Biljana Gigic

Suitability of Circulating miRNAs as Potential Prognostic Markers in Colorectal Cancer

miRNAs are crucial in cellular processes and have been shown to be abnormally expressed in cancer tissue and the circulation. Circulating miRNAs may serve as a novel class of minimally invasive biomarkers for prognosis. Within a first methodologic study, we evaluated the miRNA profile kinetics in the plasma of patients with colorectal cancer after surgical tumor removal to identify potential suitability as prognostic biomarkers. This pilot study is based on the ColoCare Study, a cohort study of newly diagnosed patients with stage I–IV colorectal cancer. Colorectal cancer pre- and postsurgical blood (2–7 days after surgery) and 6 months follow-up blood from 35 patients were examined and candidate miRNAs were investigated in the plasma. miRNA levels were measured by two-step qRT-PCR. Statistical analysis was performed using log-transformed normalized CT values using SAS 9.3. Comparing pre- and postsurgical miRNA levels revealed a statistically significant decrease of nine circulating miRNAs after surgery (miR92a, miR18a, miR320a, miR106a, miR16-2, miR20a, miR223, miR17, and miR143). Analyses of plasma levels over all three time points demonstrated a statistically significant decrease from presurgery to postsurgery and re-increase from postsurgery to the six months follow-up time point of four circulating miRNAs (miR92a, miR320a, miR106a, and miR18a). We were able to show for the first time that in plasma miRNA profiles change within days after colorectal cancer surgery. Our results underscore the role of the investigated miRNAs in colorectal cancer and their potential utility as prognostic biomarkers. See all the articles in this CEBP Focus section, “Biomarkers, Biospecimens, and New Technologies in Molecular Epidemiology.” Cancer Epidemiol Biomarkers Prev; 23(12); 2632–7. ©2014 AACR.

Impact of genetic polymorphisms on adenoma recurrence and toxicity in a COX2 inhibitor (celecoxib) trial: results from a pilot study.

Objective Chemoprevention trials have shown that celecoxib reduces adenoma recurrence but can cause cardiovascular toxicity. In this pilot study, we evaluated associations between genetic variation in several candidate pathways (e.g. prostaglandin synthesis) and adenoma recurrence and cardiovascular and gastrointestinal toxicities. Methods Genotyping analysis was carried out on 117 Israeli colorectal adenoma patients who participated in the Prevention of Colorectal Sporadic Adenomatous Polyps trial. Reassessment followed after 3 years on celecoxib and after 2 years from termination of treatment with celecoxib. Efficacy (absence of colorectal adenomas) was measured by colonoscopy at years 1, 3, and 5. Toxicities were assessed by investigators during celecoxib treatment and by self-report post-treatment. A linkage disequilibrium-based selection algorithm (r2≥0.90, MAF≥4%) identified 255 tagSNPs in 25 analyzed candidate genes. Genotyping was performed by using Illumina GoldenGate technology. Results Multiple genetic variants were associated with adenoma recurrence and toxicity. Genetic variability in COX1, COX2, and ALOX12/15 genes played a role in adenoma recurrence, particularly among patients on placebo. More gene variants (especially variants in PGES, CRP, SRC, and GPX3) were associated with increased risk for cardiovascular toxicity and symptoms, compared with gastrointestinal toxicity and symptoms. The increased risk for cardiovascular toxicity/symptoms associated with the SRC gene variants (rs6017996, rs6018256, rs6018257) ranged from 6.61 (95% confidence interval 1.66–26.36, P<0.01) to 10.71 (95% confidence interval 1.96–58.60, P<0.01). Conclusion Genetic polymorphisms in multiple inflammation-related genes appear to interact with celecoxib on adenoma recurrence and its attendant toxicity, particularly cardiovascular toxicity/symptoms. Larger studies validating these pharmacogenetic relationships are needed.

