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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 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.

In solid tumor oncology, circulating tumor DNA (ctDNA) is poised to transform care through accurate assessment of minimal residual disease (MRD) and therapeutic response monitoring. To overcome the sparsity of ctDNA fragments in low tumor fraction (TF) settings and increase MRD sensitivity, we previously leveraged genome-wide mutational integration through plasma whole genome sequencing (WGS). We now introduce MRD-EDGE, a composite machine learning-guided WGS ctDNA single nucleotide variant (SNV) and copy number variant (CNV) detection platform designed to increase signal enrichment. MRD-EDGE uses deep learning and a ctDNA-specific feature space to increase SNV signal to noise enrichment in WGS by 300X compared to our previous noise suppression platform MRDetect. MRD-EDGE also reduces the degree of aneuploidy needed for ultrasensitive CNV detection through WGS from 1Gb to 200Mb, thereby expanding its applicability to a wider range of solid tumors. We harness the improved performance to track changes in tumor burden in response to neoadjuvant immunotherapy in non-small cell lung cancer and demonstrate ctDNA shedding in precancerous colorectal adenomas. Finally, the radical signal to noise enrichment in MRD-EDGE enables de novo mutation calling in melanoma without matched tumor, yielding clinically informative TF monitoring for patients on immune checkpoint inhibition.

Background: Early detection of cancer is key to identify tumours at a time when patients can be offered curatively intended treatments and outcomes are superior. Previous studies have presented sensitive methods for detection of ctDNA in early-stage cancer, but very few have been validated in asymptomatic individuals e.g. a true screening setting. Furthermore, many methods rely on expensive sequencing-based approaches that may not be cost-effective for population-based screening. Recently, we developed TriMeth, a simple CRC-specific ctDNA detection test based on three DNA methylation markers, which showed a sensitivity of 85% (stage I: 80%, stage II: 85%, stage III: 89%, stage IV: 88%) and a specificity of 99% when evaluated in CRC patients and controls (n=434). Objective: This study aimed to assess the performance of TriMeth cell-free DNA methylation analysis in asymptomatic individuals. Methods and materials: TriMeth was evaluated in 727 average-risk, asymptomatic individuals from the Danish population-based CRC screening program aged 50-74 years. This cohort included 105 CRC patients (stage I: n=53, stage II: n=24, stage III: n=19, stage IV: n=9) and 622 age and gender-matched controls enriched for comorbidities common to the screening population, such as, diabetes (n=47), arthritis (n=9), hypertension (n=279), arteriosclerosis (n=90), inflammatory bowel disease (n=6), adenomas (n=49), and other cancers (n=91). For comparison, TriMeth was also applied to a cohort of 43 symptomatic CRC patients and 42 cancer-free controls. Results: In the asymptomatic cohort, the specificity was consistently high (99%) despite the enrichment for controls with comorbidities in this cohort. The sensitivity was 36%, and was reduced for all disease stages compared to symptomatic cancers, though most markedly reduced for stage I CRC (stage I: 9%, stage II: 67%, stage III: 53%, stage IV: 78%). For a subset of asymptomatic individuals (n=91), an additional aliquot of plasma was analyzed, revealing a very high reproducibility of TriMeth (Spearman corr.coeff. r=0.95, p=7.6e-14). However, merging data from the paired samples did not significantly increase sensitivity. TriMeth showed consistently high accuracy in the symptomatic cohort (sensitivity: 84% and specificity: 100%). Conclusion: TriMeth is highly specific and not affected by comorbidities commonly found in 50-74 years old individuals invited for CRC screening. TriMeth showed high sensitivity for all stages when evaluated in symptomatic CRC patients, but the sensitivity was significantly decreased in asymptomatic cancers, also when stage-stratified. This indicate that asymptomatic cancers, independent of stage, shed less ctDNA than symptomatic cancers and highlights the importance of evaluating methods for early cancer detection in asymptomatic individuals. Citation Format: Sarah Østrup Jensen, Nadia Øgaard, Mai-britt Worm Ørntoft, Mads Heilskov Rasmussen, Jesper Bertram Bramsen, Christina Therkildsen, Claus Lindbjerg Andersen. Methylation analysis using TriMeth indicate that asymptomatic colorectal cancers (CRCs) release less circulating tumor DNA (ctDNA) compared to symptomatic cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3391.

