolism with the M1 phenotype storing intracellular iron, and M2 phenotype releasing iron in the tumor microenvironment, we investigated MRI to quantify iron as an imaging biomarker for TAMs in GBM patients. Methods 21 adult patients with GBM underwent a 3D single echo gradient echo MRI sequence and quantitative susceptibility maps were generated. In 3 subjects, ex vivo imaging of surgical specimens was performed on a 9.4 Tesla MRI using 3D multi-echo GRE scans, and R2* (1/T2*) maps were generated. Each specimen was stained with hematoxylin and eosin, as well as CD68, CD86, CD206, and L-Ferritin. Results Significant positive correlation was observed between mean susceptibility for the tumor enhancing zone and the L-ferritin positivity percent (r =0.56, p=0.018) and the combination of tumor’s enhancing zone and necrotic core and the L-Ferritin positivity percent (r=0.72; p=0.001). The mean susceptibility significantly correlated with positivity percent for CD68 (ρ = 0.52, p=0.034) and CD86 (r=0.7 p=0.001), but not for CD206 (ρ = 0.09; p=0.7). There was a positive correlation between mean R2* values and CD68 positive cell counts (r =0.6, p=0.016). Similarly, mean R2* values significantly correlated with CD86 (r=0.54, p=0.03) but not with CD206 (r=0.15, p=0.5). Conclusion MR quantitative susceptibility mapping can quantify the iron content of GBM and provide a non-invasive method for TAM quantification and phenotyping.

BACKGROUND KRT232 is an orally bioavailable, selective small molecule inhibitor of MDM2 that blocks the protein-protein interaction between MDM2 and p53. We performed a surgical window of opportunity trial of KRT232 in patients with recurrent GBM. METHODS The primary endpoint was to determine the tumor tissue concentration of KRT232. Prior to surgery, patients received KRT232 at either 120mg (n = 10, minimal dose that is consistently associated with alterations in serum MIC-1) or 240mg (n = 10; recommended phase 2 dose as monotherapy) for two days prior to surgical resection. Surgery was performed 3-6 hours following the last administration of KRT232. Tissue was analyzed for KRT232 concentration by LC/MS and for correlative studies. Participants with TP53 wild-type tumors were eligible to continue KRT232 following recovery from surgery at the RP2D of 240mg QD x 7 days q3weeks. RESULTS Twenty-one patients were enrolled from July 2018 to April 2020. One patient was deemed ineligible after surgery due to non-GBM tumor. Study met the prespecified criteria of target intra-tumor drug concentration of ≥ 25nM in contrast enhancing tissue in more than 50% of the patients in the 120mg cohort (67.1 ± 42.7nM in 8/10 patients) and 240mg (328.7 ± 468.1nM in 10/10 patients) cohort. Serum MIC-1 fold-changes from baseline (FCB) approximately 24 hours after a single dose of KRT232 were higher in the 240mg cohort (9.1 ± 4.1-FCB) than the 120mg cohort (3.6 ± 2.0-FCB). CDKN1A (p21), a downstream marker of p53, was significantly upregulated in analyzed participants whose GBM was TP53 wildtype, but not in TP53 mutant GBM or control samples (GBM treated with standard of care). CONCLUSION KRT232 at both 120mg and 240mg achieves adequate tumor tissue penetration and affects downstream pathways in TP53 wildtype GBM. The study has moved to Alliance to complete a phase 1 study of radiation + KRT232 in patients with newly diagnosed MGMT unmethylated GBM.

