Tumour infiltrating lymphocytes and survival after adjuvant chemotherapy in patients with gastric cancer: post-hoc analysis of the CLASSIC trial.

BACKGROUND: Only a subset of gastric cancer (GC) patients with stage II-III benefits from chemotherapy after surgery. Tumour infiltrating lymphocytes per area (TIL density) has been suggested as a potential predictive biomarker of chemotherapy benefit. METHODS: We quantified TIL density in digital images of haematoxylin-eosin (HE) stained tissue using deep learning in 307 GC patients of the Yonsei Cancer Center (YCC) (193 surgery+adjuvant chemotherapy [S + C], 114 surgery alone [S]) and 629 CLASSIC trial GC patients (325 S + C and 304 S). The relationship between TIL density, disease-free survival (DFS) and clinicopathological variables was analysed. RESULTS: YCC S patients and CLASSIC S patients with high TIL density had longer DFS than S patients with low TIL density (P = 0.007 and P = 0.013, respectively). Furthermore, CLASSIC patients with low TIL density had longer DFS if treated with S + C compared to S (P = 0.003). No significant relationship of TIL density with other clinicopathological variables was found. CONCLUSION: This is the first study to suggest TIL density automatically quantified in routine HE stained tissue sections as a novel, clinically useful biomarker to identify stage II-III GC patients deriving benefit from adjuvant chemotherapy. Validation of our results in a prospective study is warranted.

Energy balance-related factors in childhood and adolescence and risk of colorectal cancer expressing different levels of proteins involved in the Warburg-effect.

Early-life (childhood to adolescence) energy balance-related factors (height, energy restriction, BMI) have been associated with adult colorectal cancer (CRC) risk. Warburg-effect activation via PI3K/Akt-signaling might explain this link. We investigated whether early-life energy balance-related factors were associated with risk of Warburg-subtypes in CRC. We used immunohistochemistry for six proteins involved in the Warburg-effect (LDHA, GLUT1, MCT4, PKM2, P53, and PTEN) on tissue microarrays of 2399 incident CRC cases from the prospective Netherlands Cohort Study (NLCS). Expression levels of all proteins were combined into a pathway-based sum score and categorized into three Warburg-subtypes (Warburg-low/-moderate/-high). Multivariable Cox-regression analyses were used to estimate associations of height, energy restriction proxies (exposure to Dutch Hunger Winter; Second World War [WWII]; Economic Depression) and adolescent BMI with Warburg-subtypes in CRC. Height was positively associated with colon cancer in men, regardless of Warburg-subtypes, and with Warburg-low colon and Warburg-moderate rectal cancer in women. Energy restriction during the Dutch Hunger Winter was inversely associated with colon cancer in men, regardless of Warburg-subtypes. In women, energy restriction during the Hunger Winter and WWII was inversely associated with Warburg-low colon cancer, whereas energy restriction during the Economic Depression was positively associated with Warburg-high colon cancer. Adolescent BMI was positively associated with Warburg-high colon cancer in men, and Warburg-moderate rectal cancer in women. In conclusion, the Warburg-effect seems to be involved in associations of adolescent BMI with colon cancer in men, and of energy restriction during the Economic Depression with colon cancer in women. Further research is needed to validate these results.

Energy balance-related factors in childhood and adolescence and risk of colorectal cancer based on KRAS, PIK3CA, and BRAF mutations and MMR status.

KRAS mutations (KRASmut ), PIK3CAmut , BRAFmut , and deficient DNA mismatch repair (dMMR) have been associated with the Warburg effect. We previously reported differential associations between early-life energy balance-related factors (height, energy restriction, body mass index [BMI]) and colorectal cancer (CRC) subtypes based on the Warburg effect. We now investigated associations of early-life energy balance-related factors and the risk of CRC subgroups based on mutation and MMR status. Data from the Netherlands Cohort Study was used. KRASmut , PIK3CAmut, BRAFmut, and MMR status were available for 2349 CRC cases, and complete covariate data for 1934 cases and 3911 subcohort members. Multivariable-adjusted Cox regression was used to estimate associations of height, energy restriction proxies (exposure to Dutch Hunger Winter, Second World War, Economic Depression), and early adult BMI (age 20 years) with risk of CRC based on individual molecular features and combinations thereof (all-wild-type+MMR-proficient [pMMR]; any-mutation/dMMR). Height was positively associated with any-mutation/dMMR CRC but not all-wild-type+pMMR CRC, with the exception of rectal cancer in men, and with heterogeneity in associations observed for colon cancer in men (p-heterogeneity = 0.049) and rectal cancer in women (p-heterogeneity = 0.014). Results on early-life energy restriction proxies in relation to the risk of CRC subgroups did not show clear patterns. Early adult BMI was positively, but not significantly, associated with KRASmut colon cancer in men and with BRAFmut and dMMR colon cancer in women. Our results suggest a role of KRASmut , PIK3CAmut , BRAFmut , and dMMR in the etiological pathway between height and CRC risk. KRASmut might potentially play a role in associations of early adult BMI with colon cancer risk in men, and BRAFmut and dMMR in women.

