Faculty Bibliography

H3 K27M-mutant gliomas often manifest as midline gliomas, have a dismal prognosis, and have no established or effective treatments at recurrence. ONC201 is the first clinical bitopic DRD2 antagonist/ClpP agonist and is under evaluation in Phase II trials for gliomas and other cancers. We previously reported in vitro studies suggesting dysregulated dopamine receptor expression and enhanced ONC201 sensitivity among H3 K27Mmutant gliomas. Following these observations, adults with midline H3 K27M-mutant glioma patients were enrolled to a dedicated Phase II clinical trial (NCT03295396), a multi-arm Phase II trial (NCT0252569), and expanded access protocols under the Sponsor's IND. An integrated radiographic analysis with an objective response rate primary endpoint in patients who received ONC201 monotherapy with confirmed H3 K27M-mutant glioma (not primarily in the pons or spinal cord and without leptomeningeal spread) that was progressive and measurable disease by RANO criteria, >90 days from completion of prior radiation, and had KPS >60. As of December 15, 2018, 15 patients have received single agent ONC201 who meet these criteria (n=9 NCT03295396; n=5 NCT0252569; n=1 expanded access). ONC201 was orally administered at 625 mg weekly, except for one patient dosed once every 3 weeks. As midline gliomas can exhibit a mixture of contrast-enhancing and non-contrast-enhancing disease, objective response was assessed by blinded independent central review using RANO-HGG and RANO-LGG criteria for each patient. Best response to date by RANO-HGG criteria is at least 27%: 1 CR, 3 PR, 7 SD, and 4 PD; by RANO-LGG is at least 36%: 1 CR, 1 PR, 3 minor response (MR), 4 SD, 5 PD, 1 unevaluable. By RANO-HGG, median onset of response is 2.6 months (range 1.3-3.4); median duration of response has not been reached with a median follow-up of 7.7 months (range 1.8-29.8). Updated radiographic response, pharmacodynamics, safety, and other clinical outcomes will be reported

PURPOSE/OBJECTIVE: Dose constraints for re-irradiation of recurrent primary brain tumors are not well-established, especially for treatment volumes too large for stereotactic radiotherapy. This prospective trial was performed to test dose constraints for conventionally-fractionated re-irradiation of recurrent primary brain tumors MATERIALS/METHODS: A singleinstitution, prospective trial of 21 adults with recurrent brain tumors was performed. Electronic dosimetry records from the first course of radiation (RT1) were obtained and deformed onto the simulation CT for the second course of radiation (RT2). Treatment plans for RT2 were developed that met protocol-assigned dose constraints for RT2 alone and the composite dose of RT1+RT2. Dose constraints were also based on histology and interval since RT1. The primary endpoint was the rate of symptomatic brain necrosis after RT2.
RESULT(S): Twenty one adults enrolled from March 2017 to May 2018. Twelve had glioblastoma, four had oligodendroglioma, two had anaplastic astrocytoma, and one each had choroid plexus papilloma, hemangiopericytoma, and pleomorphic xanthroastrocytoma (PXA). Twenty patients were treated with VMAT and one was treated with proton CSI. Median RT1-RT2 interval was 45 months (range, 9-141 months). Median RT2 dose was 42.8 Gy (range, 17.5-60 Gy). Median PTV volume was 208 cc (range, 7-1537 cc). Median imaging followup was 9 months (range, 1-20 months). Two months after RT2, the patient with PXA developed a trapped temporal horn adjacent to the RT2 treatment volume; pathology from emergent resection revealed necrotic brain tissue. The patient recovered fully and lived another 18 months until dying of disease progression. No other patient developed symptomatic radionecrosis. Median overall survival from RT2 for all patients was 11 months (range, 3-20 months).
CONCLUSION(S): Re-irradiation can be performed with conventionally fractionated schemes. Given the low rate of symptomatic radionecrosis, the dose constraints described here are a starting point for future studies of conventionally fractionated re-irradiation