BACKGROUND GBM genomic profiling relies on sequencing a limited tumor tissue sample, which is invasive and may underrepresent molecular heterogeneity. CSF, which can be obtained less invasively, is in intimate contact with tumor lesions and may better capture the full genomic profile of the tumor. However, datasets with contemporaneously collected CSF and tissue to support this claim have been lacking. To evaluate the performance of CSF NGS, we conducted a pilot study in patients with GBM undergoing a resection for suspected recurrence following first-line chemoradiotherapy. METHODS Paired CSF and tissue samples were sequenced using a hybrid capture-based NGS assay. Clinically meaningful variants were defined as those that are potentially targetable using off-label or clinical trial options, exhibit prognostic value, or may predict response or resistance to specific treatments. RESULTS Eighteen patients were enrolled, and 13 of 18 CSF samples (72.2%) were sequenced successfully. At least one variant was detected in all CSF samples analyzed. A median of 7 variants (range 1—67) was detected per sample across 54 genes. The median variant allele fraction was 0.6% (range 0.2—72.4%). Among 38 clinically meaningful genes, 102 variants were detected; 25 (24.5%) were detected in both tissue and CSF, while 60 (58.8%) were detectable solely in CSF. Hypermutation was detected by CSF in one patient. Of the 82 variants detected in this patient’s tumor, 15 (18.3%) were identified in both tissue and CSF, 15 (18.3%) were identified only in the tissue, and 52 (63.2%) were identified only in the CSF. CONCLUSIONS CSF NGS detects clinically meaningful variants at a substantial rate and frequently identifies mutations not detected by matched tissue NGS. These results suggest that CSF may be a suitable source material for tumor profiling, overcoming the limitations of tissue, and may also provide a more comprehensive tumor profile.

PURPOSE Differentiation of tumor progression (TP) from pseudoprogression (PsP) is a major unmet need in post-treatment glioblastoma (GBM). 18F-Fluciclovine is a synthetic amino acid PET radiotracer with higher uptake in tumor tissue vs. areas of treatment-related change. We investigated the value of 18F-Fluciclovine PET for differentiating PsP from TP independent from and in combination with multi-parametric MRI. METHODS We prospectively enrolled 30 patients with GBM with a new or enlarging contrast-enhancing lesion on MRI after chemoradiotherapy who were planned for surgical resection of the lesion. Patients underwent pre-operative 18F-Fluciclovine PET and multi-parametric MRI. Following surgery, the relative percentages of viable tumor and therapy-related changes observed in histopathology were quantified. Patients were categorized as TP if viable tumor represented ≥ 50% of the specimen, mixed TP if < 50% and > 10%, and PsP if ≤ 10%. RESULTS 18 patients had TP, 4 had mixed TP, and 8 PsP. Patients with TP/mixed TP had a significantly higher 40-50 minutes SUVmax (6.64 + 1.88 vs 4.11± 1.52, p=0.009) and an SUVmax cut-off of 4.66 provided 90% sensitivity and 83% specificity for differentiation of TP/mixed TP from PsP (AUC=0.856). A maximum cerebral blood volume (CBVmax) cut-off of 3.67 provided 90% sensitivity and 71% specificity for differentiation of TP/mixed TP from PsP (AUC=0.779). Combining a 40-50 minutes SUVmax cut-off of 4.662 and a relative CBVmax cut-off of 3.67 provided 100% sensitivity and 80% specificity for differentiating TP/mixed TP from PsP (AUC=0.95). The time activity curve patterns and time to peaks were not different between the groups. Normalization of PET parameters to normal brain parenchyma were not helpful to differentiate the groups due to variability in radiotracer uptake in normal brain between subjects. CONCLUSION 18F-Fluciclovine PET uptake can accurately differentiate PsP from TP in GBM patients, with even more accurate differentiation achieved when combined with MRI.

BACKGROUND We evaluated the combination of retifanlimab, INCAGN01876, and FSRT in patients with recurrent GBM. METHODS Phase II, single-center, 2 cohort study (A: single-arm non-surgical cohort; B, two-arm neoadjuvant/surgical cohort). Cohort A patients received pre-FSRT one-time doses of retifanlimab (500mg) and INCAGN01876 (300mg), FSRT (8 Gy x 3 fractions), and post-FSRT 28-day treatment cycles (retifanlimab, day 1; INCAGN01876, days 1, 15). Cohort B patients received pre-surgery doses of retifanlimab + INCAGN01876; subsequently, patients either went directly to resection (Sub-Arm 1) or to FSRT followed by resection (Sub-Arm 2). All patients resumed immunotherapy post-operatively. Primary endpoint: ORR in Cohort A. Data cut-off for this analysis was June 30, 2022. RESULTS Thirty-two evaluable patients: Cohort A, n=16; Cohort B, n=16 (Sub-Arm 1, n=8, Sub-Arm 2, n=8). Median follow-up time: Cohort A, 13.6 months; Cohort B, 8.8 months. Forty-four percent women, median age 64 (IQR, 55–65), 56% MGMT unmethylated. Most common grade 3/4 treatment-related AEs included cerebral edema (25%), lymphopenia (16%), and cognitive disturbance (13%). Efficacy in Cohort A: no objective responses observed, best response of stable disease achieved in 9/16 patients (56%), median PFS 3.9 months (95% CI 2.1 – 6.2 months), median OS 9.8 months (95% CI 8.3 months – not reached [NR]). Efficacy in Cohort B: median PFS NR, median OS 15.1 months (95% CI 8.5 months – NR). Median PFS and OS are longer in Cohort B Sub-Arm 2 compared to Cohort B Sub-Arm 1 (PFS, NR vs. 2.2 months, p = 0.009; OS, NR vs. 8.5 months, p=0.026). Results of tissue and blood-based immune correlative analyses will be presented. CONCLUSIONS The combination of retifanlimab, INCAGN01876, and FSRT is generally well-tolerated in patients with recurrent GBM when administered with or without surgical resection. Survival outcomes in the neoadjuvant cohort are encouraging, largely driven by patients that received neoadjuvant FSRT.

