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Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma–specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma–associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3+ T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3+ T cells in the direct vicinity of MHC class II–deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma.Significance:We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell–like immunosuppressive subset of LAG3+ T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints.See related commentary by Fisher and Oh, p. 342.This article is highlighted in the In This Issue feature, p. 327

Supplementary Methods, Supplementary Figures 1-19, Supplementary References

Background Emergency physicians (EPs) perform critical actions while operating with diagnostic uncertainty. Point-of-care ultrasound (POCUS) is useful in evaluation of dyspneic patients. In prior studies, POCUS is often performed by ultrasound (US) teams without patient care responsibilities. Objectives This study evaluates the effectiveness of POCUS in narrowing diagnostic uncertainty in dyspneic patients when performed by treating EPs vs. separate US teams. Methods This multicenter, prospective noninferiority cohort study investigated the effect of a POCUS performing team in patient encounters for dyspnea. Before-and-after surveys assessing medical decision-making were administered to attending physicians. Primary outcome was change in most likely diagnosis after POCUS. This was assessed for noninferiority between encounters where the primary or US team performed POCUS. Secondary outcomes included change in differential diagnosis, confidence in diagnosis, interventions considered, and image quality. Results There were 156 patient encounters analyzed. In the primary team group, most likely diagnosis changed in 40% (95% confidence interval 28–52%) of encounters vs. 32% (95% confidence interval 22–41%) in the US team group. This was noninferior using an a priori specified margin of 20% (p Conclusion POCUS performed by primary teams was noninferior to POCUS performed by US teams for changing the most likely diagnosis, and equivalent when considering mean reduction in number of diagnoses. POCUS performed by treating EPs reduces cognitive burden in dyspneic patients.

Undergraduate students participating in the UCLA Undergraduate Research Consortium for Functional Genomics (URCFG) have conducted a two-phased screen using RNA interference (RNAi) in combination with fluorescent reporter proteins to identify genes important for hematopoiesis in Drosophila. This screen disrupted the function of approximately 3500 genes and identified 137 candidate genes for which loss of function leads to observable changes in the hematopoietic development. Targeting RNAi to maturing, progenitor, and regulatory cell types identified key subsets that either limit or promote blood cell maturation. Bioinformatic analysis reveals gene enrichment in several previously uncharacterized areas, including RNA processing and export and vesicular trafficking. Lastly, the participation of students in this course-based undergraduate research experience (CURE) correlated with increased learning gains across several areas, as well as increased STEM retention, indicating that authentic, student-driven research in the form of a CURE represents an impactful and enriching pedagogical approach.

BACKGROUND: Shared decision making (SDM) is especially important for older adults with cancer given the risks of over- and undertreatment, uncertainty regarding benefits/harms worsened by research underrepresentation, and individual preferences. We aimed to adapt the Best Case/Worst Case (BC/WC) communication tool, which improves SDM in geriatric surgery, to geriatric oncology. METHODS: We conducted focus groups with 40 stakeholders (fourteen older adults with lung cancer, twelve caregivers, fourteen medical oncologists) to elicit perspectives on using the BC/WC tool for geriatric oncology and to identify components needing refinement. During each focus group, participants viewed a BC/WC demonstration video and answered questions modified from the Decision Aid Acceptability Scale. We analyzed transcripts using deductive and inductive thematic analyses. DISCUSSION: Participants believed that the BC/WC tool could help patients understand their cancer care choices, explore tradeoffs and picture potential outcomes, and deliberate about decisions based on their goals, preferences, and values. Oncologists also reported the tool could guide conversations to address points that may frequently be skipped (e.g., alternative options, treatment goals). Participant preferences varied widely regarding discussion of the worst-case scenario and desire for statistical information. CONCLUSION: The BC/WC tool is a promising strategy that may improve SDM in geriatric oncology and patient understanding of alternative options and treatment goals. Based on participant input, adaptations will include framing cancer care as a series of decisions, eliciting patient preferences and asking permission before offering the worst-case scenario, and selection of the two most relevant options to present if multiple exist.

BackgroundMaintenance of function during cancer treatment is important to older adults. Characteristics associated with pretreatment life-space mobility and changes during non-small cell lung cancer (NSCLC) treatment remain unknown.MethodsThis mixed methods cohort study recruited adults age ≥65 with advanced NSCLC starting palliative chemotherapy, immunotherapy, and/or targeted therapy from a Comprehensive Cancer Center, Veterans Affairs, and safety-net clinic. Patients completed geriatric assessments including Life-Space Assessment (LSA) pretreatment and at 1, 2, 4, and 6 months after treatment initiation. LSA scores range from 0 to 120 (greater mobility); LSA

There are approximately 350,000 out-of-hospital cardiac arrests and 200,000 in-hospital cardiac arrests annually in the United States, with survival rates of approximately 5% to 10% and 24%, respectively. The critical factors that have an impact on cardiac arrest survival include prompt recognition and activation of prehospital care, early cardiopulmonary resuscitation, and rapid defibrillation. Advanced life support protocols are continually refined to optimize intracardiac arrest management and improve survival with favorable neurologic outcome. This article focuses on current treatment recommendations for adult nontraumatic cardiac arrest, with emphasis on the latest evidence and controversies regarding intracardiac arrest management.

Background: Maintaining functional status is important to older adults with cancer, but data are limited on how systemic treatments affect functional status. We systematically reviewed changes in functional status during systemic cancer treatments and identified characteristics associated with functional decline and improvement. Methods: We searched PubMed, Embase, Web of Science, and Cochrane Register of Controlled Trials for articles examining characteristics associated with functional changes in older adults during systemic cancer treatment published in English between database inception and January 11, 2019 (PROSPERO CRD42019123125). Findings were summarized with descriptive statistics. Study characteristics between older adult–specific and non–older adult–specific studies were compared using the Fisher exact test. Results: We screened 15,244 titles/abstracts and 519 full texts. The final analysis included 44 studies, which enrolled >8,400 patients; 39% of studies focused on older adults (1 study enrolled adults aged ≥60 years, 10 enrolled adults aged ≥65 years, and 6 enrolled adults aged ≥70 years). Almost all studies (98%) used patient-reported outcomes to measure functional status; only 20% used physical performance tests. Reporting of functional change was heterogeneous, with 48% reporting change scores. Older adult–specific studies were more likely to analyze functional change dichotomously (29% vs 4%; P=.008). Functional decline ranged widely, from 6% to 90%. The most common patient characteristics associated with functional decline were older age (n=7 studies), worse performance status (n=4), progressive disease status (n=4), pain (n=4), anemia (n=4), and worse nutritional status (n=4). Twelve studies examined functional improvement and identified 11 unique associated characteristics. Conclusions: Functional decline is increasingly recognized as an important outcome in older adults with cancer, but definitions and analyses are heterogeneous, leading to a wide range of prevalence. To identify patients at highest risk of functional decline during systemic cancer treatments, trials need to routinely analyze functional outcomes and measure characteristics associated with decline (eg, nutrition).

