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The Healthy Heart Score (HHS) is a lifestyle-based equation for predicting cardiovascular disease (CVD) risk and may serve as a tool in primordial prevention. However, its performance outside North American populations is unknown. This study assessed the performance of the HHS for estimating CVD mortality in the adult population of Spain.We analyzed data from the ENRICA cohort, comprising 10 228 participants free of chronic disease and representative of the Spanish population aged ≥ 18-years, who were recruited from 2008 to 2010 and were followed up to 2020. The HHS includes body mass index, alcohol, physical activity, smoking, and a 5-component dietary score. The HHS was calculated at baseline using the sex-specific beta coefficients from the original development cohorts. Model discrimination was assessed using the Harrell c-statistic and Gonen-Heller c-statistic for survival data, and calibration was evaluated through calibration plots.After a median follow-up of 11.8 years, 110 CVD deaths were ascertained. The discrimination of the HHS was similar for women (Harrell c, 0.91; 95%CI, 0.87-0.95; Gonen-Heller-c, 0.85; 95%CI, 0.83-0.88) and men (Harrell c, 0.91; 95%CI, 0.88-0.94; Gonen-Heller c: 0.85; 95%CI, 0.83-0.88). After recalibration by the sex-specific baseline survival function, the calibration became optimal for: a) all deciles of predicted CVD risk except the highest decile, where HHS underestimated the risk, and b) all age groups except 70 years and older, where there was an underestimation.In this Spanish cohort, the HHS showed good discrimination and calibration for predicting CVD death. The performance of HHS in other European populations and its implementation in the clinical setting warrants further investigation.

Programmed cell death protein-1 (PD-1)-targeted immunotherapy is approved for recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) treatment. Although its efficacy correlates with PD-L1 expression, response is limited even among positive cases. We employed digital spatial profiling (DSP) to discover potential biomarkers of immunotherapy outcomes in HNSCC. Fifty prospectively collected, pretreatment biopsy samples from patients with anti-PD-1–treated R/M HNSCC, were assessed using DSP, for 71 proteins in four molecularly defined compartments (tumor, leukocyte, macrophage, and stroma). Markers were evaluated for associations with progression-free (PFS) and overall survival (OS). High beta-2 microglobulin (B2M), LAG-3, CD25, and 4-1BB in tumor; high B2M, CD45, CD4 in stroma, and low fibronectin in the macrophage compartment, correlated with prolonged PFS. Improved PFS and OS were observed for cases with high B2M by quantitative and mRNA. Findings were validated in an independent cohort for PFS (HR, 0.41; 95% confidence interval, 0.19–0.93; P = 0.034). B2M-high tumors showed enrichment with immune cell and immune checkpoint markers. Our study illustrates B2M expression is associated with improved survival for immune checkpoint inhibitor (ICI)-treated HNSCC. Significance: In the current study, DSP revealed the positive association of B2M expression in the tumor compartment with immunotherapy outcomes in R/M HNSCC.

Targeting the interaction of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) and its ligands has been shown to reinstate antitumor immunity. In addition, the introduction of the LAIR-1 decoy protein, LAIR-2, sensitizes previously resistant lung tumors to programmed death-1 (PD-1) blockade, indicating the potential of LAIR-1 as an alternative marker for anti-PD-1 resistance in lung cancer. Here, we assessed LAIR-1 as compared with programmed death-ligand 1 (PD-L1) expression in various tumors, with a focus on non–small cell lung cancer (NSCLC) and its histologic subtypes using multiplexed quantitative immunofluorescence (mQIF) in 287 (discovery cohort) and 144 (validation cohort) patients with NSCLC. In addition, using multispectral imaging technology on mQIF images, we evaluated the localization of LAIR-1 on various cell types. We observed that CD14+, CD68+, and CD163+ monocytes and CK+ tumor cells predominantly expressed LAIR-1 more than other cell types. Furthermore, LAIR-1 expression in the tumor compartment was significantly higher in patients with lung adenocarcinoma (LUAD) than those with lung squamous cell carcinoma subtype (**, P = 0.003). Our results indicated that high tumor LAIR-1 expression in patients with LUAD is negatively associated with OS (overall survival, HR = 2.4; *, P = 0.02) highlighting its prognostic value in LUAD but not in other subtypes. The Pearson correlation between LAIR-1 and PD-L1 is 0.31; however, mutual exclusive staining pattern (i.e., several cases were positive for LAIR-1 and negative for PD-L1) was observed. Altogether, our data suggest that the combination therapy of anti-PD-1/PD-L1 with anti-LAIR-1 or the anti-LAIR-1 monotherapy alone may be promising cancer immunotherapeutic strategies.Significance:The spatial, quantitative assessment of LAIR-1 in NSCLC shows positive association of OS with high LAIR-1+/CD68+ cell densities and negative association of OS with high LAIR-1 expression in LUAD tumor subtype.

