Loss of enteric neuronal Ndrg4 promotes colorectal cancer via increased release of Nid1 and Fbln2.

The N-Myc Downstream-Regulated Gene 4 (NDRG4), a prominent biomarker for colorectal cancer (CRC), is specifically expressed by enteric neurons. Considering that nerves are important members of the tumor microenvironment, we here establish different Ndrg4 knockout (Ndrg4-/- ) CRC models and an indirect co-culture of primary enteric nervous system (ENS) cells and intestinal organoids to identify whether the ENS, via NDRG4, affects intestinal tumorigenesis. Linking immunostainings and gastrointestinal motility (GI) assays, we show that the absence of Ndrg4 does not trigger any functional or morphological GI abnormalities. However, combining in vivo, in vitro, and quantitative proteomics data, we uncover that Ndrg4 knockdown is associated with enlarged intestinal adenoma development and that organoid growth is boosted by the Ndrg4-/- ENS cell secretome, which is enriched for Nidogen-1 (Nid1) and Fibulin-2 (Fbln2). Moreover, NID1 and FBLN2 are expressed in enteric neurons, enhance migration capacities of CRC cells, and are enriched in human CRC secretomes. Hence, we provide evidence that the ENS, via loss of Ndrg4, is involved in colorectal pathogenesis and that ENS-derived Nidogen-1 and Fibulin-2 enhance colorectal carcinogenesis.

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.

Prognostic DNA methylation markers for hormone receptor breast cancer: a systematic review.

BACKGROUND: In patients with hormone receptor-positive breast cancer, differentiating between patients with a low and a high risk of recurrence is an ongoing challenge. In current practice, prognostic clinical parameters are used for risk prediction. DNA methylation markers have been proven to be of additional prognostic value in several cancer types. Numerous prognostic DNA methylation markers for breast cancer have been published in the literature. However, to date, none of these markers are used in clinical practice. METHODS: We conducted a systematic review of PubMed and EMBASE to assess the number and level of evidence of published DNA methylation markers for hormone receptor-positive breast cancer. To obtain an overview of the reporting quality of the included studies, all were scored according to the REMARK criteria that were established as reporting guidelines for prognostic biomarker studies. RESULTS: A total of 74 studies were identified reporting on 87 different DNA methylation markers. Assessment of the REMARK criteria showed variation in reporting quality of the studies. Eighteen single markers and one marker panel were studied in multiple independent populations. Hypermethylation of the markers RASSF1, BRCA, PITX2, CDH1, RARB, PCDH10 and PGR, and the marker panel GSTP1, RASSF1 and RARB showed a statistically significant correlation with poor disease outcome that was confirmed in at least one other, independent study. CONCLUSION: This systematic review provides an overview on published prognostic DNA methylation markers for hormone receptor-positive breast cancer and identifies eight markers that have been independently validated. Analysis of the reporting quality of included studies suggests that future research on this topic would benefit from standardised reporting guidelines.

Emerging evidence for CHFR as a cancer biomarker: from tumor biology to precision medicine.

Novel insights in the biology of cancer have switched the paradigm of a "one-size-fits-all" cancer treatment to an individualized biology-driven treatment approach. In recent years, a diversity of biomarkers and targeted therapies has been discovered. Although these examples accentuate the promise of personalized cancer treatment, for most cancers and cancer subgroups no biomarkers and effective targeted therapy are available. The great majority of patients still receive unselected standard therapies with no use of their individual molecular characteristics. Better knowledge about the underlying tumor biology will lead the way toward personalized cancer treatment. In this review, we summarize the evidence for a promising cancer biomarker: checkpoint with forkhead and ring finger domains (CHFR). CHFR is a mitotic checkpoint and tumor suppressor gene, which is inactivated in a diverse group of solid malignancies, mostly by promoter CpG island methylation. CHFR inactivation has shown to be an indicator of poor prognosis and sensitivity to taxane-based chemotherapy. Here we summarize the current knowledge of altered CHFR expression in cancer, the impact on tumor biology and implications for personalized cancer treatment.

