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Abstract PURPOSE We investigated the long term impact of SRS and WBRT in two large prospective phase III trials. METHODS Patients with 1–4 BMs +/- resection were randomized to SRS or WBRT. Cognitive deterioration was a drop of >1 standard deviation from baseline in >2/6 cognitive measures (CM). Quality of life (QOL) scores were scored 0–100 point scale. CM and QOL scores were modeled using baseline adjusted Linear Mixed Models (LMM) with uncorrelated random intercept for subject and random slopes for time. Differences over time between groups and the effect of >2 cognitive scores with >2 SD change from baseline were assessed. RESULTS 88 patients were included with median follow up of 24 months. We observed decreasing CM over time (SRS: 4/6; WBRT: 5/6). Mean CM was significantly higher in SRS for Total recall and Delayed Recall at 3, 6, 9, 12 months. More patients in WBRT arm declined 1 SD in >1 and >2 CM at the 3, 6, 9, and 12 months. A 1 SD decline in >3 CM at 1 year was 21% SRS vs 47% WBRT (p=0.02). SRS had fewer patients with a 2 SD decline in >1 CM at every time point. SRS had fewer patients with a 2 SD decline at >2 and >3 CM. WBRT had lower QOL at 3 months, but switched to SRS having lower QOL at 24 months for PWB, EWB, FWB, FactG, BR, and FactBR (p< 0.05). A 2 SD decline in cognition decreased mean FWB by 6.4 units (95% CI: -11, -1.75; p=0.007) and decreased QOL by 5.1 units (95% CI: -7.7, -2.5; p< 0.001). CONCLUSIONS We report the first pooled prospective study demonstrating the long term outcomes of patients with BMs after cranial radiation. WBRT was associated with worse cognitive outcomes. Impaired cognition is associated with worse QOL.

Abstract BACKGROUND Glioblastoma (GBM) is an infiltrative, uniformly fatal brain tumor, treated with surgery, radiation, and Temozolomide (TMZ). Chemoradiation induces a senescent-like phenotype, which contributes to disease recurrence. We recently found that, radiated GBM cells can be eliminated by inhibition of BCL-XL. However, it remains unknown whether p53 is involved in this process and whether the elimination of senescent cells by BCL-XL inhibition could be augmented by MDM2 inhibition, a negative p53 regulator. METHODS p53-mutant (GBM6/GBM123) and p53-WT (GBM39/GBM76) human GBM cells were treated with 5Gy or TMZ following 48 hours of MDM2 inhibitor (AMG232) or vehicle treatment and maintained for seven days to establish a senescent-like phenotype. We evaluated the IC50 for BCL-XL inhibitor (A1331852) in radiated vs. non-radiated cells with or without MDM2 inhibitor pre-treatment. RESULTS MDM2-inhibitor treatment prior to radiation increased the expression of p21 and lead to increased cell death when combined with BCL-XL inhibition in p53-WT GBM cells. IC50 of BCL-XL inhibitor after prior MDM2 pretreatment and radiation in GBM76 was 4.5□M compared to 33.5□M, 18.1□M, and 32.3□M in vehicle without radiation, vehicle with radiation, and MDM2 inhibitor-alone treatment groups, respectively(p=0.0036). The IC50 of BCL-XL inhibitor without MDM2-inhibition in non-irradiated and radiated, as well as pre-administration of MDM2 inhibitor in non-irradiated and radiated GBM39 cells was 5618nM, 16117nM, 3926nM and 276.5nM, respectively(p=0.0003). Conversely, MDM2 co-inhibition with BCL-XL did not lead to increased rates of cell death in p53-mutant cell(p>0.05). CONCLUSION We previously identified that BCL-XL inhibition promotes cell death in senescent GBM cells. We build upon that work, demonstrating the increased rates of cell death can be augmented by MDM2 inhibition, but only in p53-WT cells. These findings highlight a novel therapeutic target for treating latent GBM tumors prior to recurrence, with the additional of MDM2 inhibition greatly increasing the efficacy of BCL-XL targeting agents.