CT-based compartmental quantification of adipose tissue versus body metrics in colorectal cancer patients

AbstractPurposeWhile obesity is considered a prognostic factor in colorectal cancer (CRC), there is increasing evidence that not simply body mass index (BMI) alone but specifically abdominal fat distribution is what matters. As part of the ColoCare study, this study measured the distribution of adipose tissue compartments in CRC patients and aimed to identify the body metric that best correlates with these measurements as a useful proxy for adipose tissue distribution.Materials and methodsIn 120 newly-diagnosed CRC patients who underwent multidetector computed tomography (CT), densitometric quantification of total (TFA), visceral (VFA), intraperitoneal (IFA), retroperitoneal (RFA), and subcutaneous fat area (SFA), as well as the M. erector spinae and psoas was performed to test the association with gender, age, tumor stage, metabolic equivalents, BMI, waist-to-height (WHtR) and waist–to-hip ratio (WHR).ResultsVFA was 28.8 % higher in men (pVFA<0.0001) and 30.5 % higher in patients older than 61 years (pVFA<0.0001). WHtR correlated best with all adipose tissue compartments (rVFA=0.69, rTFA=0.84, p<0.0001) and visceral-to-subcutaneous-fat-ratio (VFR, rVFR=0.22, p=<0.05). Patients with tumor stages III/IV showed significantly lower overall adipose tissue than I/II. Increased M. erector spinae mass was inversely correlated with all compartments.ConclusionDensitometric quantification on CT is a highly reproducible and reliable method to show fat distribution across adipose tissue compartments. This distribution might be best reflected by WHtR, rather than by BMI or WHR.Key Points• Densitometric quantification of adipose tissue on CT is highly reproducible and reliable. • Waist-to-height ratio better correlates with adipose tissue compartments and VFR than BMI or waist-to-hip ratio. • Men have higher a higher visceral fat area than women. • Patients older than 61 years have higher visceral fat area. • Patients with tumor stages III/IV have significantly lower adipose tissue than those in stages I/II.

Smoking is associated with hypermethylation of the APC 1A promoter in colorectal cancer: the ColoCare Study

Smoking tobacco is a known risk factor for the development of colorectal cancer and for mortality associated with the disease. Smoking has been reported to be associated with changes in DNA methylation in blood and in lung tumour tissues, although there has been scant investigation of how epigenetic factors may be implicated in the increased risk of developing colorectal cancer. To identify epigenetic changes associated with smoking behaviours, we performed epigenome‐wide analysis of DNA methylation in colorectal tumours from 36 never‐smokers, 47 former smokers, and 13 active smokers, and in adjacent mucosa from 49 never‐smokers, 64 former smokers, and 18 active smokers. Our analyses identified 15 CpG sites within the APC 1A promoter that were significantly hypermethylated and 14 CpG loci within the NFATC1 gene body that were significantly hypomethylated (pLIS < 1 × 10−5) in the tumours of active smokers. The APC 1A promoter was hypermethylated in 7 of 36 tumours from never‐smokers (19%), 12 of 47 tumours from former smokers (26%), and 8 of 13 tumours from active smokers (62%). Promoter hypermethylation was positively associated with duration of smoking (Spearman rank correlation, ρ = 0.26, p = 0.03) and was confined to tumours, with hypermethylation never being observed in adjacent mucosa. Further analysis of adjacent mucosa revealed significant hypomethylation of four loci associated with the TNXB gene in tissue from active smokers. Our findings provide exploratory evidence for hypermethylation of the key tumour suppressor gene APC being implicated in smoking‐associated colorectal carcinogenesis. Further work is required to establish the validity of our observations in independent cohorts. Copyright

Discovery of novel plasma proteins as biomarkers for the development of incisional hernias after midline incision in patients with colorectal cancer: The ColoCare study

Background. Ventral incisional hernia is the most common long‐term complication after an abdominal operation. Among newly diagnosed colorectal cancer patients, we screened the preoperative plasma proteome to explore predictive markers for the development of an incisional hernia. Methods. We utilized preoperative plasma samples of 72 newly diagnosed colorectal cancer patients who underwent midline incision for tumor resection between 2010 and 2013. A total of 21 patients with incisional hernia occurrence were matched with 51 patients with at least 18 months follow‐up without an incisional hernia by sex, age, and body mass index. To assess predictive markers of incisional hernia risk, we screened the plasma proteome for >2,000 distinct proteins using a well‐validated antibody microarray test. Paired t tests were used to compare protein levels between cases and controls. A gene‐set‐enrichment analysis (Gene Ontology and Kyoto Encyclopedia of Genes and Genomes) was applied to test for differences in signaling pathways between the 2 groups. Results. The proteome screen identified 25 proteins that showed elevated or reduced plasma levels in the hernia group compared to the control group (nominal P values < .05). Several proteins were in pathways associated with wound healing (CCL21, SHBG, BRF2) or cell adhesion (PCDH15, CDH3, EPCAM). Conclusion. Our study shows that there are multiple individual and groups of plasma proteins that could feasibly predict the personal hernia risk prior to undergoing an operation. Further investigations in larger, independent sample sets are warranted to replicate findings and validate clinical utility of potential biomarkers. After validation, such a biomarker could be incorporated into a multifactorial risk model to guide clinical decision‐making.