To evaluate the longitudinal association between plant-based diet index (PDI) score and incident erectile dysfunction (ED).We conducted a prospective analysis of 21 942 men aged 40 to 75 years who were enrolled in the Health Professionals Follow-Up Study. ED was assessed with questionnaires every 4 years starting in 2000. Dietary data were collected via validated food frequency questionnaires completed every 4 years and were used to calculate total PDI scores, as well as healthy (hPDI) and unhealthy (uPDI) subscores. Multivariable Cox proportional hazards models were used to compute hazard ratios (HRs) for incident ED. All models were stratified by age (60, 60 to70, ≥70 years).Among men aged 60 to70 years, hPDI was inversely associated with incident ED. Those in the highest quintile of hPDI in that age group had an 18% lower risk of ED (HR 0.82, 95% confidence interval (CI) 0.73-0.91; P-trend0.001) compared to those in the lowest quintile. Conversely, uPDI was positively associated with ED in men aged60 years (HR 1.27, 95% CI 1.01-1.60; P-trend = 0.02).Encouraging a healthy plant-based diet may be an environmentally sustainable intervention for men interested in maintaining erectile function.

BACKGROUND: Evidence regarding dietary phytoestrogens in relation to mortality remains limited.OBJECTIVES: The objective of the study is to examine the associations of intake of isoflavones, lignans, and coumarins with total and cause-specific mortality in US males and females.METHODS: We followed 75,981 females in the Nurses' Health Study (1984-2018) and 44,001 males in the Health Professionals Follow-up Study (1986-2018), who were free of cardiovascular disease (CVD), diabetes, or cancer at baseline. Their diet was repeatedly assessed using validated food frequency questionnaires every 2-4 y. Associations with mortality were assessed using time-dependent Cox models with adjustments for demographics, dietary and lifestyle factors, and medical history.RESULTS: During 3,427,156 person-years of follow-up, we documented 50,734 deaths, including 12,492 CVD deaths, 13,726 cancer deaths, and 24,516 other non-CVD and noncancer deaths. After multivariable adjustment, the higher total phytoestrogen intake was associated with lower risk of total CVD and other non-CVD and noncancer mortality: comparing extreme quintiles, the pooled HRs (95% CIs) were 0.89 (0.87, 0.92), 0.90 (0.85, 0.96), and 0.86 (0.82, 0.90), respectively. We did not find a significant association with cancer mortality [0.97 (0.92, 1.03)]. For individual phytoestrogens in relation to total mortality, the pooled HRs (95% CIs) comparing extreme quintiles were 0.90 (0.87, 0.92) for isoflavones, 0.93 (0.90, 0.96) for lignans, and 0.93 (0.90, 0.95) for coumarins. Individual phytoestrogens were also significantly associated with lower risk of CVD mortality and other types of mortality. Primary food sources of phytoestrogens, including tofu, soy milk, whole grains, tea, and flaxseed, were also inversely associated with total mortality.CONCLUSIONS: A higher intake of total phytoestrogens, including isoflavones, lignans, and coumarins, and foods rich in these compounds was associated with lower risk of total and certain cause-specific mortality in generally healthy US adults. These data suggest that these phytochemicals and their dietary sources may be integrated into an overall healthy diet to achieve a longer life span.

The National Academy of Medicine recommends that the U.S. adopt an interpretative front-of-package food labeling system, but uncertainty remains about how this system should be designed. This study examined reactions to front-of-package food labeling systems that use positive labels to identify healthier foods, negative labels to identify unhealthier foods, or both.In August 2021, U.S. adults (N=3,051) completed an online randomized experiment. Participants were randomly assigned to 1 of 4 labeling conditions: control (calorie), positive, negative, or both positive and negative labels. Labels were adapted from designs for a 'healthy' label drafted by the Food and Drug Administration and displayed on the front of product packaging. Participants selected products to purchase, identified healthier products, and reported reactions to the labels. Analyses, conducted in 2022, examined the healthfulness of participants' selections using the Ofcom Nutrient Profiling Model score (0-100, higher scores indicate being healthier).Participants exposed to only positive labels, only negative labels, or both positive and negative labels had healthier selections than participants in the control arm (differences vs control=1.13 [2%], 2.34 [4%] vs 3.19 [5%], respectively; all p0.01). The both-positive-and-negative-labels arm outperformed the only-negative-labels (p=0.03) and only-positive-labels (p0.001) arms. The only-negative-labels arm outperformed the only-positive-labels arm (p=0.005). All the 3 interpretative labeling systems also led to improvements in the identification of healthier products and beneficial psychological reactions (e.g., attention, thinking about health effects; all p0.05).Front-of-package food labeling systems that use both positive and negative labels could encourage healthier purchases and improve understanding more than systems using only positive or only negative labels.