Abstract BACKGROUND Glioblastoma is a fatal infiltrative primary brain tumor, and standard care includes maximal safe surgical resection followed by radiation and Temozolomide (TMZ). Therapy-resistant residual cells persist in a latent state a long time before inevitable recurrence. Conventional radiation and Temozolomide (TMZ) treatment cause oxidative stress and DNA damage resulting senescent-like state of cell-cycle arrest. However, increasing evidence demonstrates escaping senescence leads to tumor recurrence. Thus, the ablation of senescent tumor cells after chemoradiation may be an avenue to limit tumor recurrence. METHODS 100uM TMZ for 7days or 10-20Gy radiation (cesium gamma radiator) was used for senescence induction in human glioblastoma in vitro and confirmed by SA-Beta gal staining and PCR. Replication arrest assessed by automated quantification of cellular confluence (Thermo Scientific Series 8000 WJ Incubator). We evaluated the IC50 for several senolytics targeting multiple SCAPs, including Dasatinib, Quercetin, AMG-232, Fisetin, Onalespib, Navitoclax, and A1331852, and in senescent vs. proliferating cells. RESULTS Among the senolytic tested, the Bcl-XL inhibitors A1331852 and Navitoclax both shown senolytic effect by selectively killing radiated, senescent tumor cells at lower concentrations as compared to 0Gy treated non-senescent cells. Across 12 GBM cell lines, IC50 for senescent cells was 6–500 times lower than non-senescent GBM(p< 0.005). Such differential sensitivity to Bcl-XL inhibition after radiation has also observed by BCL-XL knockdown in radiated glioma. CONCLUSION These findings suggest the potential to harness radiation-induced biology to ablate surviving quiescent cells and demonstrate Bcl-XL dependency as a potential vulnerability of surviving tumor cells after exposure to chemoradiation.

Abstract Patients with glioblastoma (GBM) have a median survival of 15 months despite aggressive treatment. Immunosuppressive monocytes are heavily infiltrated in these tumors and in patients’ circulation. Treatment-related pseudo-progression confounds outcome assessment by MRI. Thus, there is a need for additional non-invasive methods to assess treatment response. Extracellular vesicles (EVs) contain tumor-specific microRNA (miRNA) cargo that could serve as a liquid biopsy to distinguish true progression from treatment-related pseudo-progression. We had found significant differences in plasma EVs molecular profile (i.e. miRNA signatures) between GBM patients and healthy donors. Our overall hypothesis is that these differences reflect the EVs cell of origin. Our goal in this project was to develop a fluorescent staining paradigm by flow cytometry to distinguish EVs from different cells in vitro and determine differences in EV miRNA expression profile between GBM and monocytic cell-derived EVs. Gleolan (5-ALA) is an FDA-approved orally available agent for fluorescence-guided resection of GBM tumors. It is metabolized to protoporphyrin IX (PpIX) in GBM cells but not in non-neoplastic cells and has been reported to aid in the detection of GBM-derived EVs by flow cytometry. However, distinguishing between GBM-derived EVs and EVs from other cells of origin has not been described. We co-cultured human GBM cells (dBT114 or dBT116) and CD14+ monocytes for 72 hours in the presence or absence of 5-ALA. EVs were isolated by ultracentrifugation and stained for CD11b (myeloid cell marker). ImageStream Imaging Flow Cytometry was performed showing clear differentiation between PpIX+ EVs from GBM cells and CD11b+ EVs from monocytes. Interestingly, a small number of double-positive EVs (presumably representing monocyte-derived EVs that had taken up PpIX after phagocytizing GBM cells) were also present. Taken together, we were able to optimize a technique to distinguish EVs originating from GBM and monocytes for further characterization by short non-coding RNA sequencing.