Circulating cell‐free DNA (cfDNA) has spurred much interest as a biomarker in oncology. However, inter‐ and intra‐individual cfDNA levels vary greatly. Consequently, in order to base clinical decisions on cfDNA measurements, normal reference intervals are essential to avoid that ordinary variation is confused with clinically relevant change. The lack of reference intervals may potentially explain the ambiguous results reported in the field. Our study aimed to establish reference intervals and to evaluate the association between cfDNA and demographic and clinical variables, including colorectal cancer (CRC). Plasma samples and clinical data from 2817 subjects were collected including 1930 noncancer individuals and 887 CRC patients. cfDNA was measured using droplet digital polymerase chain reaction (PCR). The large cohort combined with robust cfDNA quantification enabled establishment of reference intervals (, What's new? Circulating cell‐free DNA (cfDNA) has spurred much interest as a potential biomarker in oncology. However, large inter‐ and intra‐individual variations have led to ambiguous study results and hampered the identification of clinically relevant changes. This study analyzes circulating cfDNA levels in a large cohort of 2817 individuals, with 887 colorectal cancer patients among them. The use of age‐stratified cfDNA reference intervals reveals that aberrant cfDNA levels are prognostic of overall survival in both cancer and non‐cancer individuals, independently of age, gender, and multimorbidity. Altogether, the results could facilitate the interpretation of cfDNA cancer research and clinical use of cfDNA biomarkers.

In post-structurally informed research, the answer to the widely documented ‘achievement gap’ among ethnic minorities has been a critique of educational institutions’ monocultural discourse and its exclusionary effects, thus highlighting a contingent, discursive conception of diversity. However, in this empirical article, 10- to 15-year-old students from two ethnically mixed schools in Denmark point to a much more concrete, social and material diversity that is laid out in terms of patterns of residence, leisure activities, and socio-economic resources at home. Over the school years, however, this social and material diversity is gradually transformed to a question of ethnicity that explains why students’ opportunities for educational participation ultimately differ. From a dialectical materialist reading of Hall’s concept of articulation, this article explores how this transformation is made possible in everyday school life, thus arguing that ethnic diversity is more than a contingent, discursive construction; it is closely connected to ingrained patterns of material inequity in educational practice. In post-structurally informed research, the answer to the widely documented ‘achievement gap’ among ethnic minorities has been a critique of educational institutions’ monocultural discourse and its exclusionary effects, thus highlighting a contingent, discursive conception of diversity. However, in this empirical article, 10- to 15-year-old students from two ethnically mixed schools in Denmark point to a much more concrete, social and material diversity that is laid out in terms of patterns of residence, leisure activities, and socio-economic resources at home. Over the school years, however, this social and material diversity is gradually transformed to a question of ethnicity that explains why students’ opportunities for educational participation ultimately differ. From a dialectical materialist reading of Hall’s concept of articulation, this article explores how this transformation is made possible in everyday school life, thus arguing that ethnic diversity is more than a contingent, discursive construction; it is closely connected to ingrained patterns of material inequity in educational practice.

Novel and minimally-invasive prostate cancer (PCa)-specific biomarkers are needed to improve diagnosis and risk stratification. Here, we investigated the biomarker potential in localized and de novo metastatic PCa (mPCa) of methylated circulating tumor DNA (ctDNA) in plasma. Using the Marmal-aid database and in-house datasets, we identified three top candidates specifically hypermethylated in PCa tissue: DOCK2, HAPLN3, and FBXO30 (specificity/sensitivity: 80%&ndash, 100%/75&ndash, 94%). These candidates were further analyzed in plasma samples from 36 healthy controls, 61 benign prostatic hyperplasia (BPH), 102 localized PCa, and 65 de novo mPCa patients using methylation-specific droplet digital PCR. Methylated ctDNA for DOCK2/HAPLN3/FBXO30 was generally not detected in healthy controls, BPH patients, nor in patients with localized PCa despite a positive signal in 98%&ndash, 100% of matched radical prostatectomy tissue samples. However, ctDNA methylation of DOCK2, HAPLN3, and/or FBXO30 was detected in 61.5% (40/65) of de novo mPCa patients and markedly increased in high- compared to low-volume mPCa (89.3% (25/28) vs. 32.1% (10/31), p &lt, 0.001). Moreover, detection of methylated ctDNA was associated with significantly shorter time to progression to metastatic castration resistant PCa, independent of tumor-volume. These results indicate that methylated ctDNA (DOCK2/HAPLN3/FBXO30) may be potentially useful for identification of hormone-na&iuml, ve mPCa patients who could benefit from intensified treatment.