Purpose The Supplemental Nutrition Assistance Program (SNAP) Online Purchasing Pilot (OPP) allows for the online purchase of groceries using SNAP benefits. First implemented in California in late April 2020, little is known about program usage. This study assessed initial implementation of SNAP Online in California using SNAP OPP transaction data from April - October 2020. Insights can identify usage differences by demographics, store availability, and rurality to help inform future pilot programs and nutrition initiatives. Design Using generalized estimating equations, we modeled county-level associations between transactions and county-level demographics, rurality, and retailer availability. Setting Transaction data from California’s Department of Social Services (CDSS) was linked with publicly-available, county-level demographics. Subjects Anonymized county-level data on SNAP Online transactions and CalFresh households. Measures The primary outcome was successful SNAP Online food transactions per county. Analysis Generalized estimating equation models with clustering by county was used. Results During the first 7 months, median SNAP Online transactions per county per month was 665; 2.7% of total SNAP redemptions were from SNAP Online. Counties with more female-led, disabled, Latino, or Asian CalFresh households had fewer Amazon transactions. Each additional Walmart per county corresponded to 260.7 more Walmart transactions (P < .001). Each percent increase in county zip codes covered by Amazon Fresh corresponded to 45.4 fewer Walmart transactions (P < .05) and 37.3 more Amazon transactions (P < .001). Conclusion Number of stores per county was associated with greater online grocery transactions, whereas rurality was not. County-level SNAP demographics correlated with transactions at particular retailers.

The efficacy of the antibody drug conjugate (ADC) Trastuzumab deruxtecan (T-DXd) in HER2 low breast cancer patients suggests that the historical/conventional assays for HER2 may need revision for optimal patient care. Specifically, the conventional assay is designed to distinguish amplified HER2 from unamplified cases but is not sensitive enough to stratify the lower ranges of HER2 expression. Here we determine the optimal dynamic range for unamplified HER2 detection in breast cancer and then redesign an assay to increase the resolution of the assay to stratify HER2 expression in unamplified cases. We used the AQUA™ method of quantitative immunofluorescence to test a range of antibody concentrations to maximize the sensitivity within the lower range of HER2 expression. Then, using a cell line microarray with HER2 protein measured by mass spectrometry we determined the amount of HER2 protein in units of attomols/mm

Immune checkpoint blockade with programmed cell death (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors has resulted in significant progress in the treatment of various cancer types. However, not all patients respond to PD-1/PD-L1 blockade, underscoring the importance of identifying new potential targets for immunotherapy. One promising target is the immune system modulator Siglec-15. In this study, we assess Siglec-15 expression in solid tumors, with a focus on lung, breast, head and neck squamous and bladder cancers. Using quantitative immunofluorescence (QIF) with a previously validated antibody, we found increased Siglec-15 expression in both tumor and immune cells in all the four cancer types. Siglec-15 was seen to be predominantly expressed by the stromal immune cells (83% in lung, 70.1% in breast, 95.2% in head and neck squamous cell and 89% in bladder cancers). Considerable intra-tumoral heterogeneity was noted across cancer types. As previously described for non-small cell lung cancer (NSCLC), Siglec-15 expression was seen to be mutually exclusive to PD-L1 in all the four cancer types, although this differential expression was maintained but somewhat diminished in head and neck squamous cell carcinoma (HNSCC). Siglec-15 was not prognostic either for overall survival (OS) or progression-free survival (PFS). In summary, we show broad expression of this potential immune modulatory target in a wide range of cancer types. These data suggest potential future clinical trials in these tumor types.

Background: Long-term noise exposure is associated with cardiovascular disease (CVD), including acute cardiovascular events such as myocardial infarction and stroke. However, longitudinal cohort studies of long-term noise and CVD are almost exclusively from Europe, and few modelled nighttime and daytime noise separately. We aimed to examine the prospective association of outdoor long-term nighttime and daytime noise from anthropogenic sources with incident CVD using a US-based, nationwide cohort of women. Methods: We linked L50 (median) nighttime and L50 daytime modelled anthropogenic noise estimates from a US National Park Service model to geocoded residential addresses of 114,116 participants in the Nurses’ Health Study. We used time-varying Cox proportional hazards models to estimate risk of incident CVD, coronary heart disease (CHD), and stroke associated with long-term average noise exposure, adjusted for potential individual- and area-level confounders and CVD risk factors (1988-2018). We assessed effect modification by population density, region, air pollution, vegetation cover, and neighborhood socioeconomic status, and explored mediation by self-reported average nightly sleep duration. Results: Over 2,544,035 person-years, there were 10,331 incident CVD events. In fully-adjusted models, the hazard ratios for each interquartile range increase in L50 nighttime noise (3.67 dBA) and L50 daytime noise (4.35 dBA), respectively, were 1.04 (95% CI 1.02, 1.06) and 1.04 (95% CI 1.02, 1.07). Similar associations were observed for CHD and stroke. Stratified analyses suggested that associations of nighttime and daytime noise with CVD did not differ by prespecified effect modifiers. We found no evidence that inadequate sleep (< 5 hours per night) mediated associations of noise and CVD. Discussion: Outdoor median nighttime and daytime noise at the residential address was associated with a small increase in CVD risk in a cohort of adult female nurses.