Bovine Meat and Milk Factor-like Sequences Are Frequently Detected in Renal Cell Carcinoma Tissues.

Previous studies have indicated a potential role of diet in the pathogenesis of renal cell carcinoma (RCC). Recently, circular bovine meat and milk factor (BMMF) DNAs have been identified in peritumoral tissues of human colon and breast cancers. Here, we investigated the prevalence of the DNA of these novel human pathogenic infectious agents in RCC and adjacent peritumoral renal tissues. DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) RCC and peritumoral kidney tissues, including a test (n = 11) and a validation (n = 152) collection. BMMF1 and BMMF2 consensus primers were designed to screen for the presence of BMMF1- and BMMF2-like DNA. In addition, BMMF-specific PCR was performed on selected cases to test for the presence of additional regions of BMMF1 and BMMF2 genomes. A reference collection of hepatocellular carcinomas (HCCs; n = 60) and adjacent peritumoral liver tissues (n = 50) was also included. Our results demonstrated that BMMF1 and BMMF2 DNAs are frequently found in human RCC tissues and are particularly more prevalent in peritumoral kidney tissues. Of note, BMMF1 and BMMF2 genotype heterogeneity was higher in peritumoral kidney tissues compared to RCC tissues. This is the first study to directly test human FFPE tissues for BMMF1- and BMMF2-like DNA using consensus PCR and demonstrate BMMF DNA in neoplastic and peritumoral kidney tissues. The findings are in line with the recently proposed indirect etiopathogenetic role of BMMFs in, e.g., colorectal carcinogenesis. Follow-up studies are needed to explore the potential role of BMMFs in the etiopathogenesis of RCC.

The CpG island methylator phenotype in colorectal cancer: progress and problems.

In recent years, attention has focused on the biology and potential clinical importance of the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). While it is generally well accepted that etiologically and clinically distinct subgroups exist in this disease, a precise definition of CIMP remains to be established. Here, we summarize existing literature that documents the prevalence of CIMP in CRC, with particular attention to the various methods and definitions used to classify a tumor as CIMP positive. Through a systematic review on both case-series and population based studies, we examined only original research articles reporting on sporadic CRC and/or adenomas in unselected cases. Forty-eight papers published between January 1999 and August 2011 met the inclusion criteria. We describe the use of multiple gene panels, marker threshold values, and laboratory techniques which results in a wide range in the prevalence of CIMP. Because there is no universal standard or consensus on quantifying the phenotype, establishing its true prevalence is a challenge. This bottleneck is becoming increasingly evident as molecular pathological epidemiology continues to offer possibilities for clear answers regarding environmental risk factors and disease trends. For the first time, large, unselected series of cases are available for analysis, but comparing populations and pooling data will remain a challenge unless a universal definition of CIMP and a consensus on analysis can be reached, and the primary cause of CIMP identified.

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.

Histologic re­evaluation of a population­based series of renal cell carcinomas from The Netherlands Cohort Study according to the 2022 ISUP/WHO classification.

The aim of the present study was to re-evaluate 457 renal cell carcinoma (RCC) cases from the Netherlands Cohort Study on Diet and Cancer (NLCS), a large population-based cohort, according to the new 2022 ISUP, Genitourinary Pathology Society and World Health Organisation (WHO) classifications to assess whether newly recognized subtypes of RCC could be found among these cases. These cases were initially evaluated according to the 2004 WHO classification, the Fuhrman grading system and the 3rd version of the Tumor-Node-Metastasis (TNM). Data on tumor size, laterality and date of diagnosis, among other clinicopathological characteristics, were obtained through record linkage with the Netherlands Cancer Registry and the Pathologisch-Anatomisch Landelijk Geautomatiseerd Archief. Digital slides from the NLCS were reviewed by two urogenital pathologists according to the new ISUP grading and the 2022 WHO classification (5th edition). Immunohistochemistry staining for carbonic anhydrase IX was performed on cases with ambiguous morphology. A total of 373 cases of clear cell RCC (ccRCC), 61 cases of papillary RCC (pRCC), 13 cases of chromophobe RCC, 3 cases of collecting duct carcinoma and 4 cases of oncocytoma were identified. The subtyping showed no discrepancy with the previous diagnoses. A comparison of the WHO/ISUP grading to the original Fuhrman grading showed a similar grading in 245 (56.5%) cases of the total ccRCC and pRCC cases. The staging according to the novel TNM classification 8th edition showed a restaging in 286 cases (65.5%). Lymphovascular (microvascular) invasion (LVI) and tumor necrosis (TN) were present in 14.4% and 33.5% of the total number of cases, respectively. Furthermore, the presence of sarcomatoid differentiation in 5.1% and rhabdoid differentiation in 4.2% of the cases was observed. In conclusion, none of the newly accepted and emerging/provisional RCC entities were identified in the NLCS cases, which could be attributed to the high mean age (71.4 years) at diagnosis of the patients included in the present study. A restaging of the NLCS cases using the TNM 8th edition and regrading using ISUP grading was performed, which showed that it is possible to report on newer features, such as sarcomatoid differentiation and LVI, even in an old sample collection.