Background: The B-vitamin folate is among the most studied bioactive food compound, and a dietary intake meeting the daily requirements has been found to reduce the risk of cancer and cardiovascular diseases as well as preventing neural tube defects during fetal development. Several countries have therefore introduced dietary fortification with folic acid. However, clinical and animal studies suggest that folic acid has a dual role in cancer development. Methods: During the period of initial tumour progression, MMTV-PyMT (MMTV-polyoma virus middle T) transgenic mice were fed with normal diet and high folic acid diet. Results: We found that PyMT-induced breast tumours highly express the cancer-specific folate receptor (FR), a feature they share with several human epithelial cancers in which expression of FRα correlates with tumour grade. Mice receiving a high folic acid diet displayed a significantly increased tumour volume compared with mice receiving normal diet. In the largest tumours, only found in mice on high folic acid diet, STAT3 was activated. In primary cells from PyMT tumours, STAT3 was activated upon treatment with folic acid in culture. Conclusions: Our results offer a novel molecular explanation for folic acid-induced growth of existing tumours.

Background Most existing DNA methylation-based methods for detection of circulating tumor DNA (ctDNA) are based on conversion of unmethylated cytosines to uracil. After conversion, the 2 DNA strands are no longer complementary; therefore, targeting only 1 DNA strand merely utilizes half of the available input DNA. We investigated whether the sensitivity of methylation-based ctDNA detection strategies could be increased by targeting both DNA strands after bisulfite conversion. Methods Dual-strand digital PCR assays were designed for the 3 colorectal cancer (CRC)–specific methylation markers KCNQ5, C9orf50, and CLIP4 and compared with previously reported single-strand assays. Performance was tested in tumor and leukocyte DNA, and the ability to detect ctDNA was investigated in plasma from 43 patients with CRC stages I to IV and 42 colonoscopy-confirmed healthy controls. Results Dual-strand assays quantified close to 100% of methylated control DNA input, whereas single-strand assays quantified approximately 50%. Furthermore, dual-strand assays showed a 2-fold increase in the number of methylated DNA copies detected when applied to DNA purified from tumor tissue and plasma from CRC patients. When the results of the 3 DNA methylation markers were combined into a ctDNA detection test and applied to plasma, the dual-strand assay format detected 86% of the cancers compared with 74% for the single-strand assay format. The specificity was 100% for both the dual- and single-strand test formats. Conclusion Dual-strand assays enabled more sensitive detection of methylated ctDNA than single-strand assays.

Background Early detection plays an essential role to reduce colorectal cancer (CRC) mortality. While current screening methods suffer from poor compliance, liquid biopsy-based strategies for cancer detection is rapidly gaining promise. Here, we describe the development of TriMeth, a minimal-invasive blood-based test for detection of early-stage colorectal cancer. The test is based on assessment of three tumour-specific DNA methylation markers in circulating cell-free DNA. Results A thorough multi-step biomarker discovery study based on DNA methylation profiles of more than 5000 tumours and blood cell populations identified CRC-specific DNA methylation markers. The DNA methylation patterns of biomarker candidates were validated by bisulfite sequencing and methylation-specific droplet digital PCR in CRC tumour tissue and peripheral blood leucocytes. The three best performing markers were first applied to plasma from 113 primarily early-stage CRC patients and 87 age- and gender-matched colonoscopy-verified controls. Based on this, the test scoring algorithm was locked, and then TriMeth was validated in an independent cohort comprising 143 CRC patients and 91 controls. Three DNA methylation markers, C9orf50, KCNQ5, and CLIP4, were identified, each capable of discriminating plasma from colorectal cancer patients and healthy individuals (areas under the curve 0.86, 0.91, and 0.88). When combined in the TriMeth test, an average sensitivity of 85% (218/256) was observed (stage I: 80% (33/41), stage II: 85% (121/143), stage III: 89% (49/55), and stage IV: 88% (15/17)) at 99% (176/178) specificity in two independent plasma cohorts. Conclusion TriMeth enables detection of early-stage colorectal cancer with high sensitivity and specificity. The reported results underline the potential utility of DNA methylation-based detection of circulating tumour DNA in the clinical management of colorectal cancer.