Teachers’ belief systems about the inclusion of students with special needs may explain gaps between policy and practice. We investigated three inter-related aspects of teachers’ belief systems: teachers’ cognitive appraisals (e.g., attitudes), emotional appraisal (e.g., feelings), and self-efficacy (e.g., agency to teach inclusive classrooms). To date, research in this field has produced contradictory findings, resulting in a sparse understanding of why teachers differ in their belief systems about inclusive education, and how teachers’ training experiences contribute to their development of professional beliefs. We used meta-analysis to describe the level and range of teachers’ beliefs about inclusive education, and examine factors that contribute to variation in teachers’ beliefs, namely (1) the point in teachers’ career (pre-service versus in-service), (2) training in special versus regular education, and (3) the effects of specific programs and interventions. We reviewed 102 papers (2000–2020) resulting in 191 effect sizes based on research with 40,898 teachers in 40 countries. On average, teachers’ cognitive appraisals, emotional appraisals, and efficacy about inclusion were found to be in the mid-range of scales, indicating room for growth. Self-efficacy beliefs were higher for preservice (M = 3.69) than for in-service teachers (M = 3.13). Teachers with special education training held more positive views about inclusion than regular education teachers (d = 0.41). Training and interventions related to improved cognitive appraisal (d = 0.63), emotional appraisal (d = 0.63), and self-efficacy toward inclusive practices (d = 0.93). The training was particularly effective in encouraging reflection of beliefs and, eventually, facilitating belief change when teachers gained practical experience in inclusive classrooms. Six key findings direct the next steps.

Dietary patterns (DP) rich in plant foods are associated with improved health and reduced non-communicable disease risk. In October 2021, the Nutrition Society hosted a member-led conference, held online over 2 half days, exploring the latest research findings examining plant-rich DP and health. The aim of the present paper is to summarise the content of the conference and synopses of the individual speaker presentations are included. Topics included epidemiological analysis of plant-rich DP and health outcomes, the effects of dietary interventions which have increased fruit and vegetable (FV) intake on a range of health outcomes, how adherence to plant-rich DP is assessed, the use of biomarkers to assess FV intake and a consideration of how modifying behaviour towards increased FV intake could impact environmental outcomes, planetary health and food systems. In conclusion, although there are still considerable uncertainties which require further research, which were considered as part of the conference and are summarised in this review, adopting a plant-rich DP at a population level could have a considerable impact on diet and health outcomes, as well as planetary health.

Background: The 2018 physical activity guidelines for Americans recommend a minimum of 150 to 300 min/wk of moderate physical activity (MPA), 75 to 150 min/wk of vigorous physical activity (VPA), or an equivalent combination of both. However, it remains unclear whether higher levels of long-term VPA and MPA are, independently and jointly, associated with lower mortality. Methods: A total of 116 221 adults from 2 large prospective US cohorts (Nurses’ Health Study and Health Professionals Follow-up Study, 1988–2018) were analyzed. Detailed self-reported leisure-time physical activity was assessed with a validated questionnaire, repeated up to 15 times during the follow-up. Cox regression was used to estimate the hazard ratio and 95% CI of the association between long-term leisure-time physical activity intensity and all-cause and cause-specific mortality. Results: During 30 years of follow-up, we identified 47 596 deaths. In analyses mutually adjusted for MPA and VPA, hazard ratios comparing individuals meeting the long-term leisure-time VPA guideline (75–149 min/wk) versus no VPA were 0.81 (95% CI, 0.76–0.87) for all-cause mortality, 0.69 (95% CI, 0.60–0.78) for cardiovascular disease (CVD) mortality, and 0.85 (95% CI, 0.79–0.92) for non-CVD mortality. Meeting the long-term leisure-time MPA guideline (150–299 min/wk) was similarly associated with lower mortality: 19% to 25% lower risk of all-cause, CVD, and non-CVD mortality. Compared with those meeting the long-term leisure-time physical activity guidelines, participants who reported 2 to 4 times above the recommended minimum of long-term leisure-time VPA (150–299 min/wk) or MPA (300–599 min/wk) showed 2% to 4% and 3% to 13% lower mortality, respectively. Higher levels of either long-term leisure-time VPA (≥300 min/wk) or MPA (≥600 min/wk) did not clearly show further lower all-cause, CVD, and non-CVD mortality or harm. In joint analyses, for individuals who reported Conclusions: The nearly maximum association with lower mortality was achieved by performing ≈150 to 300 min/wk of long-term leisure-time VPA, 300 to 600 min/wk of long-term leisure-time MPA, or an equivalent combination of both.