BACKGROUND: While glioblastoma (GBM) is more prevalent in males, studies show that females with GBM tend to have longer overall survival (OS) than males. Pretreatment neutrophil to lymphocyte ratio (NLR) has also proven to be prognostic in GBM, with lower NLR having favorable outcomes. This secondary analysis of a prospective randomized trial of proton vs. photon intensity modulated radiotherapy aims to explore the interaction of gender and NLR on GBM outcomes.
METHOD(S): Analysis was performed on the full patient population. Kaplan-Meier methods estimated OS with censoring at last follow-up for those who were alive. Univariate (UVA) and multivariate (MVA)Cox proportional hazards models assessed predictors of OS.
RESULT(S): Of 90 patients, 77 were included (43 males; 34 females) with median age of 52 years (range: 26-82 years). Median OS was longer for females than males (30.7 vs 18.2 months, p=0.004). On UVA, patients with NLR below median value (NLR= 3.1) tended to have longer OS than those above median, though not meeting statistical significance (23.1 vs. 17.9 months, p=0.051). Difference in OS was statistically significant in females (OS 36.4 months NLR >median vs. 16.7 months NLR< median, p=0.002), but not in males (OS 17.8 months NLR >median vs. 19.1 NLR< median, p=0.95). MVA analysis was consistent, with female gender predicting reduced hazard ratio (HR) (0.28, p=0.034) and females with below median NLR showing a reduced HR over those with above median (0.28, p=0.005). Again, males did not benefit (HR 0.90, p=0.77).
CONCLUSION(S): Consistent with prior publications, females and all patients with lower pre-treatment NLR with newly diagnosed GBM had longer OS. However, combining these two factors revealed that the benefits from lower pre-treatment NLR were conferred only in females with no impact on males. This different impact of NLR between genders may suggest innate immune differences in gender during response to malignancy

BACKGROUND AND PURPOSE/OBJECTIVE:microRNAs are small noncoding RNAs that play important roles in cancer regulation. In this study, we investigated the expression, functional effects and mechanisms of action of microRNA-29a (miR-29a) in glioblastoma (GBM). METHODS:miR-29a expression levels in GBM cells, stem cells (GSCs) and human tumors as well as normal astrocytes and normal brain were measured by quantitative PCR. miR-29a targets were uncovered by target prediction algorithms, and verified by immunoblotting and 3' UTR reporter assays. The effects of miR-29a on cell proliferation, death, migration and invasion were assessed with cell counting, Annexin V-PE/7AAD flow cytometry, scratch assay and transwell assay, respectively. Orthotopic xenografts were used to determine the effects of miR-29a on tumor growth. RESULTS:Mir-29a was downregulated in human GBM specimens, GSCs and GBM cell lines. Exogenous expression of miR-29a inhibited GSC and GBM cell growth and induced apoptosis. miR-29a also inhibited GBM cell migration and invasion. PDGFC and PDGFA were uncovered and validated as direct targets of miR-29a in GBM. miR-29a downregulated PDGFC and PDGFA expressions at the transcriptional and translational levels. PDGFC and PDGFA expressions in GBM tumors, GSCs, and GBM established cell lines were higher than in normal brain and human astrocytes. Mir-29a expression inhibited orthotopic GBM xenograft growth. CONCLUSIONS:miR-29a is a tumor suppressor miRNA in GBM, where it inhibits cancer stem cells and tumor growth by regulating the PDGF pathway.

Ionizing radiation (IR) and chemotherapy are standard-of-care treatments for glioblastoma (GBM) patients and both result in DNA damage, however, the clinical efficacy is limited due to therapeutic resistance. We identified a mechanism of such resistance mediated by phosphorylation of PTEN on tyrosine 240 (pY240-PTEN) by FGFR2. pY240-PTEN is rapidly elevated and bound to chromatin through interaction with Ki-67 in response to IR treatment and facilitates the recruitment of RAD51 to promote DNA repair. Blocking Y240 phosphorylation confers radiation sensitivity to tumors and extends survival in GBM preclinical models. Y240F-Pten knockin mice showed radiation sensitivity. These results suggest that FGFR-mediated pY240-PTEN is a key mechanism of radiation resistance and is an actionable target for improving radiotherapy efficacy.

