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Introduction: Blood-based, cancer-associated biomarkers are susceptible to a variety of well-known preanalytical factors. The influence of bowel preparation before a diagnostic colonoscopy on biomarker levels is, however, poorly investigated. The present study assessed the influence of bowel preparation on colorectal cancer-associated biomarkers. In addition, the effect of single versus double centrifugation of plasma biomarkers was assessed., Methods: Blood samples were collected pre- and post-bowel preparation from 125 subjects scheduled for first time diagnostic colonoscopy due to symptoms attributable to CRC. The samples were separated into serum and EDTA plasma, and analyzed by four independent collaborators for: 1) the proteins AFP, CA19-9, CEA, hs-CRP, CyFra21-1, Ferritin, Galectin-3 and TIMP-1, 2) the proteins BAG4, IL6ST, vWF, CD44 and EGFR, 3) the glycoprotein Galectin-3 ligand, and 4) cell-free DNA (cfDNA). Statistical analysis of biomarker data has been performed using mixed modelling, including repeated measures., Results: The biomarkers generally showed negligible variation between pre- and post-bowel preparation except for CyFra21-1, Ferritin, BAG4 and cfDNA. CyFra21-1 levels were systematically reduced with 29% (95% CI 21-36%) by bowel preparation (p ≤ 0.0001). Ferritin was not significantly different between pre- and post-bowel preparation (p = 0.07), however the estimated difference (increase) was 18%. BAG4 was systematically reduced by 12% (95% CI 1-22%, p = 0.04), while cfDNA showed a significant increase of 28% (95% CI 17-39%, p < 0.0001). Double centrifugation compared to single centrifugation showed reduced vWF (ratio 0.86, p ≤ 0.0001) and CD44 (ratio 0.85, p = 0.016), but increased IL6ST levels (ratio 1.18, p = 0.014)., Conclusions: Results of the present study demonstrated systematic, statistically significant differences between pre-bowel and post-bowel preparation levels for three independent blood-based biomarkers (BAG4, CyFra21-1, cfDNA), illustrating the importance of timing of sample collection for biomarker analyses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The burden of colorectal cancer (CRC) is considerable-approximately 1.8 million people are diagnosed each year with CRC and of these about half will succumb to the disease. In the case of CRC, there is strong evidence that an early diagnosis leads to a better prognosis, with metastatic CRC having a 5-year survival that is only slightly greater than 10% compared with up to 90% for stage I CRC. Clearly, biomarkers for the early detection of CRC would have a major clinical impact. We implemented a coherent gel-based proteomics biomarker discovery platform for the identification of clinically useful biomarkers for the early detection of CRC. Potential protein biomarkers were identified by a 2D gel-based analysis of a cohort composed of 128 CRC and site-matched normal tissue biopsies. Potential biomarkers were prioritized and assays to quantitatively measure plasma expression of the candidate biomarkers were developed. Those biomarkers that fulfilled the preset criteria for technical validity were validated in a case-control set of plasma samples, including 70 patients with CRC, adenomas, or non-cancer diseases and healthy individuals in each group. We identified 63 consistently upregulated polypeptides (factor of four-fold or more) in our proteomics analysis. We selected 10 out of these 63 upregulated polypeptides, and established assays to measure the concentration of each one of the ten biomarkers in plasma samples. Biomarker levels were analyzed in plasma samples from healthy individuals, individuals with adenomas, CRC patients, and patients with non-cancer diseases and we identified one protein, tropomyosin 3 (Tpm3) that could discriminate CRC at a significant level ( p = 0.0146). Our results suggest that at least one of the identified proteins, Tpm3, could be used as a biomarker in the early detection of CRC, and further studies should provide unequivocal evidence for the real-life clinical validity and usefulness of Tpm3.