Despite the clinical efficacy of immune checkpoint inhibitors (ICIs) in NSCLC, only approximately 20% of patients remain disease-free at 5 years. Here, we use digital spatial profiling to find candidate biomarker proteins associated with ICI resistance.Pretreatment samples from 56 patients with NSCLC treated with ICI were analyzed using the NanoString GeoMx digital spatial profiling method. A panel of 71 photocleavable oligonucleotide-labeled primary antibodies was used for protein detection in four molecular compartments (tumor, leukocytes, macrophages, and immune stroma). Promising candidates were orthogonally validated with quantitative immunofluorescence. Available pretreatment samples from 39 additional patients with NSCLC who received ICI and 236 non-ICI-treated patients with operable NSCLC were analyzed to provide independent cohort validation.Biomarker discovery using the protein-based molecular compartmentalization strategy allows 284 protein variables to be assessed for association with ICI resistance by univariate analysis using continuous log-scaled data. Of the 71 candidate protein biomarkers, CD66b in the CD45+CD68 molecular compartment (immune stroma) predicted significantly shorter overall survival (OS) (hazard ratio [HR] 1.31, p = 0.016) and was chosen for validation. Orthogonal validation by quantitative immunofluorescence illustrated that CD66b was associated with resistance to ICI therapy but not prognostic for poor outcomes in untreated NSCLC (discovery cohort [OS HR 2.49, p = 0.026], validation cohort [OS HR 2.05, p = 0.046], non-ICI-treated cohort [OS HR 1.67, p = 0.06]).Using the technique, we have discovered that CD66b expression is indicative of resistance to ICI therapy in NSCLC. Given that CD66b identifies neutrophils, further studies are warranted to characterize the role of neutrophils in ICI resistance.

Siglec-15, a member of sialic-acid binding immunoglobulin type lectins, is normally expressed by myeloid cells and upregulated in some human cancers and represents a promising new target for immunotherapy. While PD-L1 blockade is an important strategy for immunotherapy, its effectiveness is limited. The expression of Siglec-15 has been demonstrated to be predominantly mutually exclusive to PD-L1 in certain cancer histologies. Thus, there is significant opportunity for Siglec-15 as an immunotherapeutic target for patients that do not respond to PD-1/PD-L1 inhibition. The aim of this study was to prospectively develop an immunohistochemical (IHC) assay for Siglec-15 to be used as a companion diagnostic for future clinical trials. Here, we create and validate an IHC assay with a novel recombinant antibody to the cytoplasmic domain of Siglec-15. To find an enriched target, this antibody was first used in a quantitative fluorescence (QIF) assay to screen a broad range of tumor histologies to determine tumor types where Siglec-15 demonstrated high expression. Based on this and previous data, we focused on development of a chromogenic IHC assay for lung cancer. Then we developed a scoring system for this assay that has high concordance amongst pathologist readers. We then use this chromogenic IHC assay to test the expression of Siglec-15 in two cohorts of NSCLC. We found that this assay shows a higher level of staining in both tumor and immune cells compared to previous QIF assays utilizing a polyclonal antibody. However, similar to that study, only a small percentage of positive Siglec-15 cases showed high expression for PD-L1. This validated assay for Siglec-15 expression may support development of a companion diagnostic assay to enrich for patients expressing the Siglec-15 target for therapy.

