The relationship between Stroma AReactive Invasion Front Areas (SARIFA), Warburg-subtype and survival: results from a large prospective series of colorectal cancer patients.

BACKGROUND: Stroma AReactive Invasion Front Areas (SARIFA) is a recently identified haematoxylin & eosin (H&E)based histopathologic biomarker in gastrointestinal cancers, including colorectal cancer (CRC), defined as direct contact between tumour cells and adipocytes at the tumour invasion front. The current study aimed at validating the prognostic relevance of SARIFA in a large population-based CRC series as well as at investigating the relationship between SARIFA-status and previously established Warburg-subtypes, both surrogates of the metabolic state of the tumour cells. METHODS: SARIFA-status (positive versus negative) was determined on H&E slides of 1,727 CRC specimens. Warburg-subtype (high versus moderate versus low) data was available from our previous study. The associations between SARIFA-status, Warburg-subtype, clinicopathological characteristics and CRC-specific as well as overall survival were investigated. RESULTS: 28.7% (n=496) CRC were SARIFA-positive. SARIFA-positivity was associated with more advanced disease stage, higher pT category, and more frequent lymph node involvement (all p<0.001). SARIFA-positivity was more common in Warburg-high CRC. 44.2% (n=219) of SARIFA-positive CRCs were Warburg-high compared to 22.8% (n=113) being Warburg-low and 33.1% (n=164) being Warburg-moderate (p<0.001). In multivariable-adjusted analysis, patients with SARIFA-positive CRCs had significantly poorer CRC-specific (HRCRC-specific 1.65; 95% CI 1.41-1.93) and overall survival (HRoverall survival 1.46; 95% CI 1.28-1.67) independent of clinically known risk factors and independent of Warburg-subtype. Combining the SARIFA-status and the Warburg-subtype to a combination score (SARIFA-negative/Warburg-high versus SARIFA-positive/Warburg-low versus SARIFA-positive/Warburg-high, and so on) did not improve the survival prediction compared to the use of SARIFA-status alone (SARIFA-negative + Warburg-high: HRCRC-specific 1.08; 95% CI 0.84-1.38; SARIFA-positive + Warburg-low: HRCRC-specific 1.79; 95% CI 1.32-2.41; SARIFA-positive + Warburg-high: HRCRC-specific 1.58; 95% CI 1.23-2.04). CONCLUSIONS: Our current study is the by far largest external validation of SARIFA-positivity as a novel independent negative prognostic H&E-based biomarker in CRC. In addition, our study shows that SARIFA-positivity is associated with the Warburg-high subtype. Further research is warranted to provide a more mechanistic understanding of the underlying tumour biology. Based on our data, we conclude SARIFA-status should be implemented in pathologic routine practice to stratify CRC patients.

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 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.

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.

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.

Polymorphisms in the mTOR-PI3K-Akt pathway, energy balance-related exposures and colorectal cancer risk in the Netherlands Cohort Study.

BACKGROUND: The mTOR-PI3K-Akt pathway influences cell metabolism and (malignant) cell growth. We generated sex-specific polygenic risk scores capturing natural variation in 7 out of 10 top-ranked genes in this pathway. We studied the scores directly and in interaction with energy balance-related factors (body mass index (BMI), trouser/skirt size, height, physical activity, and early life energy restriction) in relation to colorectal cancer (CRC) risk in the Netherlands Cohort Study (NLCS) (n=120,852). The NLCS has a case-cohort design and 20.3 years of follow-up. Participants completed a baseline questionnaire on diet and cancer in 1986 when 55-69 years old. ~75% of the cohort returned toenail clippings used for DNA isolation and genotyping (n subcohort=3,793, n cases=3,464). To generate the scores, the dataset was split in two and risk alleles were defined and weighted based on sex-specific associations with CRC risk in the other dataset half, because there were no SNPs in the top-ranked genes associated with CRC risk in previous genome-wide association studies at a significance level p<1*10-5. RESULTS: Cox regression analyses showed positive associations between the sex-specific polygenic risk scores and colon but not rectal cancer risk in men and women, with hazard ratios for continuously modeled scores close to 1.10. There was no modifying effect observed of the scores on associations between the energy balance-related factors and CRC risk. However, BMI (in men), non-occupational physical activity (in women), and height (in men and women) were associated with the risk of CRC, in particular (proximal and distal) colon cancer, in the direction as expected in the lower tertiles of the sex-specific polygenic risk scores. CONCLUSIONS: Current data suggest that the mTOR-PI3K-Akt pathway may be involved in colon cancer development. This study thereby sheds more light on colon cancer etiology through use of genetic variation in the mTOR-PI3K-Akt pathway.