Background: Annually, colorectal cancer (CRC) is diagnosed in >1.4 million subjects worldwide and incidence is increasing. Much effort has therefore been focused on screening, which has proven to reduce cancer-related mortality. The Sept9 DNA-methylation assay is among the most well studied blood-based screening markers. However, earlier reported performances may be misleading: the Sept9 test was recently examined in two screening based cohorts and yielded performances lower than expected. We hypothesize that comorbidities and/or demographic characteristics affect the results of the Sept9 test., Methods: Using a retrospective nested case-control study design, we studied plasma from 150 cancer and 150 controls selected from a well-characterized cohort of 4698 subjects referred for diagnostic colonoscopy due to CRC-related symptoms. The cases and controls were matched on age and gender, and moreover cases were stratified on tumor-site and tumor-stage. The selected cohort included a wide range of comorbidities. Plasma Sept9 levels were assessed using a commercially available PCR based assay (Epi-proColon)., Results: Clinical sensitivity for CRC stages I-IV was 37 %, 91 %, 77 %, and 89 %, and the overall sensitivity 73 % (95 % CI, 64-80 %) and specificity 82 % (95 % CI, 75-88 %), respectively. Age >65 was associated with both increased false positive and false negative results (p < 0.05). Arthritis was associated with a higher false negative rate (p = 0.005) whereas Arteriosclerosis was associated with a higher false positive rate (p = 0.007). Diabetes was associated with Sept9 positivity with an OR of 5.2 (95 % CI 1.4-19.1). When the performance of Sept9 was adjusted for these parameters in a final multivariate regression model, the OR for a positive Sept9 test to be associated with CRC increased from 8.25 (95 % CI 4.83-14.09) to 29.46 (95 % CI 12.58-69.02)., Conclusions: The results indicate that the performance of the Sept9 assay is negatively affected by several factors commonly associated with CRC screening populations: early-stage disease, age > 65 years, diabetes, arthritis, and arteriosclerosis. This should be taken into account if the Sept9 assay is used as a single marker for CRC screening, but may also have a wider impact, as it is likely that such factors may affect other blood based DNA markers as well.

Circulating tumor DNA (ctDNA) has emerged as a promising tool for early cancer detection and minimal residual disease monitoring. However, the biology underlying ctDNA release and its variation across cancer types and histologies remains poorly understood. This study investigated the biology behind ctDNA shedding in colorectal cancer. The study included a local cohort of 747 stage I-III colorectal cancer patients. All patients had ctDNA measurement prior to treatment and extensive clinical data. Primary tumor RNA sequencing and whole exome sequencing was performed in 95 and 652 patients respectively. Additionally, the study evaluated 89 non-small cell lung cancer patients from the TRACERx cohort, comprising primary tumor RNA sequencing and ctDNA measurement. We found tumor size and proliferative capacity to be key factors associated with ctDNA shedding in colorectal cancer. Furthermore, we found that the secretory and CMS3 colorectal cancer subtypes exhibited lower ctDNA shedding, while microsatellite instability (MSI) tumors had higher levels of ctDNA. Mutational analysis did not reveal any genes or pathways associated with ctDNA shedding in colorectal cancer. A comparison of transcriptomic profiles across multiple cancer types demonstrated that colorectal cancer and lung squamous cell carcinoma tumors shared a high-proliferative ctDNA shedding phenotype, while lung adenocarcinoma tumors displayed a distinct low-proliferative subgroup. Additionally, proliferation levels correlated with ctDNA detection sensitivity across multiple cancer types. These findings suggest that tumor size and proliferative capacity are drivers of ctDNA release in colorectal cancer and provide insights into the biology of ctDNA shedding on a pan-cancer level. • Colorectal cancer (CRC) tumors are ubiquitous circulating tumor DNA (ctDNA) shedders. • Tumor size and proliferation are drivers of ctDNA shedding in CRC. • Cancer mutations and clinical factors do not impact ctDNA shedding. • CRC comprises proliferative ctDNA shedding tumors comparable to lung adenocarcinoma • Proliferation correlates with ctDNA detection sensitivity across multiple cancer types [ABSTRACT FROM AUTHOR]