Background/UNASSIGNED:Virtually all low-grade gliomas (LGGs) will progress to high-grade gliomas (HGGs), including glioblastoma, the most common malignant primary brain tumor in adults. A key regulator of immunosuppression, fibrinogen-like protein 2 (FGL2), may play an important role in the malignant transformation of LGG to HGG. We sought to determine the mechanism of FGL2 on tumor progression and to show that inhibiting FGL2 expression had a therapeutic effect. Methods/UNASSIGNED:We analyzed human gliomas that had progressed from low- to high-grade for FGL2 expression. We modeled FGL2 overexpression in an immunocompetent genetically engineered mouse model to determine its effect on tumor progression. Tumors and their associated microenvironments were analyzed for their immune cell infiltration. Mice were treated with an FGL2 antibody to determine a therapeutic effect. Statistical tests were two-sided. Results/UNASSIGNED:We identified increased expression of FGL2 in surgically resected tumors that progressed from low to high grade (n = 10). The Cancer Genome Atlas data showed that LGG cases with overexpression of FGL2 (n = 195) had statistically significantly shorter survival (median = 62.9 months) compared with cases with low expression (n = 325, median = 94.4 months, P < .001). In a murine glioma model, HGGs induced with FGL2 exhibited a mesenchymal phenotype and increased CD4+ forkhead box P3 (FoxP3)+ Treg cells, implicating immunosuppression as a mechanism for tumor progression. Macrophages in these tumors were skewed toward the immunosuppressive M2 phenotype. Depletion of Treg cells with anti-FGL2 statistically significantly prolonged survival in mice compared with controls (n = 11 per group, median survival = 90 days vs 62 days, P = .004), shifted the phenotype from mesenchymal HGG to proneural LGG, and decreased M2 macrophage skewing. Conclusions/UNASSIGNED:FGL2 facilitates glioma progression from low to high grade. Suppressing FGL2 expression holds therapeutic promise for halting malignant transformation in glioma.

Cancer stem cells (CSCs) are a heterogeneous and dynamic self-renewing population that stands at the top of tumor cellular hierarchy and contribute to tumor recurrence and therapeutic resistance. As methods of CSC isolation and functional interrogation advance, there is a need for a reliable and accessible quantitative approach to assess heterogeneity and state transition dynamics in CSCs. We developed a high-throughput automated single cell imaging analysis (HASCIA) approach for the quantitative assessment of protein expression with single-cell resolution and applied the method to investigate spatiotemporal factors that influence CSC state transition using glioblastoma (GBM) CSCs (GSCs) as a model system. We were able to validate the quantitative nature of this approach through comparison of the protein expression levels determined by HASCIA to those determined by immunoblotting. A virtue of HASCIA was exemplified by detection of a subpopulation of SOX2-low cells, which expanded in fraction size during state transition. HASCIA also revealed that GSCs were committed to loose stem cell state at an earlier time point than the average SOX2 level decreased. Functional assessment of stem cell frequency in combination with the quantification of SOX2 expression by HASCIA defined a stable cutoff of SOX2 expression level for stem cell state. We also developed an approach to assess local cell density and found that denser monolayer areas possess higher average levels of SOX2, higher cell diversity, and a presence of a sub-population of slowly proliferating SOX2-low GSCs. HASCIA is an open source software that facilitates understanding the dynamics of heterogeneous cell population such as that of GSCs and their progeny. It is a powerful and easy-to-use image analysis and statistical analysis tool available at https://hascia.lerner.ccf.org. © 2019 International Society for Advancement of Cytometry.