Background. Multi-omic data, i.e., clinical measures, radiomic, and genetic data, capture multi-faceted tumor characteristics, contributing to a comprehensive patient risk assessment. Here, we investigate the additive value and independent reproducibility of integrated diagnostics in prediction of overall survival (OS) in newly diagnosed, treatment-naïve, IDH-wildtype GBM patients, by combining conventional and deep learning methods.Methods. Conventional radiomics and deep learning features were extracted from pre-operative multi-parametric MRI of 516 GBM patients. SVM classifiers were trained on the discovery cohort (n=404) to categorize patient groups of high-risk (OSradiomics) was produced. Multivariate Cox-PH models were generated for the replication cohort, first based on clinical measures solely, and then by layering on radiomics and molecular information.Results. Evaluation of the high-risk and low-risk classifiers in the discovery/replication cohorts revealed AUCs of 0.78 (95%CI:0.70–0.85)/0.75 (95%CI:0.64–0.79) and 0.75 (95%CI: 0.65–0.84)/0.63 (95%CI: 0.52–0.71), respectively. Cox-PH modeling showed a concordance index of 0.65 (95%CI:0.6–0.7) for clinical data, 0.70 (95%CI:0.65–0.75) for clinical and radiomics, 0.72 (95%CI:0.68–0.77) for clinical, MGMT methylation, and radiomics, and 0.75 (95%CI:0.72–0.79) for the combination of all omics, i.e., clinical, MGMT methylation, radiomics, and genomics.Conclusions. This study signifies the value of integrated diagnostics for improved prediction of OS in GBM. Our multi-omic survival prediction tool is easily scalable and can be used for more effective clinical trial stratification.

BACKGROUND We have previously demonstrated that high baseline plasma cfDNA concentration is associated with poor survival in patients with newly diagnosed GBM. The mechanism of this association remains unknown. To explore whether differences in the immune landscape between high- vs. low-cfDNA patients may play a role in their divergent clinical outcomes, we phenotyped tumors from patients with high vs. low cfDNA using mass cytometry by time of flight (CyTOF). METHODS We performed CyTOF on frozen tumor infiltrate suspension from a pilot cohort of patients with previously untreated GBM with known baseline plasma cfDNA concentration (Bagley, Clin Cancer Res 2020). CyTOF was used to simultaneously measure expression of 39 molecules related to immune cell lineage, differentiation state, and function. Differences in immune cell infiltrates between high- and low-cfDNA patients were assessed using Mann-Whitney U tests. RESULTS Four patients with high cfDNA (median 57, range 33-90 ng/mL) were compared to six patients with low cfDNA (median 12, range 7-16 ng/mL). Immune cell infiltrates with increased adaptive cells (high monocytes and T cells, p=0.05) were present in high-cfDNA compared to low-cfDNA patients. While > 70% of the infiltrating T cells were exhausted in both groups, the pattern of exhaustion was significantly different in high- vs. low-cfDNA patients, with less CXCR5+CD69+ and more CXCR5-CD69- (p=0.008) progenitor exhausted T cells in cfDNA-high patients. CONCLUSIONS In this GBM pilot study, we demonstrated differences in the tumor immune infiltrate in patients with high vs. low baseline plasma cfDNA concentration. Preclinical studies will be needed to determine if this explains the association between high plasma cfDNA and poor outcomes previously observed in patients. Our results may have implications for the use of cfDNA concentration as a predictive biomarker for immunotherapy, as tumors with more intermediate progenitor (CXCR5-CD69-) exhausted T cells may respond better to PD-1 checkpoint blockade.

