Early detection and intervention are likely to be the most effective means for reducing morbidity and mortality of human cancer. However, development of methods for noninvasive detection of early-stage tumors has remained a challenge. We have developed an approach called targeted error correction sequencing (TEC-Seq) that allows ultrasensitive direct evaluation of sequence changes in circulating cell-free DNA using massively parallel sequencing. We have used this approach to examine 58 cancer-related genes encompassing 81 kb. Analysis of plasma from 44 healthy individuals identified genomic changes related to clonal hematopoiesis in 16% of asymptomatic individuals but no alterations in driver genes related to solid cancers. Evaluation of 200 patients with colorectal, breast, lung, or ovarian cancer detected somatic mutations in the plasma of 71, 59, 59, and 68%, respectively, of patients with stage I or II disease. Analyses of mutations in the circulation revealed high concordance with alterations in the tumors of these patients. In patients with resectable colorectal cancers, higher amounts of preoperative circulating tumor DNA were associated with disease recurrence and decreased overall survival. These analyses provide a broadly applicable approach for noninvasive detection of early-stage tumors that may be useful for screening and management of patients with cancer.

It is well established that lncRNAs are aberrantly expressed in cancer where they have been shown to act as oncogenes or tumor suppressors. RNA profiling of 314 colorectal adenomas/adenocarcinomas and 292 adjacent normal colon mucosa samples using RNA‐sequencing demonstrated that the snoRNA host gene 16 (SNHG16) is significantly up‐regulated in adenomas and all stages of CRC. SNHG16 expression was positively correlated to the expression of Wnt‐regulated transcription factors, including ASCL2, ETS2, and c‐Myc. In vitro abrogation of Wnt signaling in CRC cells reduced the expression of SNHG16 indicating that SNHG16 is regulated by the Wnt pathway. Silencing of SNHG16 resulted in reduced viability, increased apoptotic cell death and impaired cell migration. The SNHG16 silencing particularly affected expression of genes involved in lipid metabolism. A connection between SNHG16 and genes involved in lipid metabolism was also observed in clinical tumors. Argonaute CrossLinking and ImmunoPrecipitation (AGO‐CLIP) demonstrated that SNHG16 heavily binds AGO and has 27 AGO/miRNA target sites along its length, indicating that SNHG16 may act as a competing endogenous RNA (ceRNA) “sponging” miRNAs off their cognate targets. Most interestingly, half of the miRNA families with high confidence targets on SNHG16 also target the 3′UTR of Stearoyl‐CoA Desaturase (SCD). SCD is involved in lipid metabolism and is down‐regulated upon SNHG16 silencing. In conclusion, up‐regulation of SNHG16 is a frequent event in CRC, likely caused by deregulated Wnt signaling. In vitro analyses demonstrate that SNHG16 may play an oncogenic role in CRC and that it affects genes involved in lipid metabolism, possible through ceRNA related mechanisms.

Colorectal cancer (CRC) is characterized by major inter-tumor diversity that complicates the prediction of disease and treatment outcomes. Recent efforts help resolve this by sub-classification of CRC into natural molecular subtypes; however, this strategy is not yet able to provide clinicians with improved tools for decision making. We here present an extended framework for CRC stratification that specifically aims to improve patient prognostication. Using transcriptional profiles from 1,100 CRCs, including >300 previously unpublished samples, we identify cancer cell and tumor archetypes and suggest the tumor microenvironment as a major prognostic determinant that can be influenced by the microbiome. Notably, our subtyping strategy allowed identification of archetype-specific prognostic biomarkers that provided information beyond and independent of UICC-TNM staging, MSI status, and consensus molecular subtyping. The results illustrate that our extended subtyping framework, combining subtyping and subtype-specific biomarkers, could contribute to improved patient prognostication and may form a strong basis for future studies.

Norager, C. B., M. B. Jensen, M. R. Madsen, and S. Laurberg. Caffeine improves endurance in 75-yr-old citizens: a randomized, double-blind, placebo-controlled, crossover study. J Appl Physiol 99: 2302-2306, 2005. First published August 4, 2005 doi:10.1152/japplphysiol.00309.2005.-This study investigated the effect of caffeine on physical performance in healthy citizens aged Ն70 yr. The randomized, double-blind, placebo-controlled, crossover study was conducted in 15 men and 15 women recruited by their general practitioner. Participants abstained from caffeine for 48 h and were randomized to receive one capsule of placebo and then caffeine (6 mg/kg) or caffeine and then placebo with 1 wk in between. One hour after intervention, we measured reaction and movement times, postural stability, walking speed, cycling at 65% of expected maximal heart rate, perceived effort during cycling, maximal isometric arm flexion strength, and endurance. Analysis was by intention to treat, and P Ͻ 0.05 was regarded as significant. Caffeine increased cycling endurance by 25% [95% confidence interval (CI): 13-38; P ϭ 0.0001] and isometric arm flexion endurance by 54% (95% CI: 29 -83; P ϭ 0.0001). Caffeine also reduced the rating of perceived exertion after 5 min of cycling by 11% (95% CI: 5-17; P ϭ 0.002) and postural stability with eyes open by 25% (95% CI: 2-53; P ϭ 0.03). Caffeine ingestion did not affect muscle strength, walking speed, reaction, and movement times. At the end of the study, 46% of participants correctly identified when they received caffeine and placebo. Caffeine increased exercise endurance in healthy citizens aged Ն70 yr, but the participants' reasons for stopping the test may have varied between subjects, as the cycling test was done at ϳ55% of maximal oxygen consumption. Further studies are required to investigate whether caffeine can be utilized to improve the physical performance of elderly citizens. fatigue; perceived exertion; postural stability; elderly CAFFEINE INGESTION INCREASES the endurance of young people exercising at 60 -85% of their maximal oxygen uptake (12,13,38). It also seems to improve endurance as measured by repeated submaximal isometric contraction (34) and decreases the rate of perceived exertion during exercise (6, 9, 37). Typically, doses of ϳ6 mg/kg caffeine (equivalent to 4 cups of coffee) were used in these studies.With a growing number of elderly with a physically active lifestyle and many elderly participating in rehabilitation programs, the endurance-enhancing effect of caffeine is of increasing interest in this age group. However, the enduranceenhancing effect of caffeine found in young subjects has not previously been examined in the elderly.The aim of this study was to investigate whether 6 mg/kg caffeine improves physical performance and reduces the perceived effort during work in healthy citizens aged Ն70 yr. The main hypothesis was that caffeine would improve cycling endurance at 65% of expected maximal heart rate, and the secondary hypothesis was that caffeine would improv...

BackgroundEarly 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.ResultsA 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.ConclusionTriMeth 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.