Abstract INTRODUCTION Gliomas are the most common and deadly adult brain cancers with a median survival time of less than 18 months. Many therapeutics have failed translating into effective therapies due to incomplete understanding of the disease and heterogeneity of tumors between patients. There is a need for methods that allow for continual access and analysis of glioma biomarkers. We sought to establish a reliable method for quantifying the onco-metabolite, D2HG. After optimizing this method, we used microdialysate and microperfusate to test our hypothesis that analysis of D2HG in response to therapy can indicate therapeutic efficacy sooner than current methods permit. METHODS Microdialysate and microperfusate were collected from the tumor centers and lateral ventricles of athymic nude mice implanted with IDH1 mutant PDX line GBM196. We performed microdialysis and microperfusion on consecutive days to determine baseline D2HG concentration. Following a five day period of TMZ treatment, we collected another round of microdialysis and microperfusion one week after treatment and again two weeks after treatment. We then used our optimized D2HG enzymatic assay to quantify the changes in D2HG in response to therapy. RESULTS Using our enzymatic D2HG assay, we were able to quantify D2HG as low as 30nM (30fmol/uL). We found a ~20% decrease in D2HG concentration in the tumor center and right lateral ventricle after 1 week of TMZ treatment (n=2), and ~36% decrease after 2 weeks of TMZ treatment (n=2). CONCLUSION Using a D2HG quantification assay to monitor changes in concentration of D2HG in microdialysate/microperfusate can predict sensitivity to therapy.

AbstractBackgroundPain affects 60% of the autosomal dominant polycystic kidney disease (ADPKD) population. Despite being an early and debilitating symptom, it is poorly characterized and management is suboptimal. This study aimed to develop an ADPKD-specific pain assessment tool (APAT) to facilitate pain research.MethodsFollowing a systematic review of PATs used in ADPKD studies and against international recommendations for pain trials, our multi-disciplinary team of clinical experts and patients constructed an ADPKD-pain conceptual framework of key pain evaluation themes. We compiled a new APAT covering domains prioritized within our framework using components of questionnaires validated in other chronic pain disorders. The APAT was administered longitudinally within a randomized high-water intake trial (NCT02933268) to ascertain feasibility and provide pilot data on ADPKD pain.ResultsThirty-nine ADPKD participants with chronic kidney disease Stages 1–4 provided 129 APAT responses. Each participant completed a median of 3 (range 1–10) assessments. Respondents’ mean ± standard deviation age was 47 ± 13 years; 59% (23) were female; and 69% (27) had enlarged kidneys with median time from diagnosis 14.2 (interquartile range 7.0–25.9) years. Pain (52%) and associated analgesic use (29%) were common. Pain severity was associated with increasing age [odds ratio (OR) = 1.07, P = 0.009], female gender (OR = 4.34, P = 0.018), estimated glomerular filtration rate <60 mL/min/1.73 m2 (OR = 5.45, P = 0.021) and hypertension (OR = 12.11, P = 0.007), but not with kidney size (P = 0.23). The APAT achieved good internal consistency (Cronbach’s alpha coefficient = 0.91) and test–retest reliability (domain intra-class correlation coefficients ranging from 0.62 to 0.90).ConclusionsThe APAT demonstrated good acceptability and reliability, and following further validation in a larger cohort could represent an invaluable tool for future ADPKD pain studies.

AbstractObjectiveMAPT mutations typically cause behavioral variant frontotemporal dementia with or without parkinsonism. Previous studies have shown that symptomatic MAPT mutation carriers have frontotemporal atrophy, yet studies have shown mixed results as to whether presymptomatic carriers have low gray matter volumes. To elucidate whether presymptomatic carriers have lower structural brain volumes within regions atrophied during the symptomatic phase, we studied a large cohort of MAPT mutation carriers using a voxelwise approach.MethodsWe studied 22 symptomatic carriers (age 54.7 ± 9.1, 13 female) and 43 presymptomatic carriers (age 39.2 ± 10.4, 21 female). Symptomatic carriers’ clinical syndromes included: behavioral variant frontotemporal dementia (18), an amnestic dementia syndrome (2), Parkinson’s disease (1), and mild cognitive impairment (1). We performed voxel‐based morphometry on T1 images and assessed brain volumetrics by clinical subgroup, age, and mutation subtype.ResultsSymptomatic carriers showed gray matter atrophy in bilateral frontotemporal cortex, insula, and striatum, and white matter atrophy in bilateral corpus callosum and uncinate fasciculus. Approximately 20% of presymptomatic carriers had low gray matter volumes in bilateral hippocampus, amygdala, and lateral temporal cortex. Within these regions, low gray matter volumes emerged in a subset of presymptomatic carriers as early as their thirties. Low white matter volumes arose infrequently among presymptomatic carriers.InterpretationA subset of presymptomatic MAPT mutation carriers showed low volumes in mesial temporal lobe, the region ubiquitously atrophied in all symptomatic carriers. With each decade of age, an increasing percentage of presymptomatic carriers showed low mesial temporal volume, suggestive of early neurodegeneration.