Robust prediction of gene regulation in colorectal cancer tissues from DNA methylation profiles

ABSTRACT DNA methylation is recognized as one of several epigenetic regulators of gene expression and as potential driver of carcinogenesis through gene-silencing of tumor suppressors and activation of oncogenes. However, abnormal methylation, even of promoter regions, does not necessarily alter gene expression levels, especially if the gene is already silenced, leaving the exact mechanisms of methylation unanswered. Using a large cohort of matching DNA methylation and gene expression samples of colorectal cancer (CRC; n = 77) and normal adjacent mucosa tissues (n = 108), we investigated the regulatory role of methylation on gene expression. We show that on a subset of genes enriched in common cancer pathways, methylation is significantly associated with gene regulation through gene-specific mechanisms. We built two classification models to infer gene regulation in CRC from methylation differences of tumor and normal tissues, taking into account both gene-silencing and gene-activation effects through hyper- and hypo-methylation of CpGs. The classification models result in high prediction performances in both training and independent CRC testing cohorts (0.92<AUC<0.97) as well as in individual patient data (average AUC = 0.82), suggesting a robust interplay between methylation and gene regulation. Validation analysis in other cancerous tissues resulted in lower prediction performances (0.69<AUC<0.90); however, it identified genes that share robust dependencies across cancerous tissues. In conclusion, we present a robust classification approach that predicts the gene-specific regulation through DNA methylation in CRC tissues with possible transition to different cancer entities. Furthermore, we present HMGA1 as consistently associated with methylation across cancers, suggesting a potential candidate for DNA methylation targeting cancer therapy.

Abstract 5325: Associations between adipose tissue compartments and the plasma metabolome in colorectal cancer patients: Results from the ColoCare Study

BACKGROUND Obesity is associated with colorectal cancer (CRC) risk and prognosis. We investigated associations between plasma metabolites and multiple dimensions of body fatness in early- and late-stage CRC patients enrolled in the ColoCare Study, a multicenter international cohort. METHODS Pre-operatively collected plasma samples of newly diagnosed CRC patients [n=212; (stage I-IV)] from the ColoCare Study were utilized to perform targeted metabolomics by mass spectrometry using the AbsoluteIDQ p180 Kit assay (Biocrates; intra-plate CVs RESULTS A total of 127 metabolites from 5 different compound classes (i.e., amino acids, biogenic amines, glycerophospholipids, sphingomyelins, acylcarnitines) were included for statistical analysis. Overall obesity (BMI) and VAT were not associated with any metabolites in early-stage or late-stage tumors. SAT (L3/L4 and L4/L5) was inversely associated with 3 glycerophospholipids in analyses restricted to late-stage, but not early-stage tumors: PC-ae-C34_0 (pFDR=0.02), PC-ae-C36_0 (pFDR=0.03), PC-ae-C36_1 (pFDR=0.03). A doubling of concentration in selected glycerophospholipids was associated with a significant increase in risk for death in late-stage (III and IV) CRC. CONCLUSIONS We observed a negative association between subcutaneous abdominal adiposity and glycerophospholipids in late-stage CRC. Glycerophospholipids are major components of cellular membranes and are pertinent to cancer cells that undergo progression and metastasis. Our results suggest that a metabolic shift in glycerophospholipid metabolism in late-stage tumors may take place and have potential impact on survival. Note: This abstract was not presented at the meeting. Citation Format: Jennifer Ose, Tengda Lin, Nina Habermann, David Achaintre, Pekka Keski-Rahkonen, Augustin Scalbert, Juergen Boehm, Biljana Gigic, Dominique Scherer, Johanna Nattenmueller, Mariam Salou, Lin Zielske, Alexis Ulrich, Jewel Samadder, Hanno Glimm, Stephen Hursting, Hans-Ulrich Kauczor, Cornelia M. Ulrich. Associations between adipose tissue compartments and the plasma metabolome in colorectal cancer patients: Results from the ColoCare Study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5325. doi:10.1158/1538-7445.AM2017-5325