Chymase: a multifunctional player in pulmonary hypertension associated with lung fibrosis.

Limited literature sources implicate mast-cell mediator chymase in the pathologies of pulmonary hypertension and pulmonary fibrosis. However, there is no evidence on the contribution of chymase to the development of pulmonary hypertension associated with lung fibrosis, which is an important medical condition linked with increased mortality of patients who already suffer from a life-threatening interstitial lung disease.The aim of this study was to investigate the role of chymase in this particular pulmonary hypertension form, by using a bleomycin-induced pulmonary hypertension model.Chymase inhibition resulted in attenuation of pulmonary hypertension and pulmonary fibrosis, as evident from improved haemodynamics, decreased right ventricular remodelling/hypertrophy, pulmonary vascular remodelling and lung fibrosis. These beneficial effects were associated with a strong tendency of reduction in mast cell number and activity, and significantly diminished chymase expression levels. Mechanistically, chymase inhibition led to attenuation of transforming growth factor ß1 and matrix-metalloproteinase-2 contents in the lungs. Furthermore, chymase inhibition prevented big endothelin-1-induced vasoconstriction of the pulmonary arteries.Therefore, chymase plays a role in the pathogenesis of pulmonary hypertension associated with pulmonary fibrosis and may represent a promising therapeutic target. In addition, this study may provide valuable insights on the contribution of chymase in the pulmonary hypertension context, in general, regardless of the pulmonary hypertension form.

Pollination of Specklinia by nectar-feeding Drosophila: the first reported case of a deceptive syndrome employing aggregation pheromones in Orchidaceae.

BACKGROUND AND AIMS: The first documented observation of pollination in Pleurothallidinae was that of Endrés, who noticed that the 'viscid sepals' of Specklinia endotrachys were visited by a 'small fly'. Chase would later identify the visiting flies as being members of the genus Drosophila. This study documents and describes how species of the S. endotrachys complex are pollinated by different Drosophila species. METHODS: Specimens of Specklinia and Drosophila were collected in the field in Costa Rica and preserved in the JBL and L herbaria. Flies were photographed, filmed and observed for several days during a 2-year period and were identified by a combination of non-invasive DNA barcoding and anatomical surveys. Tissue samples of the sepals, petals and labellum of Specklinia species were observed and documented by SEM, LM and TEM. Electroantennogram experiments were carried out on Drosophila hydei using the known aggregation pheromones ethyl tiglate, methyl tiglate and isopropyl tiglate. Floral compounds were analysed by gas chromatography-mass spectometry using those same pheromones as standards. KEY RESULTS: Flowers of S. endotrachys, S. pfavii, S. remotiflora and S. spectabilis are visited and pollinated by several different but closely related Drosophila species. The flies are arrested by aggregation pheromones, including ethyl tiglate, methyl tiglate and isopropyl tiglate, released by the flowers, and to which at least D. hydei is very sensitive. Visible nectar drops on the adaxial surface of sepals are secreted by nectar-secreting stomata, encouraging male and female Drosophila to linger on the flowers for several hours at a time. The flies frequently show courtship behaviour, occasionally copulating. Several different Drosophila species can be found on a single Specklinia species. CONCLUSIONS: Species of the S. endotrachys group share a similar pollination syndrome. There seem to be no species-specific relationships between the orchids and the flies. It is not expected that Specklinia species will hybridize naturally as their populations do not overlap geographically. The combination of pheromone attraction and nectar feeding is likely to be a generalized pollination syndrome in Pleurothallidinae.

Association between Long-term Energy Balance-Related Factors and Survival in Colorectal Cancer Overall and by Metabolic Warburg-Subtypes.