PURPOSE Decision making about the best course of treatment for glioblastoma patients becomes challenging when a new enhancing lesion appears in the vicinity of the surgical bed on follow-up MRI (after maximal safe tumor resection and chemoradiation), raising concerns for tumor progression (TP). Literature indicates 30-50% of these new lesions describe primarily treatment-related changes (TRC). We hypothesize that quantitative analysis of specific and sensitive features extracted from multi-parametric MRI (mpMRI) via machine learning (ML) techniques may yield non-invasive imaging signatures that distinguish TP from TRC and facilitate better treatment personalization. METHODS We have generated an ML model on a retrospective cohort of 58 subjects, and prospectively evaluated on an independent cohort of 58 previously unseen patients who underwent second resection for suspicious recurrence and had availability of advanced mpMRI (T1, T1-Gd, T2, T2-FLAIR, DTI, DSC). The features selected by our retrospective model, representing principal components analysis of intensity distributions, morphological, statistical, and texture descriptors, were extracted from the mpMRI of the prospective cohort. Integration of these features revealed signatures distinguishing between TP, mixed response, and TRC. Independently, a board-certified neuropathologist evaluated the resected tissue by blindly classifying it in the above three categories, based on mitotic figures, pseudopalisading necrosis, geographic necrosis, dystrophic calcification, vascular changes, and Ki67. RESULTS Tissues classified as TRC by the neuropathologist were associated with imaging phenotypes of lower angiogenesis (DSC-derived features), lower cellularity (DTI-derived features), and higher water concentration (T2, T2-FLAIR features). Our ML model characterized TP with 78% accuracy (sensitivity:86%, specificity:70%, AUC:0.80 (95%CI, 0.68-0.92)) and TRC with 81% accuracy (sensitivity:80%, specificity:81%, AUC:0.87 (95%CI, 0.72-1.00)). CONCLUSION Our proposed ML model reveals distinct non-invasive markers of TP and TRC, directly associated with histopathological changes in prospective glioblastoma patients. Reliable stratification of TP and TRC entities may help to noninvasively determine whether the course of treatment should change.

PURPOSE Multi-omics data integration captures tumor characteristics at multiple scales [i.e., microscopic (genomics and epigenetics), macroscopic (radiomics), clinical manifestation], provides a more comprehensive assessment of patient’s risk, and facilitates personalized therapies. In this work, we investigated the synergistic value of such multiple data sources for risk stratification and prediction of overall survival in IDH-wildtype glioblastoma tumors. METHODS Quantitative conventional and deep radiomics were extracted from pre-operative multi-parametric structural MRI (T1, T1Gd, T2, T2-FLAIR) of 501 patients with newly diagnosed glioblastoma. 389/501 and 112/501 patients formed our discovery and replication cohorts, respectively. Conventional radiomics were extracted from CaPTk, and deep radiomics from a pre-trained VGG-19 model. Multivariate SVM classification was performed on the discovery cohort to stratify patients in high, medium, and low-risk groups, using recursive feature elimination and 5-fold cross-validation. This model was independently tested on the replication cohort, and a radiomic-based survival prediction index (SPIradiomics) was calculated for each patient. Multi-stage integration of omics data, i.e., clinical (age, gender, extent of resection (EOR)), SPIradiomics, epigenetics (MGMT promoter methylation), and genomics (27 clinically relevant gene mutations via next-generation sequencing (NGS)), was performed using multivariate Cox proportional hazards (Cox-PH) model for stratification of the risk in the replication cohort. RESULTS Cox-PH modeling resulted in a concordance index (c-index) of 0.65 (95% CI:0.6–0.7) for clinical data, 0.67 (95% CI:0.62–0.72) for clinical and epigenetics, 0.70 (95% CI:0.65–0.75) for clinical and radiomics, 0.72 (95% CI:0.68–0.77) for clinical, epigenetics, and radiomics, and 0.75 (95% CI:0.71 – 0.78) for the multi-omics combination of all data; highlighting the added value of each layer of information in prediction of the patient’s risk. CONCLUSION Our results reinforce the synergistic value of integrated diagnostic methods for improving risk assessment of patients with glioblastoma that may pave the path towards a more personalized treatment planning.