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.

Empirical Investigation of Genomic Clusters Associated With Height and the Risk of Postmenopausal Breast and Colorectal Cancer in the Netherlands Cohort Study.

We empirically investigated genomic clusters associated with both height and postmenopausal breast cancer (BC) or colorectal cancer (CRC) (or both) in the Netherlands Cohort Study to unravel shared underlying mechanisms between height and these cancers. The Netherlands Cohort Study (1986-2006) includes 120,852 participants (case-cohort study: nsubcohort = 5,000; 20.3 years of follow-up). Variants in clusters on chromosomes 2, 4, 5, 6 (2 clusters), 10, and 20 were genotyped using toenail DNA. Cluster-specific genetic risk scores were modeled in relation to height and postmenopausal BC and CRC risk using age-adjusted linear regression and multivariable-adjusted Cox regression, respectively. Only the chromosome 10 cluster risk score was associated with all 3 phenotypes in the same sex (women); that is, it was associated with increased height (ßcontinuous = 0.34, P = 0.014), increased risk of hormone-receptor-positive BC (for estrogen-receptor-positive BC, hazard ratio (HRcontinuous score) = 1.10 (95% confidence interval (CI): 1.02, 1.20); for progesterone-receptor-positive BC, HRcontinuous score = 1.15 (95% CI: 1.04, 1.26)), and increased risk of distal colon (HRcontinuous score = 1.13, 95% CI: 1.01, 1.27) and rectal (HRcontinuous score = 1.14, 95% CI: 0.99, 1.30) cancer. The chromosome 10 cluster variants were all annotated to the zinc finger MIZ-type containing 1 gene (ZMIZ1), which is involved in androgen receptor activity. This suggests that hormone-related growth mechanisms could influence both height and postmenopausal BC and CRC.

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.

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.

Nut and peanut butter intake and the risk of colorectal cancer and its anatomical and molecular subtypes: the Netherlands Cohort Study.

Nut intake has been associated with reduced total cancer-related mortality, but evidence for colorectal cancer (CRC) risk is inconclusive. We investigated the associations between nut and peanut butter intake and anatomical CRC subtypes. To account for molecular heterogeneity, associations between nut and peanut butter intake and colorectal tumors harboring APC, KRAS or BRAF mutations, p53 overexpression or microsatellite instability were examined in secondary analyses. In the Netherlands Cohort Study (n = 120 852), lifestyle habits were measured with a questionnaire in 1986. After 20.3 years follow-up, 3567 CRC cases were included in case-cohort analyses. For the analyses of molecular CRC subtypes, 574 cases were included after 7.3 years follow-up. In categorical analyses, total nut intake was not significantly associated with CRC [HR (95% CI) 10+ g/day versus non-consumers = 0.94(0.78-1.15) in men; 0.96(0.75-1.22) in women]. In restricted cubic spline analyses, significant non-linear inverse associations with rectal cancer were observed for total nut, peanut and peanut butter intake in women, and borderline significant non-linear inverse associations for total nut and peanut intake in men. Regarding the molecular CRC subtypes, peanut butter intake was significantly associated with an increased risk of colorectal tumors that did not develop through the serrated neoplasia pathway in men [HR (95% CI) per 5 g/day increment = 1.22(1.07-1.38)]. Nut and peanut butter intake are non-linearly inversely associated with rectal cancer risk in women. In men, nut intake is borderline significantly non-linearly associated with a reduced rectal cancer risk. Peanut butter is associated with an increased risk of colorectal tumors that do not develop through the serrated neoplasia pathway in men.

Investigation of sirtuin 1 polymorphisms in relation to the risk of colorectal cancer by molecular subtype.