Purpose: Interferon (IFN) signaling in the tumor microenvironment is a critical determinant of both response and resistance of cancer to immune checkpoint inhibitors (ICIs). We hypothesized that distinct patterns of IFN signaling in melanoma are associated with clinical response or resistance to ICIs. Experimental Design: Two Tissue Microarrays (TMAs) containing samples from 97 patients with metastatic melanoma who received nivolumab, pembrolizumab, or a combination of ipilimumab and nivolumab at Yale New Haven Hospital between 2011-2017 were randomized into discovery and validation cohorts. Samples were stained and visualized using multiplexed immunofluorescence microscopy for STAT1, STAT1 phosphorylated at Y701 (pSTAT1Y701), and PD-L1, and signals were quantified using the AQUA method of quantitative immunofluorescence. Treatment response was assessed using RECIST criteria, and overall survival was analyzed. For in vitro studies, human melanoma cell lines were stimulated with IFNγ and IFNβ, and Western blotting was performed. Results: Pre-treatment STAT1 levels were higher in responders to ICIs (CR/PR/SD for > 6 months) than in non-responders (SD < 6 months/PD). Higher pre-treatment STAT1 levels were associated with improved survival after ICIs in both the discovery and validation cohorts. Western blot analysis of human melanoma cell lines stimulated with IFN demonstrated distinct patterns of upregulation of STAT1 compared with pSTAT1Y701 and PD-L1. When combining STAT1 and PD-L1 markers, patients with STAT1highPD-L1low tumors had improved survival compared with those with STAT1lowPD-L1high tumors. >Conclusions: STAT1 may better predict melanoma response to ICIs than current strategies, and combined STAT1 and PD-L1 biomarkers may provide insight into IFN-responsive versus IFN-resistant states.

We assessed the predictive value of an image analysis-based tumor-infiltrating lymphocytes (TILs) score for pathologic complete response (pCR) and event-free survival in breast cancer (BC). About 113 pretreatment samples were analyzed from patients with stage IIB-IIIC HER-2-negative BC randomized to neoadjuvant chemotherapy ± bevacizumab. TILs quantification was performed on full sections using QuPath open-source software with a convolutional neural network cell classifier (CNN11). We used easTILs% as a digital metric of TILs score defined as [sum of lymphocytes area (mm2)/stromal area(mm2)] × 100. Pathologist-read stromal TILs score (sTILs%) was determined following published guidelines. Mean pretreatment easTILs% was significantly higher in cases with pCR compared to residual disease (median 36.1 vs.14.8%, p r = 0.606, p

BackgroundHigher baseline intakes of flavonoid-rich foods and beverages are associated with a lower risk of chronic disease and mortality in observational studies. However, associations between changes in intakes and mortality remain unclear. We aimed to evaluate associations between 8-year changes in intakes of (1) individual flavonoid-rich foods and (2) a composite measure (termed the ‘flavodiet’) of foods and beverages that are known to be main contributors to flavonoid intake and subsequent total and cause-specific mortality.MethodsWe evaluated associations between 8-year changes in intakes of (1) individual flavonoid-rich foods and (2) a novel ‘flavodiet’ score and total and cause-specific mortality. We included 55,786 females from the Nurses’ Health Study (NHS) and 29,800 males from the Health Professionals Follow-up Study (HPFS), without chronic disease at baseline in our analyses. Using multivariable-adjusted Cox proportional hazard models, we examined associations of 8-year changes in intakes of (1) flavonoid-rich foods and (2) the flavodiet score with subsequent 2-year lagged 6-year risk of mortality adjusting for baseline intakes. Data were pooled using fixed-effects meta-analyses.ResultsWe documented 15,293 deaths in the NHS and 8988 deaths in HPFS between 1986 and 2018. For blueberries, red wine and peppers, a 5%, 4% and 9% lower risk of mortality, respectively, was seen for each 3.5 servings/week increase in intakes while for tea, a 3% lower risk was seen for each 7 servings/week increase [Pooled HR (95% CI) for blueberries; 0.95 (0.91, 0.99); red wine: 0.96 (0.93, 0.99); peppers: 0.91 (0.88, 0.95); and tea: 0.97 (0.95, 0.98)]. Conversely, a 3.5 servings/week increase in intakes of onions and grapefruit plus grapefruit juice was associated with a 5% and 6% higher risk of total mortality, respectively. An increase of 3 servings per day in the flavodiet score was associated with an 8% lower risk of total mortality [Pooled HR: 0.92 (0.89, 0.96)], and a 13% lower risk of neurological mortality [Pooled HR: 0.87 (0.79, 0.97)], after multivariable adjustments.ConclusionsEncouraging an increased intake of specific flavonoid-rich foods and beverages, namely tea, blueberries, red wine, and peppers, even in middle age, may lower early mortality risk.