BACKGROUND: Long-term energy balance-related factors (i.e., lifestyle and physiologic factors that influence the equilibrium between energy intake and energy expenditure over an extended period) such as body mass index (BMI) are linked to colorectal cancer risk, but their impact on colorectal cancer survival is unclear. We explored associations between these long-term energy balance-related factors and survival and examined potential differences across metabolic Warburg-subtypes. METHODS: Associations between long-term energy balance-related factors and survival in the total series of patients with colorectal cancer (n = 2,347) obtained from the prospective Netherlands Cohort Study, as well as according to Warburg-subtype (Warburg-low: n = 652, Warburg-moderate: n = 802, Warburg-high: n = 797), were investigated using Cox regression analysis. RESULTS: Among the long-term energy balance-related factors studied, only increasing prediagnostic BMI was associated with a borderline significant poorer overall survival in patients with colorectal cancer [HR5kg/m2, 1.07; 95% confidence interval (CI), 0.99-1.15]. Stratified analyses showed that prediagnostic weight gain (HR5kg, 1.04; 95% CI, 0.99-1.09) and potentially increased height (HR5cm, 1.04; 95% CI, 0.98-1.11) were associated with poor overall survival only in patients with Warburg-high colorectal cancer. Increasing prediagnostic BMI was associated with poor survival only in patients with Warburg-moderate colorectal cancer (colorectal cancer-specific: HR5kg/m2, 1.12; 95% CI, 0.96-1.32; overall: HR5kg/m2, 1.20; 95% CI, 1.05-1.36). No consistent patterns were observed across energy restriction proxies. CONCLUSIONS: Maintaining a healthy prediagnostic BMI may be beneficial for colorectal cancer survival. Moreover, associations between prediagnostic BMI, weight change, early-life energy restriction, height, and colorectal cancer survival differed according to Warburg-subtypes. IMPACT: Understanding the biologic pathways involved in associations between energy balance-related factors and colorectal cancer survival could help refine prevention strategies in the future.

Relationship between the Warburg effect in tumour cells and the tumour microenvironment in colorectal cancer patients: Results from a large multicentre study.

Colorectal cancer (CRC) remains one of the most prevalent and deadly cancers worldwide. The tumour-node-metastasis stage (TNM) is currently the most clinically important tool to predict prognosis for CRC patients. However, patients with the same TNM stage can have different prognoses. The metabolic status of tumour cells (Warburg-subtype) has been proposed as potential prognostic factor in CRC. However, potential biological mechanisms underlying the relationship between Warburg-subtype and prognosis have not been investigated in detail. One potential mechanism could be that the metabolic status of tumour cells affects the tumour microenvironment (TME). Our objective was to investigate the relationship between Warburg-subtypes and the TME. Haematoxylin/Eosin stained tumour tissue microarray cores from 2171 CRC patients from the Netherlands Cohort Study were semi quantitatively assessed for tumour infiltrating lymphocytes (TILs) and relative tumour stroma content. 5745 cores were assessed by putting each core in one of four categories for both TILs and stroma. The relationship between Warburg-subtype, TILs, and tumour stroma content was investigated. The frequency of CRC in the different TIL categories was (n, %): very low (2538, 44.2), low (2463, 42.9), high (722, 12.6), and very high (22, 0.4). The frequency of CRC in the different tumour stroma content categories was: ≤ 25% (2755, 47.9), > 25% ≤ 50% (1553, 27) > 50% ≤ 75% (905, 15.8), and > 75% (532, 9.3). There was neither an association between Warburg-subtype and tumour stroma content (p = 0.229) nor between Warburg-subtype and TILs (p = 0.429). This is the first study to investigate the relationship between Warburg-subtypes and the TME in a large population-based series of CRC patients. Our data suggest that the prognostic value of Warburg-subtypes cannot be directly attributed to differences in TILs or tumour stroma content. Our results require confirmation in an independent series.

Association between adjuvant therapy and survival in colorectal cancer patients according to metabolic Warburg-subtypes.