Abstract Background/Introduction The realisation that subjects with rheumatoid arthritis (RA) are at increased risk of cardiovascular disease (CVD) has led to a growing interest in risk factor control in such people, but whether this has influenced the management of dyslipidaemia and hypertension (HT) is uncertain. Purpose To describe differences in lipid and blood pressure (BP) levels among patients with RA from five world regions. Furthermore, to evaluate attainment of guideline recommended targets for lipid lowering and antihypertensive treatment. Methods The SUrvey of CVD Risk Factors in patients with RA (SURF-RA) was conducted at 53 centres in 19 countries from 2014 to 2019. Data including demographics, RA disease characteristics, CVD comorbidity, risk factors and use of preventive treatment was collected. HT was defined as self-reported HT, and/or measured BP >140/90 mmHg, and/or use of anti HT medication (a-HT). The treatment goal of a-HT was BP <140/90 mmHg. The 10-year risk of a fatal CVD event was calculated by the European CVD risk calculator, the Systematic COronary Risk Evaluation (SCORE), and was thereafter multiplied with 1.5 as recommended by the European League Against Rheumatism. Patients were classified in a high or very high CVD risk group according to the 2012 European Society of Cardiology guidelines, with low density lipoprotein cholesterol (LDL-c) goal at <2.6 and <1.8 mmol/L, respectively. Results In total, 14503 RA patients were included. The mean age was 59.8±13.6 years, most of whom (74%) were female. Nearly 2/3 of the patients were hypertensive. Use of a-HT in the total population differed substantially between the cohorts with limited use in West Europe and Latin America (17.4% and 24.8%), in contrast to North America and East Europe (46.8% and 57.0%). On average, half of those with HT were at the recommended BP goal. The lowest BP goal attainment was seen in Asia, West and East Europe (40.8–43.1%), and the highest in North America (63.5%). Overall 51.5% had an indication for lipid lowering therapy (LLT), and of these only 43.5% were taking LLT. Only 34.0% of patients with an indication for LLT were at recommended LDL-c goals. The proportion of RA patients on target for LDL-c varied greatly between regions, from 23.1% in East Europe to 51.0% in North America. The LDL-c goal attainment was higher in RA patients at high risk (45.1%) compared to those at very high risk of CVD (18.0%). Conclusion(s) This large international survey on RA patients revealed considerable geographical differences in CVD preventive treatment. Only one half of subjects were at blood pressure goals, and achievement of lipid goals was even poorer at one third of those eligible for treatment, which is lower than what is reported for subjects with coronary heart disease. We conclude that there is a substantial need for improvement in CVD preventive measures in RA patients. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Unrestricted research collaboration with Lilly

