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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (Krange= 0.67-0.75 and Krange= 0.61-0.69, respectively), and "moderate" for the cytoplasmic IHC marker (Krange= 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 (Knuclear = 0.74; Kmembranous = 0.73; Kcytopasmic = 0.57). Interobserver agreement between non-pathologists was "substantial" (Knuclear = 0.78; Kmembranous = 0.72; Kcytopasmic = 0.61). Intraobserver agreement of non-pathologists was "substantial" to "almost perfect" (Knuclear,range = 0.83-0.87; Kmembranous,range = 0.75-0.82; Kcytopasmic = 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Transient activation of the renin-angiotensin system (RAS) induces irreversible renal damage causing sustained elevation in blood pressure (BP) in Cyp1a1-Ren2 transgenic rats. In our current study we hypothesized that activation of the AT1-receptor (AT1R) leads to a T-cell response causing irreversible impairment of renal function and hypertension. Cyp1a1-Ren2 rats harbor a construct for activation of the RAS by indole-3-carbinol (I3C). Rats were fed a I3C diet between 4–8 weeks of age to induce hypertension. Next, I3C was withdrawn and rats were followed-up for another 12 weeks. Additional groups received losartan (20 mg/kg/day) or hydralazine (100 mg/kg/day) treatment between 4–8 weeks. Rats were placed for 24h in metabolic cages before determining BP at week 8, 12 and 20. At these ages, subsets of animals were sacrificed and the presence of kidney T-cell subpopulations was investigated by immunohistochemistry and molecular marker analysis. The development of sustained hypertension was completely prevented by losartan, whereas hydralazine only caused a partial decrease in BP. Markers of renal damage: KIM-1 and osteopontin were highly expressed in urine and kidney samples of I3C-treated rats, even until 20 weeks of age. Additionally, renal expression of regulatory-T cells (Tregs) was highly increased in I3C-treated rats, whereas the expression of T-helper 1 (Th1) cells demonstrated a strong decrease. Losartan prevented these effects completely, whereas hydralazine was unable to affect these changes. In young Cyp1a1-Ren2 rats AT1R activation leads to induction of an immune response, causing a shift from Th1-cells to Tregs, contributing to the development of irreversible renal damage and hypertension. [ABSTRACT FROM AUTHOR]