Smoking is the number one risk factor for cancer mortality but only 15–20% of heavy smokers develop lung cancer. It would, therefore, be of great benefit to identify those at high risk early on so that preventative measures can be initiated. To investigate this, we evaluated the effects of smoking on inflammatory markers, innate and adaptive immune responses to bacterial and viral challenges and blood cell composition. We found that plasma samples from 30 heavy smokers (16 men and 14 women) had significantly higher CRP, fibrinogen, IL-6 and CEA levels than 36 non-smoking controls. Whole blood samples from smokers, incubated for 7 h at 37 °C in the absence of any exogenous stimuli, secreted significantly higher levels of IL-8 and a number of other cytokines/chemokines than non-smokers. When challenged for 7 h with E. coli, whole blood samples from smokers secreted significantly lower levels of many inflammatory cytokines/chemokines. However, when stimulated with HSV-1, significantly higher levels of both PGE2 and many cytokines/chemokines were secreted from smokers’ blood samples than from controls. In terms of blood cell composition, red blood cells, hematocrits, hemoglobin levels, MCV, MCH, MCHC, Pct and RDW levels were all elevated in smokers, in keeping with their compromised lung capacity. As well, total leukocytes were significantly higher, driven by increases in granulocytes and monocytes. In addition, smokers had lower NK cells and higher Tregs than controls, suggesting that smoking may reduce the ability to kill nascent tumor cells. Importantly, there was substantial person-to person variation amongst smokers with some showing markedly different values from controls and others showing normal levels of many parameters measured, indicating the former may be at significantly higher risk of developing lung cancer.

OBJECTIVES: Since lowering carbohydrate (CHO) intake has been hypothesized to reduce cancer risk, we investigated whether low-CHO diets could prevent lung cancer in A/J mice, induced by the tobacco-specific carcinogen, 4-(N-methyl-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK), and, if so if this corresponded to changes in gut microbiome composition. METHODS: We compared the effect of different quantities and types of CHO (easily digestible vs resistant), protein (casein vs. soy) and fat (fish vs. coconut vs. a mixture of oils) in modulating lung nodule formation and gut microbiome composition in A/J mice. Mice were fed either the Western diet or the low CHO diets, composed of different types of CHO, protein and fat for two weeks before injections with NNK to initiate lung formation. After 20 weeks, their feces were collected for amplification and sequencing of the bacterial 16S RNA gene V4 regions and the lung nodules were counted. RESULTS: Diets low in easily digestible starch, high in fish oil and soy protein were the most effective at preventing the formation of NNK-induced lung nodules. Changing protein, CHO and fat type in the diets all resulted in significant differences in the fecal microbiome composition of the NNK-injected mice. We also found a reduced abundance of the Streptococacceae and Clostridiaceae families in mice with low lung nodule numbers. CONCLUSIONS: We suggest that it is possible that the diets reduced lung nodule formation, at least in part, via alterations in the microbiomes of the mice. FUNDING SOURCES: Lotte & John Hecht Memorial Foundation.

OBJECTIVES: Since cancer cells typically rely more on glycolysis than normal cells, we hypothesized that lowering carbohydrate intake may reduce cancer risk. We aimed to investigate the efficacy of low-carbohydrate (CHO) diets in preventing and treating a tobacco-specific carcinogen-induced lung cancer in female A/J mice. METHODS: We evaluated the role of different types of CHO (easily digestible vs resistant), protein (casein vs. soy) and fat (fish vs. coconut vs. a mixture of oils) in modulating 4-(N-methyl-N-nitrosamino)-1-(3- pyridyl)-1-butanone (NNK)-induced lung nodule formation in these mice. To assess the efficacy of these diets in preventing NNK-induced lung nodule formation, we put these mice in the different diets for 2 weeks, intraperitoneally-injected NNK once a week for two weeks to initiate lung nodule formation. After 5 months, the lung nodules in these mice were counted. RESULTS: The lowering of easily digestible CHO significantly reduced constitutive blood glucose levels and lung nodule formation in the mice. Interestingly, diets low in easily digestible starch, high in fish oil (FO) and soy protein (15%Amylose/Soy/FO) were the most effective at preventing the formation of NNK-induced lung nodules. To determine if this 15%Amylose/Soy/FO is also effective at slowing tumor progression, we fed NNK-injected A/J mice a Western diet until tumors were established (5 months post NNK) and then either switched them to the 15%Amylose/Soy/FO or kept them on the Western diet for 5 additional months. The 15%Amylose/Soy/FO diet prevented the formation of additional lung tumor nodules and reduced the size of the tumors, although no significant difference was observed in tumor stage. The reduction in size of the lung tumors on the 15%Amylose/Soy/FO diet was not due to a lower tumor proliferation (Ki67 index) but an increase in apoptosis, as determined by TUNEL assays. CONCLUSIONS: We conclude that a diet change that lowers glucose intake, incorporates FO and soy protein may be effective not only in preventing lung cancer formation but also in slowing the growth of established lung tumors. FUNDING SOURCES: Lotte & John Hecht Memorial Foundation.

Sympathomimetic drugs include a vast array of medicinal compounds and drugs of abuse that act to either mimic or modify the actions of catecholamines (epinephrine, norepinephrine, dopamine) within the sympathetic nervous system. (Table 8.1) Some act as direct agonists at peripheral alpha- and/or beta-adrenergic receptors (e.g. phenylephrine, dobutamine). Others activate the sympathetic nervous system indirectly by either enhancing the release of neurotransmitters (e.g. ephedrine, methamphetamine), or inhibiting neurotransmitter reuptake (e.g. cocaine). Depending on the drug, these effects may predominate in either the central nervous system or at peripheral nerve synapses. Furthermore, many of these drugs modulate activity at serotonin receptors in the central nervous system (e.g. MDMA).