Technical considerations in PCR-based assay design for diagnostic DNA methylation cancer biomarkers.

BACKGROUND: DNA methylation biomarkers for early detection, risk stratification and treatment response in cancer have been of great interest over the past decades. Nevertheless, clinical implementation of these biomarkers is limited, as only < 1% of the identified biomarkers is translated into a clinical or commercial setting. Technical factors such as a suboptimal genomic location of the assay and inefficient primer or probe design have been emphasized as important pitfalls in biomarker research. Here, we use eleven diagnostic DNA methylation biomarkers for colorectal cancer (ALX4, APC, CDKN2A, MGMT, MLH1, NDRG4, SDC2, SFRP1, SFRP2, TFPI1 and VIM), previously described in a systematic literature search, to evaluate these pitfalls. RESULTS: To assess the genomic assay location, the optimal genomic locations according to TCGA data were extracted and compared to the genomic locations used in the published assays for all eleven biomarkers. In addition, all primers and probes were technically evaluated according to several criteria, based on literature and expert opinion. Both assay location and assay design quality varied widely among studies. CONCLUSIONS: Large variation in both assay location and design hinders the development of future DNA methylation biomarkers as well as inter-study comparability.

Evaluation of a seven gene mutational profile as a prognostic factor in a population-based study of clear cell renal cell carcinoma.

In this study, we investigate the influence of the seven genes (VHL, PBRM1, SETD2, BAP1, KDM5C, MTOR and TP53) most frequently mutated in clear cell renal cell cancer (ccRCC) on cancer-specific survival (CSS) in the prospective Netherlands Cohort Study on diet and cancer. DNA isolated from routinely archived formalin-fixed paraffin-embedded tumour blocks from 252 incident ccRCC cases was available for targeted next generation sequencing. Based on the sequencing quality and the completeness of information on clinical characteristics and follow-up, we could use 110 cases for survival analysis. The association with CSS for each mutated gene in these cases was tested using multivariable Cox proportional hazards models to estimate hazards ratios (HR) and confidence intervals (CIs), and we observed mutations in one or more of the seven genes in 64 out of 110 cases (58%). In the multivariable-adjusted analyses, mutations in VHL and PBRM1 were associated with better CSS (HRs (95% CI) 0.34 (0.13‒0.89) and 0.17 (0.04-0.66), respectively), although these results were not statistically significant after multiple testing correction. No association was observed for the other five genes, which may be attributable to limited power.

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 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.

Lessons From a Systematic Literature Search on Diagnostic DNA Methylation Biomarkers for Colorectal Cancer: How to Increase Research Value and Decrease Research Waste?