Sirtuin 1 (SIRT1), a histone deacetylase, is involved in maintenance of genetic stability, inflammation, immune response, metabolism (energy-sensing molecule) and colorectal tumorigenesis. We investigated SIRT1's specific role in colorectal tumorigenesis by studying SIRT1 polymorphisms in relation to colorectal cancer (CRC) risk by microsatellite instability (MSI) and CpG island methylator phenotype (CIMP) status. The Netherlands Cohort study (NLCS) was initiated in 1986 and includes 120,852 participants in a case-cohort design. CRC tumour samples were available for incident cases between 1989 and 1993. Toenail deoxyribonucleic acid (DNA) was used for genotyping of two SIRT1 tagging variants (rs10997870 and rs12778366). Excluding the first 2.3 years of follow-up, subcohort members and CRC cases with no toenail DNA available and those with low sample call rates, and CRC cases with no tumour DNA available left 3478 subcohort members and 533 CRC cases. Cox regression was utilised to estimate hazard ratios (HRs) for MSI and CIMP positive and negative tumours by SIRT1 genotypes. The results were that the rs12778366 TC/CC versus TT genotype was inversely associated with MSI CRC (HR = 0.41, 95% confidence interval: 0.20, 0.88), while no association was found with the risk of an MSS tumour (TC/CC versus TT carriers: HR = 1.13, 95% CI: 0.89, 1.44). No significant associations were found between other SIRT1 genotypes and CRC subtypes. In conclusion, the results suggest a role for SIRT1 polymorphisms in colorectal tumorigenesis, particularly MSI CRC.

Diabetes mellitus, genetic variants in the insulin-like growth factor pathway and colorectal cancer risk.

Genetic variation in the insulin-like growth factor (IGF) pathway may further increase the risk of colorectal cancer (CRC) associated with type 2 diabetes mellitus (T2DM). Joint effects of T2DM and genetic variation in the IGF pathway on CRC risk can increase mechanistic insights. Participants from the Netherlands Cohort Study (n = 120, 852) completed a baseline questionnaire in 1986 when 55-69 years old (case-cohort, nsubcohort = 5,000, ncases = 3,441 after 16.3 years follow-up). Self-reported DM at baseline with onset at ≥30 years was classified as T2DM. Eighteen single nucleotide polymorphisms (SNPs) from the IGF pathway were aggregated in a genetic risk score (GRS). Cox proportional hazard ratios (HRs) for CRC were estimated according to combinations of T2DM status with GRS tertiles and categories of an IGF1 19-CA repeat polymorphism. Baseline T2DM prevalence was 3.1% in subcohort members and 3.8% in CRC cases. Comparison of combined categories with non-T2DM individuals in the lowest GRS tertile as reference showed that those in the highest GRS tertiles with and without T2DM had significantly increased CRC risks, particularly those with T2DM (HR = 2.28, 95% CI: 1.11, 4.66). As compared to IGF1 19-CA wild-type carriers without T2DM, carrying two IGF1 19-CA variant repeat alleles were associated with a significantly decreased CRC risk in those without T2DM (HR = 0.76, 95% CI: 0.63-0.91). This association was absent when T2DM was present. Our study of joint effects indicated that the presence of unfavorable alleles in the IGF pathway may further increase the risk of CRC associated with T2DM.

Associations of adult-attained height and early life energy restriction with postmenopausal breast cancer risk according to estrogen and progesterone receptor status.

Adult-attained height is a marker for underlying mechanisms, such as cell growth, that may also influence postmenopausal breast cancer (BC) risk, perhaps specifically hormone-sensitive BC subtypes. Early life energy restriction may inhibit these mechanisms, resulting in shorter height and a reduced postmenopausal BC risk. Women (62,573) from the Netherlands Cohort Study completed a self-administered questionnaire in 1986 when 55-69 years old, and were followed-up for 20.3 years (case-cohort: Nsubcohort = 2,438; Ncases = 3,354). Cox multivariable-adjusted hazard ratios (HR) and 95% confidence intervals (CI) were estimated for BC risk overall and by estrogen and progesterone receptor subtypes in relation to height and early life energy restriction during the Hunger Winter, War Years, and Economic Depression. Although energy restriction can only influence longitudinal growth in women exposed before and/or during the growth spurt, it may also influence BC risk when occurring after the growth spurt, possibly through different growth processes. Therefore, Cox analyses were additionally conducted according to timing of energy restriction in relation to the growth spurt. Height was associated with an increased BC risk (HRper 5cm = 1.07, 95%CI:1.01-1.13), particularly hormone receptor-positive BC. Energy restriction before and/or during the growth spurt was associated with a decreased hormone receptor-positive BC risk. Energy restriction during the Hunger Winter increased the estrogen receptor-negative BC risk regardless of the timing of energy restriction. In conclusion, height and energy restriction before and/or during the growth spurt were both associated with hormone receptor-positive BC risk, in the direction as expected, indicating critical exposure windows for hormonal growth-related mechanisms.