Obesity has reached epidemic proportions and is often accompanied by elevated levels of pro-inflammatory cytokines that promote many chronic diseases, including cancer. However, not all obese people develop these diseases and it would be very helpful to identify those at high risk early on so that preventative measures can be instituted. We performed an extensive evaluation of the effects of obesity on inflammatory markers, on innate and adaptive immune responses, and on blood cell composition to identify markers that might be useful in distinguishing those at elevated risk of cancer. Plasma samples from 42 volunteers with a BMI>35 had significantly higher CRP, PGE2, IL-1RA, IL-6 and IL-17 levels than 34 volunteers with normal BMIs. Of the cytokines and chemokines tested, only IL-17 was significantly higher in men with a BMI>35 than women with a BMI>35. As well, only IL-17 was significantly higher in those with a BMI>35 that had type 2 diabetes versus those without type 2 diabetes. Whole blood samples from participants with a BMI>35, when challenged with E. coli, produced significantly higher levels of IL-1RA while HSV-1 challenge resulted in significantly elevated IL-1RA and VEGF, and a non-significant increase in G-CSF and IL-8 levels. T cell activation of PBMCs, via anti-CD3 plus anti-CD28, resulted in significantly higher IFNγ production from volunteers with a BMI>35. In terms of blood cells, red blood cell distribution width (RDW), monocytes, granulocytes, CD4+T cells and Tregs were all significantly higher while, natural killer (NK) and CD8+ T cells were all significantly lower in the BMI>35 cohort, suggesting that obesity may reduce the ability to kill nascent tumor cells. Importantly, however, there was considerable person-to-person variation amongst participants with a BMI>35, with some volunteers showing markedly different values from controls and others showing normal levels of many parameters measured. These person-to-person variations may prove useful in identifying those at high risk of developing cancer.

We recently found that a diet composed of 15% of total calories as carbohydrate (CHO), primarily as amylose, 35% soy protein and 50% fat, primarily as fish oil (FO) (15%Amylose/Soy/FO) was highly effective at preventing lung nodule formation in a nicotine-derived nitrosamine ketone (NNK)-induced lung cancer model. We asked herein whether adopting such a diet once cancers are established might also be beneficial. To test this, NNK-induced lung nodules were established in mice on a Western diet and the mice were then either kept on a Western diet or switched to various low CHO diets. Since we previously found that sedentary mice develop more lung nodules than active mice, we also compared the effect of exercise in this cancer progression model. We found that switching to a 15%Amylose/Soy/FO diet reduced lung nodules and slowed tumor growth with both ‘active’ and ‘sedentary’ mice. Ki67, cleaved caspase 3 and Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling assays suggested that the efficacy of the 15%Amylose/Soy/FO in lowering tumor nodule count and size was not due to a reduction in tumor cell proliferation, but to an increase in apoptosis. The 15%Amylose/Soy/FO diet also significantly lowered liver fatty acid synthase and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 expression, pointing to a global metabolic switch from glycolysis to fatty acid oxidation. Mice fed the 15%Amylose/Soy/FO diet also had significantly reduced plasma levels of interleukin (IL)-1β, IL-6 and tumor necrosis factor α. These results suggest that the 15%Amylose/Soy/FO diet may slow tumor growth by suppressing proinflammatory cytokines, inducing a metabolic switch away from glycolysis and inducing apoptosis in tumors.

The RAW264.7 mouse macrophage cell line is used extensively to carry out in vitro screens for immunomodulators. Compounds that are effective at reducing the expression of pro-inflammatory cytokines or nitric oxide (NO) from lipopolysaccharide (LPS)-stimulated RAW264.7 cells are often considered candidate anti-inflammatory agents for humans. There is, however, very little data on the reliability of this screen to identify bona fide human immunomodulators. We compared the efficacy of 37 purported immunomodulators to modulate LPS or E. coli-induced inflammatory responses in RAW264.7 cell, whole human blood and human peripheral blood mononuclear cell (PBMC) assays. Interestingly, there was no significant correlation (R=0.315) between the responses obtained with RAW264.7 cells and the whole blood assay (WBA), suggesting that compounds demonstrating efficacy in RAW264.7 cells may be ineffective in humans, and, more importantly, compounds that are effective in humans may be missed with a RAW264.7 screen. Interestingly, there was also no significant correlation between the WBA and human PBMCs when the latter were cultured with 10% FCS, but a moderate correlation was seen when the PBMCs were cultured with 25% autologous plasma. The presence of plasma thus contributes to the overall inflammatory response observed in the WBA. We then asked if RAW264.7 cells, given that they are mouse macrophage-like cells, respond in a manner similar to primary murine derived macrophages. Intriguingly, there was no significant correlation (R=0.012) with the 37 putative immunomodulators, pointing to distinct inflammatory response mechanisms in the two model systems. We conclude that the use of a WBA to confirm potential immunomodulators obtained from high throughput screening with RAW264.7 cells is advisable and that future screens be carried out using a WBA.

Introduction: Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a transcription factor family that regulates gene expression programs contributing to inflammation and cell survival. NF-κB signaling occurs via two branches: classical and alternative, and is often enriched in somatic mutations of key pathway members in several lymphoid malignancies. Here, we reveal deregulation and constitutive activation of the alternative NF-κB pathway in a subset of DLBCL patients with recurrent genomic loss of the gene encoding tumor necrosis factor receptor-associated factor 3 (TRAF3), a regulator of the NF-κB signaling pathway. Methods and Results: To uncover novel driver mutations of DLBCL pathogenesis and tumor maintenance, we performed Affymetrix SNP6.0 copy number analysis on 347 de novo DLBCL samples from patients uniformly treated with rituximab, cyclophosphamide, doxorubicin, vincristine and prednisone (R-CHOP). We observed frequent, focal genomic loss of chr:14q32.31-32 which included TRAF3 and RCOR1 (7%, 22/313) in the minimally deleted region and an enrichment of activated B-cell-like (ABC) subtype cases over germinal center B-cell-like (GCB) subtype cases, confirming previously published data (Chan et al, Blood 2014). RNAseq of these DLBCL samples revealed a significant reduction of TRAF3 mRNA in chr:14q32.31-32 deleted cases compared to copy number neutral cases (p=0.002). Next, we focused on characterizing the phenotypic consequences of TRAF3 loss in DLBCL. We used CRISPR/Cas9 gene editing to knock out TRAF3 in 2 GCB-DLBCL (DOHH2, OCI-LY1) and 2 ABC-DLBCL (HBL1, OCI-LY3) cell lines. We performed immunoblotting analysis of NF-κB pathway members on cell fractionated samples of TRAF3 knockout cells and found increased levels of the NF-κB inducing kinase NIK (a direct target of TRAF3-mediated ubiquitin-proteasome degradation) and a concomitant increased nuclear translocation of NF-κB transcription factor complex subunits RelB and p52. Proteasome blockade restored RelB cytoplasmic localization and reduced processed p52 protein in TRAF3 knockout GCB-DLBCL lines only, indicating other factors may contribute to alternative NF-κB activation in ABC-DLBCL. Moreover, classical NF-κB activation remained unaffected, highlighting the specific role of TRAF3 regulation on the alternative NF-κB pathway in DLBCL. Consistent with these findings, TRAF3 knockout cells exhibited NF-κB-dependent transcriptional upregulation by luciferase reporter activity and elevated pro-inflammatory cytokine production (IL-6, TNF-β) by Luminex and ELISA. To study transcriptome changes as a result of TRAF3 loss-of-function, we performed RNAseq and differential gene expression analysis on wildtype and TRAF3 knockout DLBCL cell lines as well as primary DLBCL samples (N=347). We found enrichment of NIK and NF-κB associated pathways in TRAF3 deficient DLBCL and uncovered additional enriched gene sets including those involved in cell cycle regulation, cell division and metabolism, suggesting a potential proliferative and survival advantage. Conclusion: Our findings link TRAF3 loss-of-function to clinical and gene expression phenotypes in DLBCL and highlight alternative NF-κB activation as a pathogenically important pathway in both GCB and ABC subtypes. Future studies will be directed towards comprehensive evaluation of NF-κB inhibitors for effective blockade of constitutive alternative NF-κB activation in DLBCL. Disclosures Scott: NIH: Consultancy, Other: Co-inventor on a patent related to the MCL35 assay filed at the National Institutes of Health, United States of America.; Roche/Genentech: Research Funding; Janssen: Consultancy, Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoString, Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Seattle Genetics: Consultancy; Curis Inc: Consultancy; Juno Therapeutics: Consultancy; Bayer: Consultancy; AbbVie: Consultancy.