Abstract A26: Body fatness and adipose tissue subtypes are associated with circulating biomarkers of inflammation and angiogenesis in colorectal cancer patients: The ColoCare Study

Background: Adiposity has been linked to both risk and prognosis of colorectal cancer; however, the impact of different compartments of adipose tissue (visceral vs. subcutaneous) is unclear. In healthy individuals, adiposity is associated with elevated biomarkers of inflammation, which provides a mechanistic link between adiposity and cancer risk. For prognosis, the downstream effects of inflammation on angiogenesis may be central. We investigated associations between adiposity and biomarkers of inflammation, as well as angiogenesis, in colorectal cancer patients enrolled in the ColoCare Study, an international multicenter patient cohort. Methods: We utilized preoperatively obtained serum samples of patients with newly diagnosed colorectal cancer [n=164; (stage I-IV)] from the ColoCare Study in Heidelberg, Germany, with available diagnostic multi-detector-CT images for adipose tissue quantification. Abdominal adipose tissue was assessed by area-based quantification of visceral (VAT), and subcutaneous adipose tissue (SAT), as well as their ratio (VAT/SAT) on levels L3/L4 and L4/L5. Body mass index (BMI) was calculated (kg/m2) and demographic and clinical-surgical data were abstracted from medical records. Circulating CRP, SAA, VEGF-A, VEGF-D, sICAM-1 and sVCAM-1 levels were assessed on the Meso Scale Discoveries platform with V-plex assays at the Huntsman Cancer Institute (average intra-plate CVs 2.9%, inter-plate CVs 7.9%). Partial correlations and regression analyses, adjusting for age, sex and tumor stage were performed. Results: While overall obesity (BMI) was only significantly associated with sVCAM (r=0.23, p=0.006), visceral adiposity (VAT) was associated with both CRP and SAA (r=0.21, p=0.01 and r=0.17, p=0.04, respectively). There was no association between SAT and the measured biomarkers. Most predictive was the ratio of VAT/SAT on level L3/L4, which was associated with CRP (r=0.18, p=0.04), SAA (r=0.24, p=0.006), sICAM-1 (r=0.18, p=0.04), and particularly VEGF-A (r=0.28, p=0.0008). Similar associations were observed for the VAT/SAT ratio on level L4/5. Conclusions: We demonstrated a link between specifically visceral adiposity and biomarkers of inflammation in colorectal cancer patients. In addition, we showed that visceral adiposity also affects circulating VEGF-A levels. This protein has various effects, including the induction of angiogenesis, vasculogenesis and endothelial cell growth, as well as the promotion of cell migration, and the inhibition of apoptosis. Our findings support a mechanistic role of visceral adipose tissue in colorectal cancer risk and potentially prognosis. Citation Format: Cornelia M. Ulrich, Jurgen Bohm, Christy Warby, Tengda Lin, Mariam Salou, Biljana Gigic, Dominique Scherer, Johanna Nattenmueller, Jennifer Ose, Lin Zielske, Petra Schrotz-King, Torsten Kolsch, Erin Siegel, Christopher Li, Alexis Ulrich, Hanno Glimm, Jewel Samadder, Stephen Hursting, Hans-Ulrich Kauczor. Body fatness and adipose tissue subtypes are associated with circulating biomarkers of inflammation and angiogenesis in colorectal cancer patients: The ColoCare Study. [abstract]. In: Proceedings of the AACR Special Conference: Improving Cancer Risk Prediction for Prevention and Early Detection; Nov 16-19, 2016; Orlando, FL. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2017;26(5 Suppl):Abstract nr A26.