Background Higher baseline intakes of flavonoid-rich foods and beverages are associated with a lower risk of chronic disease and mortality in observational studies. However, associations between changes in intakes and mortality remain unclear. We aimed to evaluate associations between 8-year changes in intakes of (1) individual flavonoid-rich foods and (2) a composite measure (termed the ‘flavodiet’) of foods and beverages that are known to be main contributors to flavonoid intake and subsequent total and cause-specific mortality. Methods We evaluated associations between 8-year changes in intakes of (1) individual flavonoid-rich foods and (2) a novel ‘flavodiet’ score and total and cause-specific mortality. We included 55,786 females from the Nurses’ Health Study (NHS) and 29,800 males from the Health Professionals Follow-up Study (HPFS), without chronic disease at baseline in our analyses. Using multivariable-adjusted Cox proportional hazard models, we examined associations of 8-year changes in intakes of (1) flavonoid-rich foods and (2) the flavodiet score with subsequent 2-year lagged 6-year risk of mortality adjusting for baseline intakes. Data were pooled using fixed-effects meta-analyses. Results We documented 15,293 deaths in the NHS and 8988 deaths in HPFS between 1986 and 2018. For blueberries, red wine and peppers, a 5%, 4% and 9% lower risk of mortality, respectively, was seen for each 3.5 servings/week increase in intakes while for tea, a 3% lower risk was seen for each 7 servings/week increase [Pooled HR (95% CI) for blueberries; 0.95 (0.91, 0.99); red wine: 0.96 (0.93, 0.99); peppers: 0.91 (0.88, 0.95); and tea: 0.97 (0.95, 0.98)]. Conversely, a 3.5 servings/week increase in intakes of onions and grapefruit plus grapefruit juice was associated with a 5% and 6% higher risk of total mortality, respectively. An increase of 3 servings per day in the flavodiet score was associated with an 8% lower risk of total mortality [Pooled HR: 0.92 (0.89, 0.96)], and a 13% lower risk of neurological mortality [Pooled HR: 0.87 (0.79, 0.97)], after multivariable adjustments. Conclusions Encouraging an increased intake of specific flavonoid-rich foods and beverages, namely tea, blueberries, red wine, and peppers, even in middle age, may lower early mortality risk.

expression of immune-related and immune-checkpoint markers in B2M high vs low groups

signal to noise ratio plots for tumor, leukocyte and macrophage compartments

b2m expression range, multivariate analysis of pfs and os and association with response and disease control in Yale cohort

b2m expression range, multivariate analysis of pfs and os and association with response and disease control in Athens cohort

LAIR-1 expression in different molecular subtypes of within Storma and Tumor. (A) Umap (Uniform Manifold Approximation and Projection) plot showing the expression of LAIR-1 RNA in patient lung tumor biopsies (n=7) derived single cell RNA-seq data. (B) Quantification of LAIR-1 expression in LM22 immune cells showing highest expression of LAIR-1 in macrophages.

correlation of b2m protein with B2M mRNA expression in Athens cohort. Association of B2M mRNA levels with response and disease control

Target-protein panels included in the GeoMxTM Immuno-Oncology Human Protein Assay

association of B2M mRNA expression with OS in the non-immunotherapy treated TCGA HNSCC ccohort

Clinicopathological characteristics of NSCLC validation cohort (YTMA-250).

Programmed cell death protein‐1 (PD-1)-targeted immunotherapy is approved for recurrent or metastatic head and neck squamous cell carcinoma (R/M HNSCC) treatment. Although its efficacy correlates with PD-L1 expression, response is limited even among positive cases. We employed digital spatial profiling (DSP) to discover potential biomarkers of immunotherapy outcomes in HNSCC. Fifty prospectively collected, pretreatment biopsy samples from patients with anti-PD-1–treated R/M HNSCC, were assessed using DSP, for 71 proteins in four molecularly defined compartments (tumor, leukocyte, macrophage, and stroma). Markers were evaluated for associations with progression-free (PFS) and overall survival (OS). High beta-2 microglobulin (B2M), LAG-3, CD25, and 4-1BB in tumor; high B2M, CD45, CD4 in stroma, and low fibronectin in the macrophage compartment, correlated with prolonged PFS. Improved PFS and OS were observed for cases with high B2M by quantitative and mRNA. Findings were validated in an independent cohort for PFS (HR, 0.41; 95% confidence interval, 0.19–0.93; P = 0.034). B2M-high tumors showed enrichment with immune cell and immune checkpoint markers. Our study illustrates B2M expression is associated with improved survival for immune checkpoint inhibitor (ICI)-treated HNSCC.Significance:In the current study, DSP revealed the positive association of B2M expression in the tumor compartment with immunotherapy outcomes in R/M HNSCC.

LAIR-1 expression in different compartments and its association with survival in Validation Cohort 250 (YTMA-250). (A) LAIR-1 protein expression in tumor and stroma compartments. (B) LAIR-1 tumor and stromal protein expression in both LUAD and LUSC-NSCLC subtypes. (C) Association of LAIR-1 expression in tumor and CD68 compartments with the outcome.

Representative figures of staining by different antibody clones. Representative QIF images of different LAIR-1 clones staining targeting non-overlapping epitopes in a lung test array (YTMA–295) containing 35 lung tumor cores stained by (A) E7X61 clone, (B) NKT25 clone, (C) 1A10 clone and (D) 1E4 clone. (E) The staining quantification of the four LAIR-1 clones. CK: green, LAIR-1: red, DAPI: blue. The fluorescent channels where each marker was acquired were shown in brackets.