PURPOSE: Tumor location and tumor node metastasis (TNM) stage guide treatment decisions in colorectal cancer (CRC) patients. However, patients with the same disease stage do not benefit equally from adjuvant therapy. Hence, there remains an urgent clinical need to identify prognostic and/or predictive biomarker(s) to personalize treatment decisions. In this exploratory study, we investigated whether our previously defined metabolic Warburg-subtypes can predict which CRC patients might derive survival benefit from adjuvant therapy. METHODS: Information regarding treatment (surgery only: n = 1451; adjuvant radiotherapy: n = 82; or adjuvant chemotherapy: n = 260) and Warburg-subtype (Warburg-low: n = 485, -moderate: n = 641, or -high: n = 667) was available for 1793 CRC patients from the Netherlands Cohort Study (NLCS). Kaplan-Meier curves and Cox regression models were used to investigate survival benefit from adjuvant therapy compared to surgery-only for the different Warburg-subtypes. RESULTS: Patients with Warburg-moderate CRC (HRCRC-specific 0.64; 95% CI 0.47-0.86, HRoverall 0.61; 95% CI 0.47-0.80), and possibly Warburg-high CRC (HRCRC-specific 0.86; 95% CI 0.65-1.14, HRoverall 0.82; 95% CI 0.64-1.05), had survival benefit from adjuvant therapy. No survival benefit was observed for patients with Warburg-low CRC (HRCRC-specific 1.07; 95% CI 0.76-1.52, HRoverall 0.95; 95% CI 0.70-1.30). There was a significant interaction between Warburg-subtype and adjuvant therapy for CRC-specific survival (p = 0.049) and overall survival (p = 0.035). CONCLUSION: Our results suggest that Warburg-subtypes may predict survival benefit from adjuvant therapy in CRC patients. A survival benefit from adjuvant therapy was observed for patients with Warburg-moderate and possibly Warburg-high CRC, but not for patients with Warburg-low CRC. Future prospective studies are necessary to validate our findings.

Association between mutational subgroups, Warburg-subtypes, and survival in patients with colorectal cancer.

BACKGROUND: Previous research suggests that Warburg-subtypes are related to potentially important survival differences in colorectal cancer (CRC) patients. In the present study, we investigated whether mutational subgroups based on somatic mutations in RAS, BRAF, PIK3CA, and MET, which are known to promote the Warburg-effect, as well as mismatch repair (MMR) status, hold prognostic value in CRC. In addition, we investigated whether Warburg-subtypes provide additional prognostic information, independent of known prognostic factors like TNM stage. METHODS: CRC patients (n = 2344) from the prospective Netherlands Cohort Study (NLCS) were classified into eight mutually exclusive mutational subgroups, based on observed mutations in RAS, BRAF, PIK3CA, and MET, and MMR status: All-wild-type + MMRproficient , KRASmut  + MMRproficient , KRASmut  + PIK3CAmut  + MMRproficient , PIK3CAmut  + MMRproficient , BRAFmut  + MMRproficient , BRAFmut  + MMRdeficient , other + MMRproficient , and other + MMRdeficient . Kaplan-Meier curves and Cox regression models were used to investigate associations between mutational subgroups and survival, as well as associations between our previously established Warburg-subtypes and survival within these mutational subgroups. RESULTS: Compared to patients with all-wild-type + MMRproficient CRC, patients with KRASmut  + MMRproficient , KRASmut  + PIK3CAmut  + MMRproficient , BRAFmut  + MMRproficient , or other + MMRproficient CRC had a statistically significant worse survival (HRCRC-specific ranged from 1.29 to 1.88). In contrast, patients with other + MMRdeficient CRC had the most favorable survival (HRCRC-specific 0.48). No statistically significant survival differences were observed for the Warburg-subtypes within mutational subgroups. CONCLUSION: Our results highlight the prognostic potential of mutational subgroups in CRC. Warburg-subtypes did not provide additional prognostic information within these mutational subgroups. Future larger-scale prospective studies are necessary to validate our findings and to examine the potential clinical utility of CRC subtyping based on mutational subgroups.

Energy Balance-Related Factors and Risk of Colorectal Cancer Expressing Different Levels of Proteins Involved in the Warburg Effect.