Simple Summary Immune-checkpoint inhibitors (ICI) have been increasingly used in the management of various types of cancers. More studies and guidelines also recommended the combination of ICI with other anti-cancer agents to improve the efficacy and treatment outcome. This retrospective study showed that the combination of ICI and targeted agents increased the risk of immune-related endocrine dysfunction and hypothyroidism. Moreover, older patients on ICI had a higher risk of immune-related diabetes mellitus. ICI is safe and well-tolerated regardless of age, but close monitoring of fasting glucose is essential in older populations. Abstract The number of immune-related endocrine dysfunctions (irEDs) has concurrently increased with the widespread use of immunotherapy in clinical practice and further expansion of the approved indications for immune checkpoint inhibitor (ICI) in cancer management. A retrospective analysis was conducted on consecutive patients ≥18 years of age with advanced solid malignancies who had received at least one dose of anti-programmed cell death protein 1 (anti-PD-1) and/or anti-CTLA4 antibodies between January 2014 and December 2019 at a university hospital in Hong Kong. Patients were reviewed up to two months after the last administration of an ICI. The types, onset times and grades of irEDs, including hypothyroidism, hyperthyroidism, adrenal insufficiency and immune-related diabetes mellitus, were recorded. Factors associated with irEDs were identified using multivariate analysis. A total of 953 patients (male: 603, 64.0%; median age: 62.0 years) were included. Of these, 580 patients (60.9%) used ICI-alone, 132 (13.9%) used dual-ICI, 187 (19.6%) used an ICI combined with chemotherapy (chemo + ICI), and 54 (5.70%) used immunotherapy with a targeted agent (targeted + ICI). A significantly higher proportion of patients using targeted + ICI had irEDs and hypothyroidism; in contrast, a higher proportion of patients using dual-ICI had adrenal insufficiency. There was no significant difference in the incidence of irED between the younger (

Septic arthritis is an important but difficult to make diagnosis that leads to significant morbidity and mortality. Joint effusion is generally accepted to be a highly sensitive finding in septic arthritis; however, final diagnosis requires synovial fluid studies. Without a significant joint effusion, it is difficult to obtain synovial fluid. In this case report, we describe the presentation and diagnosis of septic arthritis in the first MTP due to mycobacterium chelonae in a 69 year old man with a history of gout and immunosuppression due to a heart transplant. There was notably no significant effusion in the joint on clinical examination or bedside ultrasound. As the patient was immunosuppressed, arthrocentesis was performed under ultrasound guidance. A needle was clearly visualized in the joint; however, minimal synovial fluid was obtained. The fluid grew Mycobacterium chelonae in culture. Subsequent joint washout revealed purulent synovial fluid that grew out the same bacteria. This case emphasizes the importance of obtaining synovial fluid to evaluate for septic arthritis, even when joint effusion is absent. Ultrasound guidance can facilitate this.

Hodgkin lymphoma is characterized by an extensively dominant tumor microenvironment (TME) composed of different types of noncancerous immune cells with rare malignant cells. Characterization of the cellular components and their spatial relationship is crucial to understanding cross-talk and therapeutic targeting in the TME. We performed single-cell RNA sequencing of more than 127,000 cells from 22 Hodgkin lymphoma tissue specimens and 5 reactive lymph nodes, profiling for the first time the phenotype of the Hodgkin lymphoma–specific immune microenvironment at single-cell resolution. Single-cell expression profiling identified a novel Hodgkin lymphoma–associated subset of T cells with prominent expression of the inhibitory receptor LAG3, and functional analyses established this LAG3+ T-cell population as a mediator of immunosuppression. Multiplexed spatial assessment of immune cells in the microenvironment also revealed increased LAG3+ T cells in the direct vicinity of MHC class II–deficient tumor cells. Our findings provide novel insights into TME biology and suggest new approaches to immune-checkpoint targeting in Hodgkin lymphoma. Significance: We provide detailed functional and spatial characteristics of immune cells in classic Hodgkin lymphoma at single-cell resolution. Specifically, we identified a regulatory T-cell–like immunosuppressive subset of LAG3+ T cells contributing to the immune-escape phenotype. Our insights aid in the development of novel biomarkers and combination treatment strategies targeting immune checkpoints. See related commentary by Fisher and Oh, p. 342. This article is highlighted in the In This Issue feature, p. 327