Targeting the interaction of leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) and its ligands has been shown to reinstate antitumor immunity. In addition, the introduction of the LAIR-1 decoy protein, LAIR-2, sensitizes previously resistant lung tumors to programmed death-1 (PD-1) blockade, indicating the potential of LAIR-1 as an alternative marker for anti-PD-1 resistance in lung cancer. Here, we assessed LAIR-1 as compared with programmed death-ligand 1 (PD-L1) expression in various tumors, with a focus on non–small cell lung cancer (NSCLC) and its histologic subtypes using multiplexed quantitative immunofluorescence (mQIF) in 287 (discovery cohort) and 144 (validation cohort) patients with NSCLC. In addition, using multispectral imaging technology on mQIF images, we evaluated the localization of LAIR-1 on various cell types. We observed that CD14+, CD68+, and CD163+ monocytes and CK+ tumor cells predominantly expressed LAIR-1 more than other cell types. Furthermore, LAIR-1 expression in the tumor compartment was significantly higher in patients with lung adenocarcinoma (LUAD) than those with lung squamous cell carcinoma subtype (**, P = 0.003). Our results indicated that high tumor LAIR-1 expression in patients with LUAD is negatively associated with OS (overall survival, HR = 2.4; *, P = 0.02) highlighting its prognostic value in LUAD but not in other subtypes. The Pearson correlation between LAIR-1 and PD-L1 is 0.31; however, mutual exclusive staining pattern (i.e., several cases were positive for LAIR-1 and negative for PD-L1) was observed. Altogether, our data suggest that the combination therapy of anti-PD-1/PD-L1 with anti-LAIR-1 or the anti-LAIR-1 monotherapy alone may be promising cancer immunotherapeutic strategies.Significance:The spatial, quantitative assessment of LAIR-1 in NSCLC shows positive association of OS with high LAIR-1+/CD68+ cell densities and negative association of OS with high LAIR-1 expression in LUAD tumor subtype.

Antibody Validation. (A) LAIR-1 mean RNA expression across nine tumor types. Representative QIF images of antibody validation using four different anti-LAIR-1 antibody clones (B) E7X61, (C) NKT25, (D) 1A10 and (E) 1E4 targeting non-overlapping epitopes in a lung test array (YTMA–295) containing 35 lung tumor cores. CK: green, LAIR-1: red, DAPI: blue. Three clones NKT25, 1A10 and 1E4 were tested earlier in the experiment hence the serial TMA sections and E7X61 was tested in a TMA a few sections away from the earlier tested TMAs later at the stage of the experiment. The fluorescent channels where each marker was acquired were shown in brackets. (F) Correlation of LAIR-1 protein expression scores measured by AQUA between four antibody clones. (G) Correlation plot showing the good reproducibility of staining by 1E4 clone between two independent experiments. (H) Correlation plot showing high expression LAIR-1 in both tumor and stroma.

Neuronal Calcium Sensor 1 (NCS-1) is a multi-functional Ca2+-binding protein that affects a range of cellular processes beyond those related to neurons. Functional characterization of NCS-1 in neuronal model systems suggests that NCS-1 may influence oncogenic processes. To this end, the biological role of NCS-1 was investigated by altering its endogenous expression in MCF-7 and MB-231 breast cancer cells. Overexpression of NCS-1 resulted in a more aggressive tumor phenotype demonstrated by a marked increase in invasion and motility, and a decrease in cell-matrix adhesion to collagen IV. Overexpression of NCS-1 was also shown to increase the efficacy of paclitaxel-induced cell death in a manner that was independent of cellular proliferation. To determine the association between NCS-1 and clinical outcome, NCS-1 expression was measured in two independent breast cancer cohorts by the Automated Quantitative Analysis method of quantitative immunofluorescence. Elevated levels of NCS-1 were significantly correlated with shorter survival rates. Furthermore, multivariate analysis demonstrated that NCS-1 status was prognostic, independent of estrogen receptor, progesterone receptor, HER2, and lymph node status. These findings indicate that NCS-1 plays a role in the aggressive behavior of a subset of breast cancers and has therapeutic or biomarker potential.Implications: NCS-1, a calcium-binding protein, is associated with clinicopathologic features of aggressiveness in breast cancer cells and worse outcome in two breast cancer patient cohorts. Mol Cancer Res; 15(7); 942–52. ©2017 AACR.

Table with Details of Patient Characteristics

APM mutation and copy number alterations

Interferon pathway mutation and copy number alterations

Methods for quantitative immunofluorescence and IFN gamma treatment of PDXs