BACKGROUND: Energy balance-related factors [body mass index (BMI), waist circumference, physical activity] have been associated with colorectal cancer risk. Warburg effect activation via PI3K/Akt signaling is one of the proposed mechanisms. We investigated whether energy balance-related factors were associated with risk of Warburg subtypes in colorectal cancer. METHODS: We investigated this using immunohistochemistry for six proteins involved in the Warburg effect (LDHA, GLUT1, MCT4, PKM2, P53, PTEN) on tissue microarrays of 2,399 incident colorectal cancer cases from the prospective Netherlands Cohort Study (ntotal = 120,852; nsubcohort = 5,000; aged 55-69 in 1986; 20.3 years follow-up). Data analyses included 3,911 subcohort members and 1,972 colorectal cancer cases with complete covariate data. Expression levels of all proteins were combined into a pathway-based sum score and categorized into three "Warburg subtypes" (Warburg-low/moderate/high). Multivariable Cox regression analyses were used to estimate associations of BMI, clothing size (waist circumference proxy), and physical activity with Warburg subtypes in colorectal cancer. RESULTS: BMI and clothing size were positively associated with Warburg-moderate and Warburg-high colon cancer risk in men (Pheterogeneity = 0.192). In women, clothing size was positively associated with Warburg-low and Warburg-high colon cancer (Pheterogeneity = 0.005). Nonoccupational physical activity was inversely associated with Warburg-low and Warburg-moderate colon cancer in women (Pheterogeneity = 0.045), but positively associated with Warburg-high rectal cancer in men (Pheterogeneity = 0.089). CONCLUSIONS: The Warburg effect might be involved in associations between adiposity and colon cancer risk, though additional mechanisms could be at play in women as well. The inverse association between physical activity and colon cancer might be explained by mechanisms other than the Warburg effect. IMPACT: Further research is needed to reproduce these results and investigate possible additional mechanisms.

Expression of proteins associated with the Warburg-effect and survival in colorectal cancer.

Previous research has suggested that the expression of proteins related to the Warburg effect may have prognostic value in colorectal cancer (CRC), but results remain inconsistent. Our objective was to investigate the relationship between Warburg-subtypes and patient survival in a large population-based series of CRC patients. In the present study, we investigated the expression of six proteins related to the Warburg effect (LDHA, GLUT1, MCT4, PKM2, p53, PTEN) by immunohistochemistry on tissue microarrays (TMAs) from 2,399 incident CRC patients from the prospective Netherlands Cohort Study. Expression levels of the six proteins were combined into a pathway-based sum-score and patients were categorised into three Warburg-subtypes (low/moderate/high). The associations between Warburg-subtypes and CRC-specific and overall survival were investigated using Kaplan-Meier curves and Cox regression models. CRC patients were classified as Warburg-low (n = 695, 29.0%), Warburg-moderate (n = 858, 35.8%) or Warburg-high (n = 841, 35.1%). Patients with Warburg-high CRC had the poorest CRC-specific [hazard ratio (HR) 1.17; 95% CI 1.00-1.38] and overall survival (HR 1.19; 95% CI 1.05-1.35), independent of known prognostic factors. In stratified analyses, this was particularly true for patients with tumour-node-metastasis (TNM) stage III CRC (HRCRC-specific 1.45; 95% CI 1.10-1.92 and HRoverall 1.47; 95% CI 1.15-1.87), and cancers located in the rectum (HRoverall 1.56; 95% CI 1.15-2.13). To our knowledge, this is the first study to identify the prognostic value of immunohistochemistry-based Warburg-subtypes in CRC. Our data suggest that Warburg-subtypes are related to potentially important differences in CRC survival. Further research is required to validate our findings and to investigate the potential clinical utility of these Warburg-subtypes in CRC.

Energy balance-related factors and risk of colorectal cancer based on KRAS, PIK3CA, and BRAF mutations and MMR status.

INTRODUCTION: KRAS mutations (KRASmut), PIK3CAmut, BRAFmut, and mismatch repair deficiency (dMMR) have been associated with the Warburg-effect. We previously observed differential associations between energy balance-related factors (BMI, clothing-size, physical activity) and colorectal cancer (CRC) subtypes based on the Warburg-effect. We now investigated whether associations between energy balance-related factors and risk of CRC differ between subgroups based on mutation and MMR status. METHODS: Information on molecular features was available for 2349 incident CRC cases within the Netherlands Cohort Study (NLCS), with complete covariate data available for 1934 cases and 3911 subcohort members. Multivariable-adjusted Cox-regression was used to estimate associations of energy balance-related factors with risk of CRC based on individual molecular features (KRASmut; PIK3CAmut; BRAFmut; dMMR) and combinations thereof (all-wild-type + MMR-proficient (pMMR); any-mutation/dMMR). RESULTS: In men, BMI and clothing-size were positively associated with risk of colon, but not rectal cancer, regardless of molecular features subgroups; the strongest associations were observed for PIK3CAmut colon cancer. In women, however, BMI and clothing-size were only associated with risk of KRASmut colon cancer (p-heterogeneityKRASmut versus all-wild-type+pMMR = 0.008). Inverse associations of non-occupational physical activity with risk of colon cancer were strongest for any-mutation/dMMR tumors in men and women, and specifically for PIK3CAmut tumors in women. Occupational physical activity was inversely associated with both combination subgroups of colon cancer in men. CONCLUSION: In men, associations did not vary according to molecular features. In women, a role of KRAS mutations in the etiological pathway between adiposity and colon cancer is suggested, and of PIK3CA mutations between physical activity and colon cancer.