Herein we demonstrate that ultraviolet light-inactivated Herpes Simplex Virus-1 (UV-HSV-1) stimulates peripheral blood mononuclear cells (PBMCs) to lyse both androgen-sensitive and androgen-independent prostate cancer (PrCA) cell lines, but not the benign prostatic hyperplastic epithelial cell line, BPH-1, and is 1000-10,000-fold more potent at stimulating this killing than ultraviolet light-inactivated Vesicular Stomatitis Virus, adenovirus, reovirus or cytomegalovirus. Among PBMCs, natural killer (NK) cells appear to be a major cell type involved in this killing and UV-HSV-1 appears to directly and potently stimulate NK cell expression of CD69, degranulation, cytokine production, and migration to IL-8 in PC3 conditioned medium. We also found that UV-HSV-1 stimulates glycolysis in PBMCs and NK cells, and that 2-deoxyglucose and the protein kinase C inhibitor, Go6976, and the NFκB inhibitor, Bay 11-7082, all abrogate UV-HSV-1 activated killing of PC3 cells by PBMCs and NK cells. Using neutralizing anti-Toll-like receptor 2 (TLR2) we found that UV-HSV-1, like HSV-1, activates NK cells via TLR2. Taken together, these results are consistent with Toll-like receptor 2 ligands on UV-HSV-1 stimulating TLR2 on NK cells to activate protein kinase C, leading to enhanced glycolysis and NFκB activation, both of which play a critical role in this anti-PrCA innate immune response. Importantly, UV-HSV-1 synergizes with IL-15 to increase the cytolytic activity of PBMCs against PC3 cells and there was considerable donor-to-donor variation in killing ability. These results support the preclinical development of UV-HSV-1 as an adjuvant, in combination with IL-15, for cell infusions of healthy, preselected NK cells to treat PrCA.

In previous studies, we found that low-carbohydrate (CHO) diets reduced the incidence of tumors in mice genetically predisposed to cancer. However, because >90% of human cancers arise via carcinogen-induced somatic mutations, we investigated, herein, the role that different types and levels of CHO, protein and lipid play in lung cancer induced by the tobacco-specific carcinogen, nicotine-derived nitrosamine ketone (NNK) in A/J mice. We found lowering CHO levels significantly reduced lung nodules and blood glucose levels. We also found that soy protein was superior to casein and that coconut oil was ineffective at reducing lung nodules. Diets containing amylose or inulin (at 15% of total calories), soy protein (at 35%) and fat (at 50%, 30% being fish oil) were the most effective at reducing lung nodules. These fish oil-containing diets increased plasma levels of the ketone body, β-hydroxybutyrate, while reducing both insulin and 8-isoprostane in plasma and bronchoalveolar interleukin-12 and lung PGE2 levels. After only 2 weeks on this diet, the levels of γ-H2AX were significantly reduced, 24 hours after NNK treatment. Housing these mice in two-tiered rat cages with exercise wheels led to similar mouse weights on the different diets, whereas keeping mice in standard mouse cages led to both significant weight differences between the low-CHO, soy protein, fish oil diet and Western diet and substantially more lung nodules than in the two-tiered cages. Our results suggest that low-CHO, soy protein, fish oil-containing diets, together with exercise, may reduce the incidence of lung cancer.

e24022 Background: Maintaining function and preventing functional decline during cancer treatment is critically important to older adults. This systematic review characterized and identified patient factors associated with functional change during systemic cancer therapy in older adults. Methods: Following PRISMA guidelines, we searched PubMed, Embase, Web of Science, and Cochrane Central Register of Controlled Trials for articles examining changes in function during systemic cancer treatment published in English through 1/11/19. Studies were eligible if they included adults age >65 and analyzed associations between patient factors and change in function. At least two independent investigators reviewed each article with discrepancies resolved by consensus. Major findings were summarized; no meta-analysis was planned a priori given the heterogeneity in studies. Results: We screened 15,244 titles/abstracts and 519 full texts. The final analysis included 69 studies, which enrolled > 11,000 patients with cancer. Most studies enrolled adults of all ages; 20% included only adults age >65 and 13% only adults age >70. A quarter of studies enrolled patients with lung cancer while 22% included all solid tumors and hematologic malignancies. The majority of studies evaluated function during chemotherapy (96%) with 9% including targeted therapy and 4% immunotherapy. Function was primarily measured with patient-reported outcomes (93% of studies). Reporting of functional change was heterogeneous with many reporting change scores or means at multiple time points. Among studies that reported the percentage of patients who developed functional decline, results ranged widely from 6% to 90%. Functional improvement occurred among 2% to 57% of patients. The most common patient factors associated with functional decline during systemic cancer therapy were older age (n = 8 studies), fatigue (n = 8), worse baseline performance status (n = 8) and physical activity (n = 5), and anemia (n = 5). Only 10 studies examined factors associated with functional recovery, identifying 12 unique patient factors. Conclusions: Among older adults with cancer, functional changes during systemic cancer therapy are common. Interventions to target modifiable patient factors associated with functional decline are needed to help patients maintain or improve function during treatment. Additionally, evaluating both functional decline and improvement is necessary to better characterize functional trajectories during systemic cancer therapy.

Lung cancer development is driven by the expression of mutant oncogenes, with EGFR and KRAS being the most frequent mutations in lung adenocarcinoma. However, these mutations alone are not sufficient for tumorigenesis, and additional factors influence tumor development and progression, including the balance of antitumor immune effector cells and protumorigenic immune suppressor cells within the tumor environment. We hypothesized that oncogene signaling regulates the production of cytokines by tumor cells in order to modulate the immune microenviroment and promote lung tumor development. We used CIBERSORT to quantify 22 immune cell types in over 300 human lung adenocarcinomas (LUAD) and 100 matched normal lung tissues. Cells associated with inflammatory or antitumor response, macrophages (M1, M0), T follicular helper cells, and plasma cells, were enriched in LUAD compared to normal lung tissue. Additionally, immunosuppressive regulatory T cells (Tregs) were significantly enriched in LUAD tumors, even at the early stages. To identify cytokines that could be induced by oncogenic signaling early in lung tumorigenesis, we used normal cells expressing doxycycline-inducible mutant KRASG12V mutant EGFRL858R or wild-type EGFR and analyzed cytokine production with a multiplex assay (LUMINEX). Induction of oncogenic signaling in normal cells rapidly increased production of cytokines CCL5 and CCL2. These are capable of recruiting a variety of cells types, including Tregs. In KRAS mutant lung cancer cells, disruption of oncogene signaling with a MEK inhibitor (trametinib) decreased CCL5 production. We used transgenic mice that spontaneously develop lung tumors in response to tetracycline-inducible expression of mutant EGFRDelEx19 or mutant KRASG12V in type II alveolar cells to investigate immune cell populations that may change in response to oncogene-driven lung tumorigenesis. We found that Tregs were increased in the lungs of tumor-bearing mice. These Tregs express the CCL5 receptor, CCR5. Several cytokines were elevated in bronchioalveolar lavage fluid or in lung lysates of tumor-bearing mice, including IL-12(p40), CXCL1, CCL2, CCL3 and CCL5. Murine Lewis lung carcinoma (LLC) cells harbor a KRAS mutation and express high levels of CCL5. We are currently investigating the effects of disrupting oncogene signaling or CCL5 expression in LLC cells on cytokine production and immune cell recruitment in vivo using syngeneic implantation of the cells into mice. Our data suggest that oncogenic signaling regulates expression of cytokines in lung tumor cells. Targeted inhibition of cytokines directly or indirectly through oncogenic signaling may represent therapeutic strategies to block the recruitment of immune-suppressive cells to the tumor microenvironment, thereby enhancing the antitumor immune response. Citation Format: Elizabeth Franks, Etienne Melese, Bryant Harbourne, Liz Halvorsen, Rachel Cederberg, Jenna Collier, Natalie Firmino, Jennifer Luu, Arun Unni, Min Hee Oh, Vivian Lam, Gerry Krystal, Ninan Abraham, Kevin Bennewith, William Lockwood. Oncogenes drive production of immunosuppressive cytokines to facilitate lung cancer progression [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A38.