OBJECTIVES: To improve colorectal cancer (CRC) survival and lower incidence rates, colonoscopy and/or fecal immunochemical test screening are widely implemented. Although candidate DNA methylation biomarkers have been published to improve or complement the fecal immunochemical test, clinical translation is limited. We describe technical and methodological problems encountered after a systematic literature search and provide recommendations to increase (clinical) value and decrease research waste in biomarker research. In addition, we present current evidence for diagnostic CRC DNA methylation biomarkers. METHODS: A systematic literature search identified 331 diagnostic DNA methylation marker studies published before November 2020 in PubMed, EMBASE, Cochrane Library, and Google Scholar. For 136 bodily fluid studies, extended data extraction was performed. STARD criteria and level of evidence were registered to assess reporting quality and strength for clinical translation. RESULTS: Our systematic literature search revealed multiple issues that hamper the development of DNA methylation biomarkers for CRC diagnosis, including methodological and technical heterogeneity and lack of validation or clinical translation. For example, clinical translation and independent validation were limited, with 100 of 434 markers (23%) studied in bodily fluids, 3 of 434 markers (0.7%) translated into clinical tests, and independent validation for 92 of 411 tissue markers (22%) and 59 of 100 bodily fluids markers (59%). DISCUSSION: This systematic literature search revealed that major requirements to develop clinically relevant diagnostic CRC DNA methylation markers are often lacking. To avoid the resulting research waste, clinical needs, intended biomarker use, and independent validation should be better considered before study design. In addition, improved reporting quality would facilitate meta-analysis, thereby increasing the level of evidence and enabling clinical translation.

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.

Loss of SerpinA5 protein expression is associated with advanced-stage serous ovarian tumors.

Epithelial ovarian cancer, the most lethal neoplasm of the female genital tract, is usually diagnosed at an advanced stage as obvious symptoms are absent at early stages. This disease is believed to originate from malignant transformation of the ovarian surface epithelium or fallopian tube. Histologically, several subtypes are being recognized, with serous histology accounting for the majority of cases. Serous tumors include serous borderline tumors and serous carcinomas. A better understanding of the tumor biology and molecular mechanisms involved in these tumors is needed, as both patient management and prognosis differ substantially. Previous microarray analysis identified SerpinA5, a uPA inhibitor, as key regulator for indolent borderline behavior. As carcinomas are characterized by loss of SerpinA5 mRNA expression, we hypothesized that SerpinA5 protein expression is reduced or lost in carcinomas when compared with borderline tumors. We performed SerpinA5 immunohistochemical staining on 32 serous borderline tumors, 187 primary serous carcinomas and 62 serous omental metastases. Reduced or absent SerpinA5 protein staining was observed in carcinomas when compared with borderline tumors (P<0.001). SerpinA5 protein expression was significantly lowered in the omental metastases (P<0.001) when compared with the matching primary carcinoma. Interestingly, SerpinA5 protein expression was reduced in advanced-stage borderline tumors, often characterized by micropapillary growth and/or microinvasion, when compared with early-stage borderline tumors (P=0.015). In conclusion, SerpinA5 expression is significantly reduced in advanced-stage serous borderline tumors and serous carcinomas when compared with the early-stage counterparts, and reduction of expression is linked to more aggressive features of borderline tumors.

Prognostic significance of Gremlin1 (GREM1) promoter CpG island hypermethylation in clear cell renal cell carcinoma.

Gremlin1 (GREM1), a bone morphogenetic protein antagonist and putative angiogenesis-modulating gene, is silenced by promoter hypermethylation in human malignancies. Here we study GREM1 methylation in clear cell renal cell carcinoma (ccRCC) and its impact on tumor characteristics and clinical outcome. Three GREM1 promoter CpG island regions (i, ii, iii) were analyzed by methylation-specific PCR and/or bisulfite sequencing in ccRCC cell lines and ccRCCs from two independent patient series. Results were correlated with clinicopathological and angiogenic parameters. Bisulfite sequencing of ccRCC cell lines showed GREM1 methylation, associated with absence of GREM1 mRNA. GREM1 methylation prevalence in ccRCCs varied between regions: 55%, 24%, and 20% for regions i, ii, and iii, respectively. GREM1 region iii methylation was associated with increased tumor size (P = 0.02), stage (P = 0.013), grade (P = 0.04), tumor (P = 0.001), and endothelial cell (P = 0.0001) proliferation and decreased mean vessel density (P = 0.001) in a hospital-based ccRCC series (n = 150). In univariate analysis, GREM1 region iii methylated ccRCCs had a significant worse survival when compared with unmethylated ccRCCs (hazard ratio [HR] = 2.35, 95% confidence interval [CI]:1.29 to 4.28), but not in multivariate analysis (HR = 0.88, 95% CI: 0.45 to 1.74). In a population-based validation series (n = 185), GREM1 region iii methylation was associated with increased Fuhrman grade (P = 0.03) and decreased overall survival (P = 0.001) in univariate and multivariate analysis (HR = 2.32, 95% CI: 1.52 to 3.53 and HR = 2.27, 95% CI: 1.44 to 3.59, respectively). The strong correlation between GREM1 region iii promoter methylation and increased malignancy and its correlation with active angiogenesis indicates a role for GREM1 in ccRCC carcinogenesis and tumor angiogenesis.