Abstract 4921: Plasma proteomics for the discovery of biomarkers of incisional hernia in colorectal cancer patients in the ColoCare Study

Background Incisional hernia is the most common long-term complication after laparotomy for colorectal cancer resection with an incidence of 9-20%. Predisposing factors have been identified. Nevertheless, there are limited approaches to precisely predict individual risk, creating a need for predictive biomarkers of incisional hernia development. Methods We utilized pre-operative plasma samples of patients with newly diagnosed colorectal cancer [n = 72; (stage I-IV)] from the ColoCare Study in Heidelberg, Germany, who underwent laparoscopic tumor resection between 2010 and 2013. Patient questionnaires and telephone-interviews were used to assess the incidence of an incisional hernia, and demographic and clinical-surgical data were abstracted from medical records. 21 patients with incisional hernia occurrence were matched with 51 patients without an incisional hernia ( = controls) by gender, age, and BMI with at least 18 months follow-up. To assess predictive markers of incisional hernia risk we screened the plasma proteome for 3061 proteins using a well-validated antibody microarray test. Paired t-tests were used to compare protein levels between cases and controls. A gene-set-enrichment analysis (Gene Ontology and KEGG) was applied to test for differences in signaling pathways between the two groups. Results The proteome screen identified 27 proteins that showed elevated or reduced plasma levels in the hernia group compared to the control group (nominal p-values Conclusion To date no predictive blood-based biomarkers of incisional hernia risk are available. We discovered several candidate protein biomarkers which, after validation in further studies, could be incorporated into a multifactorial risk model to guide clinical decision making. This could enable the initiation of prophylactic treatments such as a mesh implantation and individualized patient recommendations to prevent incisional hernia occurrence in high-risk patients. Citation Format: Jurgen Bohm, Frank Pianka, Nina Stuttgen, Biljana Gigic, Yuzheng Zhang, Petra Schrotz-King, Nina Habermann, Alexis Ulrich, Martin Schneider, Paul Lampe, Markus Diener, Cornelia M. Ulrich. Plasma proteomics for the discovery of biomarkers of incisional hernia in colorectal cancer patients in the ColoCare Study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4921.

Abstract 4282: Branched-chain amino acids in the urinary metabolic profile of colorectal cancer patients and associations with muscle mass, BMI, and physical activity in the ColoCare Study

Background Branched-chain amino acids (BCAA; e.g., valine, leucine and isoleucine) have been previously linked with survival in colorectal cancer patients. It is unclear whether BCAAs are prognostic biomarkers or a surrogate endpoint for factors related to muscle mass. Thus, we investigated the correlation between BCAAs and muscle mass, physical activity and body mass index (BMI) at multiple time points during the course of disease in a prospective cohort of patients. Methods Patients with newly diagnosed colorectal cancer [n = 197; (stage I - IV)] from the ColoCare study in Heidelberg, Germany with baseline and follow-up data, and measurements of urinary BCAAs were eligible. Skeletal muscle mass of the dorsal muscle was quantified based on abdominal computed tomography (CT)-scans (vertebral body L3/4 and L4/5). BMI (kg/m2) and physical activity [metabolic equivalent (MET) hours/week] were reported by questionnaire. In addition, minutes of moderate and vigorous physical activity/week were measured by accelerometry. We prospectively monitored the urinary metabolome of these patients at multiple time points after surgery [baseline (n = 197), 6-month follow-up (n = 107) and 12 month follow-up (n = 75)]. BCAAs (valine, leucine and isoleucine) were identified using gas chromatography mass spectrometry (GC/MS). Metabolites were normalized based on the sum intensity of all annotated metabolites for statistical analyses. Pearson and partial correlation coefficients with parameters of energy balance/muscle mass were computed for each time point, adjusted for gender and age at diagnosis. Results As expected, urinary valine, leucine and isoleucine were highly correlated, independent of time point (r>0.95, P Conclusion The present data suggests that urinary levels of BCAA in colorectal cancer patients do not reflect parameters of energy balance and muscle mass. Thus, they warrant further investigation as potential prognostic biomarkers. Citation Format: Jennifer Ose, David B. Liesenfeld, Juergen Boehm, Nina Habermann, Robert W. Owen, Biljana Gigic, Stefanie Skender, Johanna Nattenmueller, Hans-Ulrich Kauczor, Cornelia Ulrich. Branched-chain amino acids in the urinary metabolic profile of colorectal cancer patients and associations with muscle mass, BMI, and physical activity in the ColoCare Study. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4282.