BACKGROUND CAN-2409 is a replication-deficient adenovirus that delivers HSV thymidine kinase to cancer cells, resulting in local conversion of orally administered valacyclovir into a toxic metabolite. Previously, a phase 1b/2 clinical trial of CAN-2409 combined with standard-of-care (SOC) demonstrated safety and improved survival in HGG patients. Addition of CAN-2409 to nivolumab has the potential to activate locally recruited lymphocytes and teach them to recognize tumor neoantigens, changing the ‘cold’ immunosuppressive tumor microenvironment, and synergizing with the activity mediated by immune checkpoint inhibitors. This notion is supported by preclinical experiments showing that the combination of CAN-2409 with anti-PD1 therapy was more effective than either treatment alone. METHODS This ongoing phase 1 clinical trial evaluates safety and initial efficacy of CAN-2409 combined with nivolumab and SOC in newly diagnosed HGG. CAN-2409 is injected during neurosurgery into the tumor bed, followed by 14-days of valacyclovir. Radiation starts within 8 (+/-4) days of surgery. Temozolomide is administered to MGMT-methylation positive patients only. Nivolumab is given every 2 weeks, up to 52-weeks. Deep immune profiling studies are ongoing and initial results will be available shortly. RESULTS From February 2019 to March 2021, 41 patients were enrolled and 35 were evaluable for safety from the combination of CAN-2409, nivolumab and SOC: 24 male and 11 female; 34 glioblastoma, 1 diffuse astrocytoma; 33 IDH-wildtype, 2 IDH-mutant; 15 MGMT-methylated, 20 unmethylated. Median age was 62-years (range 28-79), median KPS 90 (range 80-100). With 13 months median follow-up, no unexpected serious adverse events were observed, and 23 patients are still alive. The most frequent possibly related adverse events (>10%) were nausea, fatigue, fever, headache, and increased ALT. CONCLUSIONS The combination of CAN-2409 + nivolumab + SOC was well tolerated. Clinical follow-up and extensive biomarker analyses will provide a better understanding of the therapeutic potential of this approach.

BACKGROUND Preclinical studies have demonstrated that IDH1-mutant (IDHmt) gliomas harbor a BRCAness phenotype with a defect in homologous recombination that confers PARP inhibitor sensitivity. Pamiparib (BeiGene BGB-290) is an effective PARP-trapping PARP inhibitor with demonstrated favorable brain penetration in animal models. METHODS ABTC 1801 is a study examining the safety, pharmacokinetics, and efficacy of the combination of pamiparib with low dose metronomic temozolomide in recurrent IDHmt gliomas. The Phase I component utilized a 3 + 3 design with a target DLT rate ≤ 33%. Pamiparib dose was 60 mg BID and temozolomide dose 20 mg daily, with dose de-escalation levels for anticipated hematological toxicity. RESULTS Seven patients were enrolled on the Phase I portion at dose level 1; one patient was replaced for inadequate dosing secondary to non-compliance. All patients had prior radiotherapy and temozolomide; 4/7 had received multiple lines of alkylator therapy including nitrosoureas. Median age was 45, KPS 90, and number of prior relapses 3. Four patients had anaplastic astrocytoma, 2 anaplastic oligodendroglioma, and 1 glioblastoma. One of 6 patients (16.7%) experienced DLT during the first cycle (grade 3 neutropenia and thrombocytopenia). Two additional patients had grade 2 neutropenia. Two patients remain on study treatment at 12+ and 10+ months, while a third progressed at 10.1 months (PFS-6 43%). Tumor tissue was collected from two patients in the surgical arm. In enhancing and non-enhancing tumors, the mean unbound pamiparib concentrations were 198 and 160 nmol/L (or nmol/kg), respectively, which were > 20-fold the in vitro IC50 for PARP inhibition; mean unbound tumor-to-plasma ratios were 0.65 and 0.38. CONCLUSIONS Phase I results support pamiparib 60 mg BID with temozolomide 20 mg daily as the dosages for the Phase II study. Preliminary Phase 0 data suggest that pamiparib likely achieves sufficient pharmacologically active concentrations in both enhancing and non-enhancing brain tumors.