Modified UroVysion scoring criteria increase the urothelial carcinoma detection rate in cases of equivocal urinary cytology.

AIMS: UroVysion(®) is a four-target fluorescence in situ hybridization technique for the detection of urothelial carcinoma (UC) in urinary cytology. The aim of this retrospective study was to investigate the UC detection rate of a modified UroVysion test in patients with equivocal urinary cytology. The modification comprised the addition of a cytological prescreening technique and different evaluation criteria. METHODS AND RESULTS: Thin-layer slides were prepared from the residual urine samples of 82 patients with equivocal urinary cytology, prestained and prescreened to confirm the presence of atypical urothelial cells. The same slides were used for the UroVysion test, and scored according to different evaluation criteria. The results were compared with the outcomes of cystoscopic and histological findings. UroVysion detected 68% of the UCs when the manufacturer's evaluation criteria were applied. In cases of altered evaluation criteria, the sensitivity increased to 81% when at least one copy number change of a probe target was considered to be a positive test result. The specificity only decreased from 84% to 82%. CONCLUSIONS: Our data suggest that the sensitivity of the UroVysion test can be increased by the addition of a cytological pre-screening technique prior to the UroVysion test and a modification of the UroVysion evaluation criteria.

Diagnostic DNA Methylation Biomarkers for Renal Cell Carcinoma: A Systematic Review.

CONTEXT: The 5-yr survival of early-stage renal cell carcinoma (RCC) is approximately 93%, but once metastasised, the 5-yr survival plummets to 12%, indicating that early RCC detection is crucial to improvement in survival. DNA methylation biomarkers have been suggested to be of potential diagnostic value; however, their current state of clinical translation is unclear and a comprehensive overview is lacking. OBJECTIVE: To systematically review and summarise all literature regarding diagnostic DNA methylation biomarkers for RCC. EVIDENCE ACQUISITION: We performed a systematic literature review of PubMed, EMBASE, Medline, and Google Scholar up to January 2019, according to the Preferred Reporting Items for Systematic Review and Meta-Analysis of Diagnostic Test Accuracy Studies (PRISMA-DTA) guidelines. Included studies were scored according to the Standards for Reporting of Diagnostic Accuracy Studies (STARD) criteria. Forest plots were generated to summarise diagnostic performance of all biomarkers. Level of evidence (LoE) and potential risk of bias were determined for all included studies. EVIDENCE SYNTHESIS: After selection, 19 articles reporting on 44 diagnostic DNA methylation biomarkers and 11 multimarker panels were included; however, only 15 biomarkers were independently validated. STARD scores varied from 4 to 13 out of 23 points, with a median of 10 points. Large variation in subgroups, methods, and primer locations was observed. None of the reported biomarkers exceeded LoE III, and the majority of studies reported inadequately. CONCLUSIONS: None of the reported biomarkers exceeded LoE III, indicating their limited clinical utility. Moreover, study reproducibility and further development of these RCC biomarkers are greatly hampered by inadequate reporting. PATIENT SUMMARY: In this report, we reviewed whether specific biomarkers could be used to diagnose the most common form of kidney cancer. We conclude that due to limited evidence and reporting inconsistencies, none of these biomarkers can be used in clinical practice, and further development towards clinical use is hindered.