Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.
Use of Community Based Participatory Research to Design Interventions for Healthy Lifestyle in an Alternative Learning Environment
Introduction/Objectives Childhood obesity develops as the result of the interplay between individual and environmental factors. Community based participatory research (CBPR) is an effective tool for improving health of communities. There is limited research on CBPR for facilitating healthy lifestyle in community schools with an alternative learning environment. The objective of the study was to explore student and staff perspectives via focus groups on barriers and facilitators for healthy eating and physical activity in a community school with alternative learning environment and to prioritize, design, and implement suggested interventions to improve healthy lifestyle. Methods We conducted qualitative research through 8 focus groups of middle and high school students (n = 40) and 2 focus groups of school staff (n = 8). The school community and research team subsequently identified and implemented interventions for facilitating healthy lifestyle in students within the school environment. Results Barriers identified for healthy lifestyle included lack of motivation, lack of healthy food options at school, inadequate knowledge about healthy lifestyle and insufficient opportunities for physical activity. Facilitators for healthy lifestyle were support and motivation from mentors and knowledge about healthy nutrition. Key strategies implemented were addition of healthier food options, educational materials for healthy eating, creation of a walk path, standing desks in classrooms and additional equipment in the school gymnasium. Conclusions Formative feedback from students and staff was helpful in the implementation of strategies for facilitating healthy lifestyle among students within a community school with an alternative learning environment.
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)
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Study Design: Retrospective cohort study. Objective: Studies investigating the impact of interbody subsidence in ACDF suggest a correlation between subsidence and worse radiographic and patient-reported outcomes. The purpose of this study was to assess whether allograft subsidence assessed on CT is associated with worse cervical alignment. Methods: We performed a retrospective review of a prospective cohort of patients undergoing 1 to 3 level ACDF. Cervical alignment was assessed on standing radiographs performed preoperatively, less than 2 months postoperatively, and greater than 6 months postoperatively. Allograft subsidence was assessed on CT scan performed at least 6 months postoperatively. Patients with at least 1 level demonstrating greater than 4mm of cage subsidence were classified as severe subsidence. Student’s t-test was used to compare all means between groups. Results: We identified 66 patients for inclusion, including 56 patients with non-severe subsidence and 10 patients with severe subsidence. For the entire cohort, there was a significant increase in C2-7 Lordosis (p = 0.005) and Segmental Lordosis (p < 0.00 001) from preoperative to early postoperative. On comparison of severely and non-severely subsided levels, severely subsided levels demonstrated a significantly greater loss of segmental lordosis from early to mid-term follow-up than non-severely subsided levels (-4.89 versus -2.59 degrees, p < 0.0001), manifesting as a significantly lower segmental lordosis at >6 months postoperative (0.54 versus 3.82 degrees, p < 0.00 001). There were no significant differences in global cervical alignment parameters between patients with severe and non-severe subsidence. Conclusions: Severe subsidence is associated with a significant increase in loss of segmental lordosis, but has minimal effect on global cervical alignment parameters.
Characterizing benefit from temozolomide in MGMT promoter unmethylated and methylated glioblastoma: a systematic review and meta-analysis
Abstract Background The current standard of care for the management of patients with newly diagnosed glioblastoma (GBM) includes maximal safe resection followed by radiotherapy (RT) with concurrent and adjuvant temozolomide (TMZ). While it is well established that TMZ has better efficacy in patients with MGMT promoter methylation, it remains an area of debate whether TMZ should be omitted when treating GBM patients with unmethylated MGMT. Methods We conducted a systematic review and meta-analysis to provide separate estimates of median overall survival (OS) and progression-free survival (PFS) for patients with methylated and unmethylated GBM treated with RT with or without TMZ. We searched multiple databases from inception to January 13, 2020. Results The median OS for patients with unmethylated GBM treated with RT/TMZ pooled from 5 phase III studies (N = 655) was 14.11 months (95% confidence interval [CI], 13.18–15.04) with a median PFS of 4.99 months (95% CI, 4.25–5.72). In contrast, the median OS for patients with methylated GBM pooled from 6 studies (N = 753) was 24.59 months (95% CI, 22.19–26.99) with a median PFS pooled from 7 studies (N = 805) of 9.51 months (95% CI, 7.41–11.61). There is a paucity of prospective data pertaining to OS/PFS in unmethylated patients treated with RT only and therefore a direct comparison was not possible. Conclusions This meta-analysis provides estimates of survival for patients with MGMT methylated or unmethylated GBM treated with RT/TMZ. Further research is needed to delineate whether TMZ should be withheld for patients with unmethylated GBM outside of the setting of clinical trials.