Item does not contain fulltext Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer(1). However, characteristics of the origins and molecular features of cell-free DNA are poorly understood. Here we developed an approach to evaluate fragmentation patterns of cell-free DNA across the genome, and found that profiles of healthy individuals reflected nucleosomal patterns of white blood cells, whereas patients with cancer had altered fragmentation profiles. We used this method to analyse the fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric or bile duct cancer and 245 healthy individuals. A machine learning model that incorporated genome-wide fragmentation features had sensitivities of detection ranging from 57% to more than 99% among the seven cancer types at 98% specificity, with an overall area under the curve value of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation-based cell-free DNA analyses detected 91% of patients with cancer. The results of these analyses highlight important properties of cell-free DNA and provide a proof-of-principle approach for the screening, early detection and monitoring of human cancer.

Blood circulating cell-free DNA (cfDNA) is becoming popular in the search of promising predictive and prognostic biomarkers. Among these biomarkers, cfDNA methylation markers have especially gained considerable attention. A significant challenge in the utilization of cfDNA methylation markers is the limited amount of cfDNA available for analyses; reportedly, bisulfite conversion (BSC) reduce cfDNA amounts even further. Nevertheless, few efforts have focused on ensuring high cfDNA conversion efficiency and recovery after BSC. In order to compare cfDNA recovery of different BSC assays, we compared 12 different commercially available BSC kits. We tested whether DNA recovery was affected by the molecular weight and/or quantity of input DNA. We also tested BSC efficiency for each kit. We found that recovery varied for DNA fragments of different lengths: certain kits recovered short fragments better than others, and only three kits recovered DNA fragments of

3018 Background: Analyses of cell-free DNA (cfDNA) in the blood provide a noninvasive diagnostic avenue for patients with cancer. However, cfDNA analyses have largely focused on targeted sequencing of specific genes, and the characteristics of the origins and molecular features of cfDNA are poorly understood. We developed an ultrasensitive approach that allows simultaneous examination of a large number of abnormalities in cfDNA through genome-wide analysis of fragmentation patterns. Methods: We used a machine learning model to examined cfDNA fragmentation profiles of 236 patients with largely localized breast, colorectal, lung, ovarian, pancreatic, gastric, or bile duct cancer and 245 healthy individuals. Estimation of performance was determined by ten-fold cross validation repeated ten times. Results: cfDNA profiles of healthy individuals reflected nucleosomal patterns of white blood cells, while patients with cancer had altered fragmentation patterns. The degree of abnormality in fragmentation profiles during therapy closely matched levels of mutant allele fractions in cfDNA as determined using ultra-deep targeted sequencing. The sensitivity of detection ranged from 57% to > 99% among the seven cancer types at 98% specificity, with an overall AUC of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation-based cfDNA analyses detected 91% of cancer patients. Conclusions: This effort is the first study to demonstrate genome-wide cell-free DNA fragmentation abnormalities in patients with cancer. Results of these analyses highlight important properties of cfDNA and provide a facile approach for screening, early detection, and monitoring of human cancer.