INTRODUCTION: Classic Hodgkin lymphoma (cHL) is uniquely characterized by an extensively dominant microenvironment composed primarily of different types of non-cancerous immune cells with a rare population (~1%) of tumor cells. Detailed characterization of these cellular components and their spatial relationship is crucial to understand crosstalk and therapeutic targeting in the cellular ecosystem of the tumor microenvironment (TME). METHODS: In this study, we performed high dimensional and spatial profiling of immune cells in the TME of cHL. Single cell RNA sequencing (scRNA-seq) was performed with the 10x Genomics platform on cell suspensions collected from lymph nodes of 22 cHL patients, including 12 of nodular sclerosis subtype, 9 of mixed cellularity subtype and 1 of lymphocyte-rich subtype, with 5 reactive lymph nodes (RLNs) serving as normal controls. Illumina sequencing (HiSeq 2500) was performed to yield single-cell expression profiles for 127,786 cells. We also performed multicolor IHC and imaging mass cytometry (IMC) on TMA slides from the same patients. RESULTS: Unsupervised clustering using PhenoGraph identified 22 cell clusters including 12 T cell clusters, 7 B cell clusters and 1 macrophage cluster. While most immune cell populations were common between cHL and RLN, we observed an enrichment of cells from cHL in all 3 regulatory T cell (Treg) clusters. The most cHL-enriched cluster was characterized by high expression of LAG3, in addition to common Treg markers such as IL2RA (CD25) and TNFRSF18 (GITR), but lacked expression of FOXP3, consistent with a type 1 regulatory (Tr1) T cell population. LAG3+ T cells in cHL had high expression of immune-suppressive cytokines IL-10 and TGF-b . In vitro exposure of T cells to cHL cell line supernatant induced significantly higher levels of LAG3 in naïve T cells compared to co-culture with other lymphoma cell line supernatant or medium only. CD4+ LAG3+ T cells isolated by FACS also suppressed the proliferation of responder CD4+ T cells when co-cultured in vitro. Additionally, Luminex analysis revealed that cHL cell lines secrete substantial amounts of cytokines and chemokines that can promote Tr1 cell differentiation (e.g. IL-6). Our scRNA-seq analysis revealed that LAG3 expression was significantly higher in cHL cases with loss of major histocompatibility class II (MHC-II) expression on HRS cells as compared to MHC-II positive cases (P = 0.019), but was not correlated with EBV status or histological subtype. Strikingly, LAG3 was identified as the most up-regulated gene in cells from MHC-II negative cases compared to MHC-II positive cases. Topological analysis using multicolor IHC and IMC revealed that in MHC-II negative cases, HRS cells were surrounded by LAG3+ T cells. In these cases, the density of LAG3+ T cells in HRS cell-rich regions was significantly increased, and the average distance between an HRS cell and its closest LAG3+ T cell neighbor was significantly shorter. These associations were confirmed in an independent cohort of 166 cHL patients. Finally, we observed a trend towards an inferior disease-specific survival (DSS; P = 0.072) and overall survival (OS; P = 0.12) in cases with an increased number of LAG3+ T cells. A high proportion of LAG3+ T cells (> 20%) was identified as an independent prognostic factor for DSS by multivariate Cox regression. CONCLUSIONS: Our results reveal a diverse TME composition with inflammatory and immunosuppressive cellular components that are linked to MHC class II expression status on HRS cells (Figure). Unprecedented transcriptional and spatial profiling at the single cell level has established the pathogenic importance of HRS cell-induced CD4+ LAG3+ T cells as a mediator of immunosuppression in cHL, with potential implications for novel therapeutic approaches. Figure Disclosures Savage: Seattle Genetics, Inc.: Consultancy, Honoraria, Research Funding; BMS, Merck, Novartis, Verastem, Abbvie, Servier, and Seattle Genetics: Consultancy, Honoraria. Scott:Roche/Genentech: Research Funding; Celgene: Consultancy; Janssen: Consultancy, Research Funding; NanoString: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution], Research Funding. Steidl:Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: Filed patent on behalf of BC Cancer; Roche: Consultancy; Seattle Genetics: Consultancy; Bayer: Consultancy; Juno Therapeutics: Consultancy; Tioma: Research Funding.

92 Background: Functional decline during NSCLC treatment is critically important to older adults. Yet, data on functional decline—particularly during chemoIO, IO, and targeted therapy—remain limited. Additionally, the best measures to capture functional decline from the patient’s perspective remain unknown. Methods: We conducted a multisite, mixed methods prospective cohort study to characterize function during systemic NSCLC treatment and compare quantitative measures with gold standard qualitative descriptions of patients’ lived experience. Prior to and at 2 months after treatment initiation, 69 adults age >65 with advanced NSCLC starting chemo, IO, and/or targeted therapy underwent geriatric assessments including instrumental activities of daily living (IADL), EORTC QLQ-C30 Physical Functioning (PF), and Life-Space Assessment (LSA). LSA measures where a person goes in their environment, how frequently, and how independently. Pretreatment functional impairment and decline at 2 months were assessed. In a qualitative substudy, 16 purposively sampled patients with diverse pretreatment function completed two semi-structured interviews (pretreatment, 2 months) to explore treatment effects on daily functioning. Grounded theory was used to code for functional decline. Using the qualitative patient experience as the gold standard, sensitivity and specificity were calculated for each functional measure. Results: Mean age was 75 (range 65-94). NSCLC treatment included IO (36%), targeted therapy (28%), chemoIO (23%), and chemo (13%). Pretreatment functional impairment was common and ~25% of patients experienced functional decline on quantitative measures at 2 months (Table). Qualitatively, 50% described functional decline at 2 months. LSA had the highest sensitivity (71%) and similar specificity (88%) as IADL and PF. Conclusions: Functional decline during NSCLC treatment is common. LSA provides a sensitive and specific patient-centered measure of function. [Table: see text]