Alcohol drinking, ADH1B and ADH1C genotypes and the risk of postmenopausal breast cancer by hormone receptor status: the Netherlands Cohort Study on diet and cancer.

Alcohol consumption has consistently been shown to increase breast cancer (BC) risk. This association may be modified by single nucleotide polymorphisms (SNPs) in alcohol dehydrogenase (ADH) isoenzymes ADH1B and ADH1C. The Netherlands Cohort Study comprises 62 573 women, aged 55-69 years at baseline (1986). Follow-up for postmenopausal BC for 20.3 years was available. Genotyping of six tag SNPs in ADH1B and ADH1C was performed on DNA from toenails. A case-cohort approach was used for analysis (complete data available for nsubcohort = 1301; ncases = 1630). Cox regression models for postmenopausal BC were applied to determine marginal effects of alcohol intake and SNPs using a dominant genetic model, as well as multiplicative interaction of the two. Results were also obtained for subtypes by estrogen receptor (ER) and progesterone receptor (PR) status. Multiple testing was adjusted for by applying the false discovery rate (FDR). Alcohol intake (categorical) increased the risk of postmenopausal BC (Ptrend = 0.031). Trends for ER and PR subgroups followed a similar pattern. Continuous modeling of alcohol resulted in a hazard rate ratio (HR) for overall postmenopausal BC of 1.09 (95% confidence interval: 1.01-1.19) per 10 g/day of alcohol. SNPs were not associated with BC risk. No effect modification of the alcohol-BC association by SNP genotype was seen after FDR correction in overall BC and ER/PR subgroups. In conclusion, alcohol consumption was shown to increase the risk of postmenopausal BC. This association was not significantly modified by common SNPs in ADH1B and ADH1C, neither in overall BC nor in hormone receptor-defined subtypes.

Alcohol intake, ADH1B and ADH1C genotypes, and the risk of colorectal cancer by sex and subsite in the Netherlands Cohort Study.

The alcohol-colorectal cancer (CRC) association may differ by sex and ADH1B and ADH1C genotypes. ADH enzymes oxidize ethanol to acetaldehyde, both of which are human carcinogens. The Netherlands Cohort Study includes 120 852 participants, aged 55-69 years at baseline (1986), and has 20.3 years follow-up (case-cohort: nsubcohort = 4774; ncases = 4597). The baseline questionnaire included questions on alcohol intake at baseline and 5 years before. Using toenail DNA, available for ~75% of the cohort, we successfully genotyped six ADH1B and six ADH1C SNPs (nsubcohort = 3897; ncases = 3558). Sex- and subsite-specific Cox hazard ratios and 95% confidence intervals for CRC were estimated comparing alcohol categories, genotypes within drinkers and alcohol categories within genotype strata. We used a dominant genetic model and adjusted for multiple testing. Alcohol intake increased CRC risk in both sexes, though in women only in the (proximal) colon when in excess of 30 g/day. In male drinkers, ADH1B rs4147536 increased (distal) colon cancer risk. In female drinkers, ADH1C rs283415 increased proximal colon cancer risk. ADH1B rs3811802 and ADH1C rs4147542 decreased CRC risk in heavy (>30 g/day) and stable drinkers (compared to 5 years before baseline), respectively. Rs3811802 and rs4147542 significantly modified the alcohol-colon cancer association in women (Pfor interaction = 0.004 and 0.02, respectively). A difference in associations between genotype strata was generally clearer in men than women. In conclusion, men showed increased CRC risks across subsites and alcohol intake levels, while only colon cancer risk was increased in women at heavy intake levels. ADH1B rs3811802 and ADH1C rs4147542 significantly modified the alcohol-colon cancer association in women.

Lifestyle, Diet, and Colorectal Cancer Risk According to (Epi)genetic Instability: Current Evidence and Future Directions of Molecular Pathological Epidemiology.