Background: Screening for colorectal cancer (CRC) using fecal occult blood tests (FOBT) reduces CRC mortality, and many CRC screening programs therefore use FOBT. However, FOBT is not the best approach; first, because population acceptance is low due to unpleasantness of fecal sampling; second, because bowel tumors bleed only intermittently, which limits FOBT sensitivity. Development of a novel blood-based screening approach may alleviate these problems. Objective: This study aims to develop and validate novel blood-based biomarker assays to achieve high patient compliance, sensitivity and specificity. Methods and materials: CRC-specific DNA methylation biomarker candidates were identified using a genome-wide discovery strategy based on >4,000 Illumina 450K DNA methylation arrays. We designed digital droplet PCR assays to detect top biomarker candidates in circulating cell-free DNA (cfDNA) isolated from plasma. Initially, sensitivity and specificity of biomarkers were evaluated in validation cohort 1 consisting of plasma collected from 114 symptomatic CRC patients and 86 colonoscopy-confirmed healthy controls. To ensure comparable technical sensitivities for all assays in all samples, we used a fixed cfDNA input of 4,500 copies per ddPCR reaction. Next, we tested markers in validation cohort 2, a selected cohort of 8 mL plasma collected from participants in the Danish national screening program. This cohort comprised 131 asymptomatic CRCs and 869 controls enriched for comorbidities like adenomas, other cancers, diabetes, arthritis, hypertension, inflammatory bowel disease and arteriosclerosis. Results: Our discovery identified 12 DNA methylation biomarkers. Their performance was evaluated in validation cohort 1, and the three best performing markers showed a sensitivity of 89% at a specificity of 99%, which is superior to FOBT. Sensitivity increased with stage, reaching 65%, 85%, 78% and 83% for stage I-IV, respectively. When evaluated in validation cohort 2, the sensitivity was 51% for CRC samples with an input of at least 4,500 copies of cfDNA, and sensitivity was reduced equally for all disease stages compared with results from validation cohort 1. Sensitivity correlated positively with cfDNA input, and it is therefore critical to collect sufficient plasma volumes to achieve high sensitivities in clinical practice. Finally, specificity was 92.4 % even though we enriched for comorbidities in this cohort, and specificity was 95.7% in controls with a clean colon and no comorbidities. Conclusion: Our systematic biomarker discovery and validation study identified a three-gene DNA methylation panel with superior performance in plasma compared to FOBT. Citation Format: Sarah Østrup Jensen, Mai-Britt Worm Ørntoft, Nadia Øgaard, Helle Kristensen, Mads Heilskov Rasmussen, Jesper Bertram Bramsen, Peter Mouritzen, Mogens Rørbæk, Anne-Sofie Kannerup, Søren Laurberg, Hans Jørgen Nielsen, Claus Lindbjerg Andersen. Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer - A clinical biomarker discovery and validation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5604.

Analysis of circulating cell-free DNA (cfDNA) is a promising approach for non-invasive detection of human cancer. The approach has wide-spread applications including early detection of disease in asymptomatic individuals, residual disease detection and disease monitoring. A challenge for the clinical utilization of cfDNA is the small fragment size of cfDNA and the limited amount of cfDNA present in blood. For detection of circulating tumor DNA (ctDNA) highly sensitive methods for cfDNA detection e.g. targeted sequencing and digital droplet PCR (ddPCR) target tumor specific mutations or DNA methylation alterations. Detection of methylation-based biomarkers requires that cfDNA is bisulfite converted prior to biomarker detection, which is known to be associated with some loss of cfDNA. Yet, surprisingly few efforts have focused on ensuring high cfDNA bisulfite conversion (BSC) efficiency and -recovery, and few commercial kits are especially designed for this purpose. In this study we aimed to find a BSC method suited for cfDNA methylation biomarker research, with high BS conversion efficiency and minimum loss of cfDNA, in order to increase the amount of cfDNA template available for downstream analysis. We studied 13 different methods for BSC and examined both the recovery and the BSC efficiency using quantitative real-time PCR (qPCR), polyacrylamide gel electrophoresis (PAGE) and ddPCR. A significant variation in DNA recovery was observed between the 13 methods tested using both high and low molecular weight DNA templates. Especially five kits performed consistently well on all DNA templates tested, as evaluated by qPCR and PAGE. As the lengths of cfDNA is 146-167 base pairs, only methods that are able to recover fragments of this lengths is suitable for cfDNA pipelines. Results showed that few methods enabled recovery of DNA fragments ≤ 150 base pairs. However, three kits showed a higher amount of 150 base pair fragments compared to the other methods tested. In a clinical setting it is expected that the target cfDNA compose only a minor fraction of the total cfDNA in plasma. Thus, the five best performing kits were tested using ddPCR on serial dilutions of leucocyte DNA fragmented to ~ 150bp to mimic cfDNA. The best performing kit had a recovery of 69% of the initial input compared to only 13% for the poorest performing kit. Collectively, our comprehensive comparison hereby guide which BS conversion methods are suited for cfDNA methylation-based biomarker pipelines. Citation Format: Sarah Ø. Jensen, Mai-Britt W. Ørntoft, Jesper B. Bramsen, Torben F. Ørntoft, Claus L. Andersen. Comprehensive comparison of bisulfite conversion kits: A guide for optimal sensitivity and specificity of circulating cell-free DNA methylation-based biomarkers [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 3804. doi:10.1158/1538-7445.AM2017-3804