Integrating multiplex immunofluorescent and mass spectrometry imaging to map myeloid heterogeneity in its metabolic and cellular context.

Cells often adopt different phenotypes, dictated by tissue-specific or local signals such as cell-cell and cell-matrix contacts or molecular micro-environment. This holds in extremis for macrophages with their high phenotypic plasticity. Their broad range of functions, some even opposing, reflects their heterogeneity, and a multitude of subsets has been described in different tissues and diseases. Such micro-environmental imprint cannot be adequately studied by single-cell applications, as cells are detached from their context, while histology-based assessment lacks the phenotypic depth due to limitations in marker combination. Here, we present a novel, integrative approach in which 15-color multispectral imaging allows comprehensive cell classification based on multi-marker expression patterns, followed by downstream analysis pipelines to link their phenotypes to contextual, micro-environmental cues, such as their cellular ("community") and metabolic ("local lipidome") niches in complex tissue. The power of this approach is illustrated for myeloid subsets and associated lipid signatures in murine atherosclerotic plaque.

Impaired autonomic regulation of resistance arteries in mice with low vascular endothelial growth factor or upon vascular endothelial growth factor trap delivery.

BACKGROUND: Control of peripheral resistance arteries by autonomic nerves is essential for the regulation of blood flow. The signals responsible for the maintenance of vascular neuroeffector mechanisms in the adult, however, remain largely unknown. METHODS AND RESULTS: Here, we report that VEGF( partial differential/ partial differential) mice with low vascular endothelial growth factor (VEGF) levels suffer defects in the regulation of resistance arteries. These defects are due to dysfunction and structural remodeling of the neuroeffector junction, the equivalent of a synapse between autonomic nerve endings and vascular smooth muscle cells, and to an impaired contractile smooth muscle cell phenotype. Notably, short-term delivery of a VEGF inhibitor to healthy mice also resulted in functional and structural defects of neuroeffector junctions. CONCLUSIONS: These findings uncover a novel role for VEGF in the maintenance of arterial neuroeffector function and may help us better understand how VEGF inhibitors cause vascular regulation defects in cancer patients.

Validity and Reproducibility of Immunohistochemical Scoring by Trained Non-Pathologists on Tissue Microarrays.

BACKGROUND: Scoring of immunohistochemistry (IHC) staining is often done by non-pathologists, especially in large-scale tissue microarray (TMA)-based studies. Studies on the validity and reproducibility of scoring results from non-pathologists are limited. Therefore, our main aim was to assess interobserver agreement between trained non-pathologists and an experienced histopathologist for three IHC markers with different subcellular localization (nucleus/membrane/cytoplasm). METHODS: Three non-pathologists were trained in recognizing adenocarcinoma and IHC scoring by a senior histopathologist. Kappa statistics were used to analyze interobserver and intraobserver agreement for 6,249 TMA cores from a colorectal cancer series. RESULTS: Interobserver agreement between non-pathologists (independently scored) and the histopathologist was "substantial" for nuclear and membranous IHC markers (κrange = 0.67-0.75 and κrange = 0.61-0.69, respectively), and "moderate" for the cytoplasmic IHC marker (κrange = 0.43-0.57). Scores of the three non-pathologists were also combined into a "combination score" (if at least two non-pathologists independently assigned the same score to a core, this was the combination score). This increased agreement with the pathologist (κnuclear = 0.74; κmembranous = 0.73; κcytopasmic = 0.57). Interobserver agreement between non-pathologists was "substantial" (κnuclear = 0.78; κmembranous = 0.72; κcytopasmic = 0.61). Intraobserver agreement of non-pathologists was "substantial" to "almost perfect" (κnuclear,range = 0.83-0.87; κmembranous,range = 0.75-0.82; κcytopasmic = 0.69). Overall, agreement was lowest for the cytoplasmic IHC marker. CONCLUSIONS: This study shows that adequately trained non-pathologists are able to generate reproducible IHC scoring results, that are similar to those of an experienced histopathologist. A combination score of at least two non-pathologists yielded optimal results. IMPACT: Non-pathologists can generate reproducible IHC results after appropriate training, making analyses of large-scale molecular pathological epidemiology studies feasible within an acceptable time frame.