Children and adults prenatally exposed to alcohol show higher rates of mental health problems than unexposed individuals, with depression and anxiety being among the more commonly encountered disorders. Previous studies in rats showed that prenatal alcohol exposure (PAE) can indeed increase depressive- and anxiety-like behavior in adulthood; however, depression and anxiety are often observed in the context of stress and/or a dysregulated stress response system (the hypothalamic-pituitary-adrenal [HPA] axis). PAE can dysregulate the HPA axis, resulting in hyperresponsivity to stress. In turn, this may predispose individuals prenatally exposed to alcohol to the adverse effects of stress compared to unexposed individuals. We have shown previously that PAE animals may be more sensitive to the effects of chronic stress on behavior, showing increased anxiety- and depressive-like behavior following chronic unpredictable stress (CUS) exposure. Here, we investigated the independent and interactive effects of PAE and adult CUS on anxiety-like behavior and receptor systems (corticotropin-releasing hormone receptor type 1 [CRHR1], mineralocorticoid receptor [MR], and glucocorticoid receptor [GR]), and underlying stress and emotional regulation, and whether exposure to CUS differentially results in immediate or delayed effects. Adult male and female offspring from PAE, pair-fed (PF), and ad libitum-fed control (C) dams were exposed to either 10 days of CUS or left undisturbed. Behavioral testing began 1 or 14 days post-CUS, and brains were collected following testing. Anxiety-like behaviors were evaluated using the open field, elevated plus maze and dark-light emergence tests. CRHR1, MR, and GR mRNA expression were assessed in the medial prefrontal cortex (mPFC), amygdala, and hippocampal formation, brain areas key to both stress and emotional regulation. We found that PAE differentially increased anxiety-like behavior and altered GR mRNA in males and females compared to their control counterparts. Furthermore, depending on the timing of testing, CUS unmasked alterations in GR and CRHR1 mRNA expression in the mPFC and amygdala in PAE males, and MR mRNA in the hippocampal formation in PAE females compared to their C counterparts. Overall, the changes observed in these receptor systems may underlie the increase in anxiety-like behavior following PAE and CUS exposure in adulthood. That CUS differentially affected brain and behavioral outcome of PAE and C animals, and did so in a sexually-dimorphic manner, has important implications for understanding the etiology of psychopathology in individuals prenatally exposed to alcohol.

A major goal in ecosystem management is to prevent shifts to undesirable states, and there is widespread interest in understanding, mapping, and quantifying resilience to support management decisions. With rapid changes taking place on coral reefs, managers and scientists are faced with a challenging task in prioritising interventions that help strengthen reef resilience. Resilience-based management (RBM) of coral reefs requires in-depth knowledge on the various components and feedback mechanisms of the ecosystem, and uses resilience theory to inform decisions. To date, there are mechanistic models that analyse and forecast the future of reefs, but being data intensive and requiring specialist knowledge, are not applicable to managers in most locations with limited data and analytical training. Hence, there is a need to develop approaches to acquire insights on resilience using monitoring data.The global decline of reefs to varying extents, underscores the importance of understanding how biophysical factors influence and shape reef community dynamics. Multiple environmental factors affect reef functioning, either positively or negatively, and the diversity and complexity of responses make it difficult to predict future reef trajectories. My thesis addresses the need to improve retrospective analyses of time-series monitoring data to suit management goals, such as resilience, by focusing on the data collection and analytical components of long-term monitoring programmes. In this thesis, I trialled two state-of-the-art methods to extract useful ecological information and identified drivers of coral reef trajectories from long-term monitoring data collected in the Great Barrier Reef, arguably one of the most comprehensive coral reef monitoring dataset in the world.In Chapter 1, I provide a theoretical overview of the issues related to resilience and identify the quantification approaches found within the coral reef field. By examining the current state of knowledge, a simple framework of resilience-related concepts is developed, followed by an analysis of approaches used to quantify reef resilience and vulnerability. Seven broad quantification approaches were identified under the three principle concepts of (1) ecological resilience (ecological resilience, precariousness and current attractor), (2) engineering resilience (short-term recovery rate and long-term reef performance), and (3) vulnerability (absolute and relative vulnerability) respectively. Resilience-based management (RBM) is a new approach to reef management that is being adopted by governmental institutions globally. However, there are no quantitative studies on how this has changed coral reef monitoring and assessment. In Chapter 2, I conducted a critical evaluation of how RBM has changed the way we monitor our reefs. Through a comparison of metrics used in conventional programmes with those developed specifically for resilience assessments, I show that resilience metrics are much more diverse and have a strong focus on ecological processes and exposure to environmental drivers. Also, monitoring metrics focus on reef state, but seem less likely to predict the future responses of reefs. To identify key drivers of coral trajectories in the inshore and offshore GBR, regions with fundamentally different biophysical drivers, I used multivariate autoregressive state-space approach (MARSS) and generalised additive mixed models (GAMMS) within a Bayesian framework using geoadditive coefficients in Chapters 3 and 4 respectively. Key drivers identified in Chapter 3 suggest that inshore reef coral trajectories are largely governed by episodic localised disturbances, alike their offshore counterparts. Surprisingly, no water quality variables emerged as key drivers, implying that the impacts of chronic water quality are more likely to affect reef dynamics over a longer time-scale and difficult to detect with a decadal dataset. In Chapter 4, I further examined the role of different disturbances on different Acropora morphologies, novel in the context of a large scale ecological dataset. Concurrent with the general literature, the impact of storms and crown-of-thorns starfish were found to be the key drivers of coral dynamics in the mid- and off-shore GBR, but there are significant differences on the relative disturbance impacts on coral growth forms.This study has reviewed the field of coral reef resilience and captured the effects of biological and physical variables on inshore and offshore reef dynamics using a range of novel approaches. While coral reef dynamics seem to be driven mainly by acute disturbances, an important implication that stems from this research is on how one uses physical data (i.e. water quality, DHW) as predictors of resilience, and that the integration and scoring of variables into resilience assessments should be context dependent, contrary to current resilience assessments. The comparison of the last two chapters provides information to guide current reef monitoring programmes and delivers practical suggestions to improve future monitoring and resilience assessments.