Abstract 5707: A standard operating procedure for the interpretation of oncogenicity/pathogenicity of somatic mutations
Abstract Somatic variants in cancer-relevant genes are interpreted from multiple partially overlapping perspectives. When considered in discovery and translational research endeavors, it is important to determine if a particular variant observed in a gene of interest is oncogenic/pathogenic or not, as such knowledge provides the foundation on which targeted cancer treatment research is based. In contrast, clinical applications are dominated by diagnostic, prognostic, or therapeutic interpretations which in part also depends on underlying variant oncogenicity/pathogenicity. The Association for Molecular Pathology, the American Society of Clinical Oncology, and the College of American Pathologists (AMP/ASCO/CAP) have published structured somatic variant clinical interpretation guidelines which specifically address diagnostic, prognostic, and therapeutic implications. These guidelines have been well-received by the oncology community. Many variant knowledgebases, clinical laboratories/centers have adopted or are in the process of adopting these guidelines. The AMP/ASCO/CAP guidelines also describe different data types which are used to determine oncogenicity/pathogenicity of a variant, such as: population frequency, functional data, computational predictions, segregation, and somatic frequency. A second collaborative effort created the European Society for Medical Oncology (ESMO) Scale for Clinical Actionability of molecular Targets to provide a harmonized vocabulary that provides an evidence-based ranking system of molecular targets that supports their value as clinical targets. However, neither of these clinical guideline systems provide systematic and comprehensive procedures for aggregating population frequency, functional data, computational predictions, segregation, and somatic frequency to consistently interpret variant oncogenicity/pathogenicity, as has been published in the ACMG/AMP guidelines for interpretation of pathogenicity of germline variants. In order to address this unmet need for somatic variant oncogenicity/pathogenicity interpretation procedures, the Variant Interpretation for Cancer Consortium (VICC, a GA4GH driver project) Knowledge Curation and Interpretation Standards (KCIS) working group (WG) has developed a Standard Operating Procedure (SOP) with contributions from members of ClinGen Somatic Clinical Domain WG, and ClinGen Somatic/Germline variant curation WG using an approach similar to the ACMG/AMP germline pathogenicity guidelines to categorize evidence of oncogenicity/pathogenicity as very strong, strong, moderate or supporting. This SOP enables consistent and comprehensive assessment of oncogenicity/pathogenicity of somatic variants and latest version of an SOP can be found at https://cancervariants.org/wg/kcis/. Citation Format: Peter Horak, Malachi Griffith, Arpad Danos, Beth A. Pitel, Subha Madhavan, Xuelu Liu, Jennifer Lee, Gordana Raca, Shirley Li, Alex H. Wagner, Shashikant Kulkarni, Obi L. Griffith, Debyani Chakravarty, Dmitriy Sonkin. A standard operating procedure for the interpretation of oncogenicity/pathogenicity of somatic mutations [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 5707.
Evaluation of Vasopressor Exposure and Mortality in Patients With Septic Shock*
Objectives: The objectives of this study were to: 1) determine the association between vasopressor dosing intensity during the first 6 hours and first 24 hours after the onset of septic shock and 30-day in-hospital mortality; 2) determine whether the effect of vasopressor dosing intensity varies by fluid resuscitation volume; and 3) determine whether the effect of vasopressor dosing intensity varies by dosing titration pattern. Design: Multicenter prospective cohort study between September 2017 and February 2018. Vasopressor dosing intensity was defined as the total vasopressor dose infused across all vasopressors in norepinephrine equivalents. Setting: Thirty-three hospital sites in the United States ( n = 32) and Jordan ( n = 1). Patients: Consecutive adults requiring admission to the ICU with septic shock treated with greater than or equal to 1 vasopressor within 24 hours of shock onset. Interventions: None. Measurements and Main Results: Out of 1,639 patients screened, 616 were included. Norepinephrine (93%) was the most common vasopressor. Patients received a median of 3,400 mL (interquartile range, 1,851–5,338 mL) during the 24 hours after shock diagnosis. The median vasopressor dosing intensity during the first 24 hours of shock onset was 8.5 μg/min norepinephrine equivalents (3.4–18.1 μg/min norepinephrine equivalents). In the first 6 hours, increasing vasopressor dosing intensity was associated with increased odds ratio of 30-day in-hospital mortality, with the strength of association dependent on concomitant fluid administration. Over the entire 24 hour period, every 10 μg/min increase in vasopressor dosing intensity was associated with an increased risk of 30-day mortality (adjusted odds ratio, 1.33; 95% CI, 1.16–1.53), and this association did not vary with the amount of fluid administration. Compared to an early high/late low vasopressor dosing strategy, an early low/late high or sustained high vasopressor dosing strategy was associated with higher mortality. Conclusions: Increasing vasopressor dosing intensity during the first 24 hours after septic shock was associated with increased mortality. This association varied with the amount of early fluid administration and the timing of vasopressor titration.