Integrative multiomics analysis of human atherosclerosis reveals a serum response factor-driven network associated with intraplaque hemorrhage.

BACKGROUND: While single-omics analyses on human atherosclerotic plaque have been very useful to map stage- or disease-related differences in expression, they only partly capture the array of changes in this tissue and suffer from scale-intrinsic limitations. In order to better identify processes associated with intraplaque hemorrhage and plaque instability, we therefore combined multiple omics into an integrated model. METHODS: In this study, we compared protein and gene makeup of low- versus high-risk atherosclerotic lesion segments from carotid endarterectomy patients, as judged from the absence or presence of intraplaque hemorrhage, respectively. Transcriptomic, proteomic, and peptidomic data of this plaque cohort were aggregated and analyzed by DIABLO, an integrative multivariate classification and feature selection method. RESULTS: We identified a protein-gene associated multiomics model able to segregate stable, nonhemorrhaged from vulnerable, hemorrhaged lesions at high predictive performance (AUC >0.95). The dominant component of this model correlated with αSMA- PDGFRα+ fibroblast-like cell content (p = 2.4E-05) and Arg1+ macrophage content (p = 2.2E-04) and was driven by serum response factor (SRF), possibly in a megakaryoblastic leukemia-1/2 (MKL1/2) dependent manner. Gene set overrepresentation analysis on the selected key features of this model pointed to a clear cardiovascular disease signature, with overrepresentation of extracellular matrix synthesis and organization, focal adhesion, and cholesterol metabolism terms, suggestive of the model's relevance for the plaque vulnerability. Finally, we were able to corroborate the predictive power of the selected features in several independent mRNA and proteomic plaque cohorts. CONCLUSIONS: In conclusion, our integrative omics study has identified an intraplaque hemorrhage-associated cardiovascular signature that provides excellent stratification of low- from high-risk carotid artery plaques in several independent cohorts. Further study revealed suppression of an SRF-regulated disease network, controlling lesion stability, in vulnerable plaque, which can serve as a scaffold for the design of targeted intervention in plaque destabilization.

Twenty-four-hour exposure to altered blood flow modifies endothelial Ca2+-activated K+ channels in rat mesenteric arteries.

We tested the hypothesis that changes in arterial blood flow modify the function of endothelial Ca2+-activated K+ channels [calcium-activated K+ channel (K(Ca)), small-conductance calcium-activated K+ channel (SK3), and intermediate calcium-activated K+ channel (IK1)] before arterial structural remodeling. In rats, mesenteric arteries were exposed to increased [+90%, high flow (HF)] or reduced blood flow [-90%, low flow (LF)] and analyzed 24 h later. There were no detectable changes in arterial structure or in expression level of endothelial nitric-oxide synthase, SK3, or IK1. Arterial relaxing responses to acetylcholine and 3-oxime-6,7-dichlore-1H-indole-2,3-dione (NS309; activator of SK3 and IK1) were measured in the absence and presence of endothelium, NO, and prostanoid blockers, and 6,12,19,20,25,26-hexahydro-5,27:13,18:21,24-trietheno-11,7-metheno-7H-dibenzo [b,n] [1,5,12,16]tetraazacyclotricosine-5,13-diium dibromide (UCL 1684; inhibitor of SK3) or 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; inhibitor of IK1). In LF arteries, endothelium-dependent relaxation was markedly reduced, due to a reduction in the endothelium-derived hyperpolarizing factor (EDHF) response. In HF arteries, the balance between the NO/prostanoid versus EDHF response was unaltered. However, the contribution of IK1 to the EDHF response was enhanced, as indicated by a larger effect of TRAM-34 and a larger residual NS309-induced relaxation in the presence of UCL 1684. Reduction of blood flow selectively blunts EDHF relaxation in resistance arteries through inhibition of the function of K(Ca) channels. An increase in blood flow leads to a more prominent role of IK1 channels in this relaxation.

Genomic and epigenomic EBF1 alterations modulate TERT expression in gastric cancer.