Wind turbines operate within an environment of varying wind direction and magnitude leading to misalignment or yaw between the wind and the turbine rotor. Many control techniques have been applied to small horizontal axis wind turbines (HAWTs) to prevent significant yaw loads, but in reality, most of the time the rotor is under some yaw load which may be significant. A blade element of a HAWT rotor under yaw loading is similar to a sinusoidally pitching airfoil. Here a miniaturized pitching S822 airfoil with a reduced frequency k of 0.025 at a Reynolds number ( Re = 105) is considered where the maximum dynamic angle of attack is equal to the static angle where the maximum lift to drag ratio occurs. These dynamic results obtained with laser Doppler anemometry (LDA), particle image velocimetry (PIV) and a numerical simulation with the Transition SST method are compared to a constant wind-loaded blade element. Since direct load measurement for dynamic miniaturized models in wind tunnels is very challenging, the non-intrusive PIV control volume analysis approach has been applied for load determination. Unlike the rotor in an ideal unidirectional freestream flow, a rotor under yaw loads, as presented here, experiences significant unbalanced loading in one cycle and a non-uniform wake. Results may be used to predict the cyclical range of loads a blade element experiences in yaw conditions and effects on the downstream rotor wake.

To identify reliable biomarkers of age-related changes in chronic inflammation and responsiveness to bacterial and viral challenges, we evaluated endogenous and ex vivo stimulated levels of 18 inflammatory markers, using whole blood collected in EDTA and sodium heparin tubes from 41 healthy volunteers, i.e., 11 men + 10 women aged 20-35 and 10 men + 10 women aged 50-77. These studies revealed significant differences in the levels of inflammatory markers when blood was collected in EDTA versus sodium heparin and age related differences in these biomarkers were confirmed with blood collected in EDTA from 120 healthy volunteers in 3 age categories, ie, 20 men + 20 women, aged 20-35, 36-49 and 50-77. Studies with unstimulated blood samples, to measure levels of chronic inflammation, revealed a significant increase with age in IL-12p70, CRP and PGE2, consistent with the concept of "inflammaging", and a decrease in G-CSF in both men and women. Interestingly, in response to E. coli stimulation, PGE2 levels were markedly reduced in the 50-77 year old cohort while they were increased following Herpes Simplex virus-1 (HSV-1) stimulation, along with IL-8. In addition, unlike E. coli, HSV-1 potently stimulated IFNα production, but levels were dramatically reduced in the older cohort, consistent with a reduced ability to generate an anti-viral response. We also found platelets and CD8+ T cells were reduced with age while CD4+ T cells were significantly increased, resulting in a substantially higher CD4/CD8 ratio in the older cohort. Surprisingly, however, we found that the older cohort exhibited more T cell proliferation and IFNγ production in response to anti-CD3+anti-CD28 stimulation. Importantly, there was considerable person-to-person variation in these inflammatory markers in all age groups, making possible comparisons between a person's "inflammage" and chronological age. These assays should help to identify individuals at high risk of autoimmune disorders and cancer.

Synapses, the basic units of communication in the brain, require complex molecular machinery for neurotransmitter release and reception. Whereas numerous components of excitatory postsynaptic sites have been identified, relatively few proteins are known that function at inhibitory postsynaptic sites. One such component is neuroligin-2 (NL2), an inhibitory synapse-specific cell surface protein that functions in cell adhesion and synaptic organization via binding to neurexins. In this study, we used a transgenic tandem affinity purification and mass spectrometry strategy to isolate and characterize NL2-associated complexes. Complexes purified from brains of transgenic His6-FLAG-YFP-NL2 mice showed enrichment in the Gene Ontology terms cell-cell signaling and synaptic transmission relative to complexes purified from wild type mice as a negative control. In addition to expected components including GABA receptor subunits and gephyrin, several novel proteins were isolated in association with NL2. Based on the presence of multiple components involved in trafficking and endocytosis, we showed that NL2 undergoes dynamin-dependent endocytosis in response to soluble ligand and colocalizes with VPS35 retromer in endosomes. Inhibitory synapses in brain also present a particular challenge for imaging. Whereas excitatory synapses on spines can be imaged with a fluorescent cell fill, inhibitory synapses require a molecular tag. We find the His6-FLAG-YFP-NL2 to be a suitable tag, with the unamplified YFP signal localizing appropriately to inhibitory synapses in multiple brain regions including cortex, hippocampus, thalamus, and basal ganglia. Altogether, we characterize NL2-associated complexes, demonstrate regulated trafficking of NL2, and provide tools for further proteomic and imaging studies of inhibitory synapses.

11540 Background: Prognosis in NSCLC is vital for clinical decision making. With the aging US population and rapidly changing treatment landscape, we aimed to identify prognostic factors among older adults with advanced NSCLC receiving chemo-, immuno-, and/or targeted therapy. Methods: We conducted a prospective cohort study of adults age ≥65 with advanced NSCLC starting a new non-curative systemic treatment (chemo-, immuno-, and/or targeted therapy) at a Comprehensive Cancer Center, Veterans Affairs Medical Center, and safety-net hospital. Prior to treatment initiation, patients completed a geriatric assessment including cognition, function, comorbidities, mood, social support, and quality of life. Cox proportional hazards models were performed to identify prognostic factors for overall survival (OS). Results: In a sample of 51 patients, median age was 73 (range 65-94). The majority of patients had stage IVB (59%) or IVA (39%) NSCLC. Current treatment included immunotherapy (37%), targeted therapy (29%), chemoimmunotherapy (18%), and chemo (16%). Most patients had received prior NSCLC treatment (80%): chemo (51%), targeted therapy (35%), immunotherapy (22%), radiation (RT; 47%), and/or surgery (19%). At enrollment, 73% had an abnormal Montreal Cognitive Assessment score < 26 (MoCA; median score 23) and 35% had an abnormal Timed Up and Go time ≥13.5 secs (TUG; median time 12.7 secs). Median OS was 12.5 months. In univariable analyses, stage IVB disease (HR 6.99, 95% CI 1.55-31.5), prior RT (HR 2.98, 95% CI 1.08-8.21), worse MoCA score (HR 1.15 per 1 point change, 95% CI 1.03-1.28), and longer TUG time (HR 1.13 per 1 sec change, 95% CI 1.05-1.23) were associated with worse OS. Of note, age, current NSCLC treatment, line of therapy, and Karnofsky Performance Status were not associated with OS. In multivariable analysis, MoCA score was the only statistically significant prognostic factor (HR 1.15, 95% CI 1.01-1.30). Conclusions: We found that abnormal pretreatment cognition is very common and an important prognostic factor among older adults with advanced NSCLC. Pretreatment screening for cognitive impairment should be considered to inform prognostication, decision making, and treatment planning.