Mendelian randomization analyses suggest a role for cholesterol in the development of endometrial cancer
Abstract Blood lipids have been associated with the development of a range of cancers, including breast, lung and colorectal cancer. For endometrial cancer, observational studies have reported inconsistent associations between blood lipids and cancer risk. To reduce biases from unmeasured confounding, we performed a bidirectional, two‐sample Mendelian randomization analysis to investigate the relationship between levels of three blood lipids (low‐density lipoprotein [LDL] and high‐density lipoprotein [HDL] cholesterol, and triglycerides) and endometrial cancer risk. Genetic variants associated with each of these blood lipid levels ( P < 5 × 10 −8 ) were identified as instrumental variables, and assessed using genome‐wide association study data from the Endometrial Cancer Association Consortium (12 906 cases and 108 979 controls) and the Global Lipids Genetic Consortium (n = 188 578). Mendelian randomization analyses found genetically raised LDL cholesterol levels to be associated with lower risks of endometrial cancer of all histologies combined, and of endometrioid and non‐endometrioid subtypes. Conversely, higher genetically predicted HDL cholesterol levels were associated with increased risk of non‐endometrioid endometrial cancer. After accounting for the potential confounding role of obesity (as measured by genetic variants associated with body mass index), the association between genetically predicted increased LDL cholesterol levels and lower endometrial cancer risk remained significant, especially for non‐endometrioid endometrial cancer. There was no evidence to support a role for triglycerides in endometrial cancer development. Our study supports a role for LDL and HDL cholesterol in the development of non‐endometrioid endometrial cancer. Further studies are required to understand the mechanisms underlying these findings.
Structural MRI Analysis of Chronic Pain Patients Following Interdisciplinary Treatment Shows Changes in Brain Volume and Opiate-Dependent Reorganization of the Amygdala and Hippocampus
Abstract Objective The present study examined pre- to post-treatment changes in volumes for brain structures known to be associated with pain processing (thalamus, caudate, putamen, pallidum, hippocampus, amygdala, and accumbens) following an interdisciplinary pain management program. Design Twenty-one patients participating in a four-week interdisciplinary pain management program completed the study. The program consisted of individual and group therapies with the following disciplines: physical therapy, occupational therapy, pain psychology, biofeedback/relaxation training, nursing lectures, and medical management. All patients underwent functional magnetic resonance imaging of the brain before the start and at completion of the program. They also completed standard outcome measures assessing pain, symptoms of central sensitization, disability, mood, coping, pain acceptance, and impressions of change. Results Our results showed a significant increase in total brain volume, as well as increased volumes in the thalamus, hippocampus, and amygdala. As expected, we also found significant improvements in our standard outcome measures. The majority of patients rated themselves as much or very much improved. The increase in volume in the hippocampus was significantly associated with patient perceptions of change. However, the correlations were in the unexpected direction, such that greater increases in hippocampal volume were associated with perceptions of less improvement. Further exploratory analyses comparing patients by their opioid use status (use vs no use) showed differential program effects on volume increases in the hippocampus and amygdala. Conclusions These findings show that a four-week interdisciplinary pain management program resulted in changes in the brain, which adds objective findings further demonstrating program efficacy.
Multiplex TaqMan assays for a 7-gene prognostic immune response score to differentiate risk among women with ER-negative breast cancer.
10586 Background: Teschendorff et al. (Breast Cancer Res 2008) reported a 7-gene, immune response-enriched gene expression classifier using microarrays that was prognostic in women with ER-negative breast cancer. To facilitate further evaluation of the prognostic value of a classifier in archival fixed tumor tissue, we developed two multiplex TaqMan assays to quantify the expression levels of these genes, then trained and validated a 7-gene immune response score (IRS). Methods: RNA was extracted from FFPE sections of 185 ER-negative breast cancer patient samples from Mayo Clinic (n=81), Guy’s Hospital (n=50) and California Pacific Medical Center (CPMC) (n=27). Patients (N+ and N-) did not receive adjuvant therapy and were followed for a minimum of 5 years. Amplification of i) LY9, TNFRSF17, HLA-F, and IGLC2 and ii) XCL2, SPP1, C1QA and two reference genes NUP214 and PPIG were carried out in two multiplex RT-PCR TaqMan reactions and relative expression levels were determined using the DDCT method. The training set, comprised of the Mayo Clinic samples, was used to develop an IRS by using a summation of expression levels of the 7 genes. Association of the IRS with distant metastasis was assessed using a Cox proportional hazards model. The score was subsequently tested in a validation set (Guy’s + CPMC). Results: The amplification efficiency of each gene within the multiplex reactions was comparable to singleplex reactions. Duplicate amplifications showed excellent assay reproducibility and sensitivity (R2=0.98). The hazard ratio for a single standard deviation decrease in the IRS was 1.45 (95% CI 0.99-2.12, p=0.056) in the training set and 2.18 (1.32-3.61, p=0.002) in the validation set. ROC curves of continuous IRS to predict distant metastasis within 5 years had AUC of 0.74, 95% CI = 0.60-0.88. Conclusions: Robust multiplex TaqMan assays for FFPE tissue extracted RNA were developed for a 7-gene IRS. Increases in risk are associated with decreases in the IRS. Immune related gene expression clearly plays an important role in the spectrum of risk of ER-negative tumors. Additional signatures are in development that further support this conclusion.