Transcriptional reactivation of telomerase catalytic subunit (TERT) is a frequent hallmark of cancer, occurring in 90% of human malignancies. However, specific mechanisms driving TERT reactivation remain obscure for many tumor types and in particular gastric cancer (GC), a leading cause of global cancer mortality. Here, through comprehensive genomic and epigenomic analysis of primary GCs and GC cell lines, we identified the transcription factor early B cell factor 1 (EBF1) as a TERT transcriptional repressor and inactivation of EBF1 function as a major cause of TERT upregulation. Abolishment of EBF1 function occurs through 3 distinct (epi)genomic mechanisms. First, EBF1 is epigenetically silenced via DNA methyltransferase, polycomb-repressive complex 2 (PRC2), and histone deacetylase activity in GCs. Second, recurrent, somatic, and heterozygous EBF1 DNA-binding domain mutations result in the production of dominant-negative EBF1 isoforms. Third, more rarely, genomic deletions and rearrangements proximal to the TERT promoter remobilize or abolish EBF1-binding sites, derepressing TERT and leading to high TERT expression. EBF1 is also functionally required for various malignant phenotypes in vitro and in vivo, highlighting its importance for GC development. These results indicate that multimodal genomic and epigenomic alterations underpin TERT reactivation in GC, converging on transcriptional repressors such as EBF1.

Calcitonin gene-related peptide selectively relaxes contractile responses to endothelin-1 in rat mesenteric resistance arteries.

We tested the hypothesis that endothelin-1 (ET-1) modulates sensory-motor nervous arterial relaxation by prejunctional and postjunctional mechanisms. Isolated rat mesenteric resistance arteries were investigated with immunohistochemistry, wire-myography, and pharmacological tools. ET(A)- and ET(B)-receptors could be visualized on the endothelium and smooth muscle and on periarterial fibers containing calcitonin gene-related peptide (CGRP). Arterial contractile responses to ET-1 (0.25-16 nM) were not modified by blockade of ET(B)-receptors, NO-synthase, and cyclooxygenase or desensitization of transient receptor potential cation channel, subfamily V, member 1 (TRPV1) with capsaicin. ET-1 reversed relaxing responses to CGRP in depolarized arteries. This effect was inhibited by ET(A)-antagonists. It was not selective because ET-1 also reversed relaxing responses to Na-nitroprusside (SNP) and because phenylephrine (PHE; 0.25-16 microM) similarly reversed relaxing responses to CGRP or SNP. Conversely, contractile responses to ET-1 were, compared with PHE, hypersensitive to the relaxing effects of the TRPV1-agonist capsaicin and to exogenous CGRP, but not to acetylcholine, forskolin, pinacidil, or SNP. In conclusion, ET-1 does not stimulate sensory-motor nervous arterial relaxation, but ET(A)-mediated arterial contractions are selectively sensitive to relaxation by the sensory neurotransmitter CGRP. This does not involve NO, cAMP, or ATP-sensitive K(+) channels.

Functional antagonism between endogenous neuropeptide Y and calcitonin gene-related peptide in mesenteric resistance arteries.

To test the hypothesis that endogenous neuropeptide Y (NPY) counteracts the vasodilator effects of calcitonin gene-related peptide (CGRP), we used isolated mesenteric resistance arteries of rats and mice. With immunohistochemistry, we observed CGRP-containing fibers along and in the vicinity of a subset of NPY- or tyrosine hydroxylase-immunoreactive fibers. The CGRP1 receptor component calcitonin-related-like receptor was expressed by periarterial nerves and smooth muscle cells, whereas receptor activity-modifying protein 1 was observed primarily on the smooth muscle. In organ chambers, exogenous CGRP caused relaxations that were reversed by exogenous NPY. The effects were inhibited by 1-piperidinecarboxamide, N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]-carbonyl]pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)-methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl) (BIBN4096BS, a CGRP1 receptor antagonist; pK(B) = 8.54 +/- 0.52) and (R)-NZ-(diphenylacetyl)-N-[(4-hydroxyphenyl)methyl]argininamide (BIBP3226, a Y1 antagonist; pK(B) = 7.00 +/- 0.49), respectively. Pretreatment with capsaicin (1 muM; 20 min) and the presence of BIBN4096BS (20 nM) increased contractile responses to K(+) (20-40 mM) and electrical field stimulation (EFS; 1-32 Hz). NPY increased contractile responses to K(+) and BIBP3226 (400 nM) reduced contractile responses to EFS. These effects were inhibited by capsaicin and BIBN4096BS, respectively. Furthermore, the relaxing effect of exogenous CGRP (10 nM) during phenylephrine-induced contraction (30 muM) was reversed by EFS, and this effect was reduced in the presence of BIBP3226. We confirmed that bioactive concentrations of endogenous CGRP and NPY can be released from periarterial sensory-motor and sympathetic nerves, respectively, and we demonstrate for the first time functional antagonism between endogenous NPY and CGRP at the level of the smooth muscle.