Abstract Background The cardiovascular disease (CVD) risk in patients with rheumatoid arthritis (RA) is comparable to that of patients with diabetes mellitus (DM). Although several studies have indicated high prevalence's of DM in RA patients, little is known about how this affects their CVD risk. Objectives To examine indications for, and use of antihypertensive treatment (a-HT) and lipid-lowering therapy (LLT) in RA patients with DM (RA-DM) and RA patients without DM (RAwoDM). Further, to compare the prevalence of various types of CVD across RA-DM and RAwoDM. Methods The cohort was derived from the SUrvey of cardiovascular disease Risk Factor in patients with Rheumatoid Arthritis (SURF-RA), which was performed in 53 centres/17 countries in 5 world regions (West and East Europe; North and Latin America; and Asia) from 2014 - 2019. Indication for a-HT was defined as: 1) systolic/diastolic blood pressure (BP) ≥140/90 mm Hg, 2) self-reported hypertension, and/or 3) current use of a-HT. Indication for LLT was defined according to ESC guidelines. CVD risk estimates (by SCORE) were multiplied by 1.5 according to EULAR recommendations. Target treatment targets for BP and lipids were defined according to ESC guidelines applicable at the time data were recorded. Results Presence of comorbid DM was available in 10 602 (73.1%) of the 14 503 RA patients included in SURF-RA, of whom 75 and 1262 patients reported DM type 1 and type 2, respectively (total 1337 patients, 12.6%). Although less often current smokers, RA-DM patients were more often previous smokers, male sex and had higher body mass index compared to RAwoDM (p<0.0001 for all). a-HT (84.7% vs 62.3%) and LLT (100% vs 47.2%) were more frequently indicated in RA-DM than in RAwoDM patients (p<0.0001 for both). RA-DM were more likely than RAwoDM to receive a-HT on indication (60.4% vs 57.6%, p<0.0001), while the difference in LLT use on indication was not significantly different (45.7% vs 42.5%, p=0.06). Moreover, RA-DM compared to RAwoDM patients had more often reached treatment goals when on a-HT (60.7% vs 54.1%, p<0.0001) and LLT (62.8% vs 48.9%, p<0.0001). Finally, the risk of all recorded established CVD (coronary heart disease, stroke, peripheral artery disease and atrial fibrillation) was increased by a factor of 2 to 3 in RA-DM compared to RAwoDM (Figure). Conclusion The effect of RA and comorbid DM on CVD risk appears to be additive. While CVD preventive medications are more often indicated in RA-DM than in RAwoDM patients, they are also more likely to receive such therapy and to reach CVD preventive treatment goals. The latter finding may be due to more developed CVD preventive care in DM compared to RA patients. Improved CVD preventive systems for patients with RA are warranted. CVD in RA patients with and without DM Funding Acknowledgement Type of funding source: Other. Main funding source(s): Lilly

Abstract Background Patients with rheumatoid arthritis (RA) are at high risk for cardiovascular disease (CVD). Purpose The aim of this survey was to evaluate updated information on CVD risk factors, comorbidities, RA disease characteristics, RA and CVD preventive medication in patient with RA. Methods The audit is termed SUrvey of cardiovascular disease Risk Factors in patients with Rheumatoid Arthritis (SURF-RA) and was performed in 53 centres/19 countries/5 world regions in 2014–2019. SURF-RA have been performed in patients with coronary heart disease, in primary care, and now in patients with stroke, SLE and antiphophlipid syndrome. The survey was approved by the Data Protection Officer (2017/7243) and a GDPR evaluation has been performed (10/10–2018). Results Among 14 503 patients with RA in West (n=8 493) and East (n=923) Europe, Latin (n=407) and North (n=4 030) America and Asia (n=650) the mean (SD) age was 59.9 (13.6) years, and 2/3 or more were female (table). RA disease duration was comparable across the world regions, ranging from 9.9 to 12.6 years. The prevalence of atherosclerotic CVD (ASCVD) was lowest in Latin America (2.5%) and highest in East Europe (21.4%), and this pattern was similar regarding familial premature CVD. The mean prevalence (% of each entity) of blood pressure above 140/90 mmHg was 5.3%, of low density lipoprotein cholesterol >2.5 mmol/L: 63.3%. Overall, 29% used antihypertensive medication, lowest in West Europe (17.4%) and highest in East Europe (57.0%), and 26.4% used lipid lowering agent(s), lowest in Asia (7.2%) and highest in North America (31.1%). Body mass index >30 kg/m2 was present in 26.6%, with the smallest waist circumference in Asia [mean (SD): 84.1 (13.6) cm] and highest in East Europe [92.5 (15.5) cm]. The proportion of current smokers was on average: 16.2%, lowest in Asia (7.8%) and highest in East Europe (28.5%). Conclusion The high prevalence of CVD risk factors and ASCVD in patients with RA across five world regions shows that there is still an unmet need for vigilance and improved implementation of preventive measures in this high CVD risk patient population. Funding Acknowledgement Type of funding source: Other. Main funding source(s): Lilly