PURPOSE OF REVIEW: In this review, we describe molecular pathological epidemiology (MPE) studies from around the world that have studied diet and/or lifestyle factors in relation to molecular markers of (epi)genetic pathways in colorectal cancer (CRC), and explore future perspectives in this realm of research. The main focus of this review is diet and lifestyle factors for which there is evidence for an association with CRC as identified by the World Cancer Research Fund reports. In addition, we review promising hypotheses, that warrant consideration in future studies. RECENT FINDINGS: Associations between molecular characteristics of CRC have been published in relation to smoking, alcohol consumption; body mass index (BMI); waist:hip ratio; adult attained height; physical activity; early life energy restriction; dietary acrylamide, fiber, fat, methyl donors, omega 3 fatty acids; meat, including total protein, processed meat, and heme iron; and fruit and vegetable intake. SUMMARY: MPE studies help identify where associations between diet, lifestyle, and CRC risk may otherwise be masked and also shed light on how timing of exposure can influence etiology. Sample size is often an issue, but this may be addressed in the future by pooling data.

A systematic SNP selection approach to identify mechanisms underlying disease aetiology: linking height to post-menopausal breast and colorectal cancer risk.

Data from GWAS suggest that SNPs associated with complex diseases or traits tend to co-segregate in regions of low recombination, harbouring functionally linked gene clusters. This phenomenon allows for selecting a limited number of SNPs from GWAS repositories for large-scale studies investigating shared mechanisms between diseases. For example, we were interested in shared mechanisms between adult-attained height and post-menopausal breast cancer (BC) and colorectal cancer (CRC) risk, because height is a risk factor for these cancers, though likely not a causal factor. Using SNPs from public GWAS repositories at p-values < 1 × 10-5 and a genomic sliding window of 1 mega base pair, we identified SNP clusters including at least one SNP associated with height and one SNP associated with either post-menopausal BC or CRC risk (or both). SNPs were annotated to genes using HapMap and GRAIL and analysed for significantly overrepresented pathways using ConsensuspathDB. Twelve clusters including 56 SNPs annotated to 26 genes were prioritised because these included at least one height- and one BC risk- or CRC risk-associated SNP annotated to the same gene. Annotated genes were involved in Indian hedgehog signalling (p-value = 7.78 × 10-7) and several cancer site-specific pathways. This systematic approach identified a limited number of clustered SNPs, which pinpoint potential shared mechanisms linking together the complex phenotypes height, post-menopausal BC and CRC.

Nuclear inclusion bodies of mutant and wild-type p53 in cancer: a hallmark of p53 inactivation and proteostasis remodelling by p53 aggregation.

Although p53 protein aggregates have been observed in cancer cell lines and tumour tissue, their impact in cancer remains largely unknown. Here, we extensively screened for p53 aggregation phenotypes in tumour biopsies, and identified nuclear inclusion bodies (nIBs) of transcriptionally inactive mutant or wild-type p53 as the most frequent aggregation-like phenotype across six different cancer types. p53-positive nIBs co-stained with nuclear aggregation markers, and shared molecular hallmarks of nIBs commonly found in neurodegenerative disorders. In cell culture, tumour-associated stress was a strong inducer of p53 aggregation and nIB formation. This was most prominent for mutant p53, but could also be observed in wild-type p53 cell lines, for which nIB formation correlated with the loss of p53's transcriptional activity. Importantly, protein aggregation also fuelled the dysregulation of the proteostasis network in the tumour cell by inducing a hyperactivated, oncogenic heat-shock response, to which tumours are commonly addicted, and by overloading the proteasomal degradation system, an observation that was most pronounced for structurally destabilized mutant p53. Patients showing tumours with p53-positive nIBs suffered from a poor clinical outcome, similar to those with loss of p53 expression, and tumour biopsies showed a differential proteostatic expression profile associated with p53-positive nIBs. p53-positive nIBs therefore highlight a malignant state of the tumour that results from the interplay between (1) the functional inactivation of p53 through mutation and/or aggregation, and (2) microenvironmental stress, a combination that catalyses proteostatic dysregulation. This study highlights several unexpected clinical, biological and therapeutically unexplored parallels between cancer and neurodegeneration. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.