Rapid detection of Salmonella enterica in primary production samples by eliminating DNA amplification inhibitors using an improved sample pre-treatment method.

Sensitive detection of pathogens in livestock farms is an integral part of the One Health Action Plan of the European Union (EU). Ensuring this requires on-site testing devices that are compatible with complex matrices such as primary production samples. Among all, faeces are considered the most challenging matrix type that makes it difficult to identify pathogens because of complexity in sample preparation for molecular testing. We have developed a loop-mediated isothermal amplification (LAMP) based veterinary point-of-care (POC) device (VETPOD) and adapted it to detect Salmonella enterica in primary production samples. Three different sampling methods (semi-wet chicken faeces, boot socks collection and dust samples from poultry shed) were iteratively tested to assess their nature of complexity and possibility for adapting them as suitable sampling methods for on-site testing. During the study, the sample preparation method that included a two-step centrifugation combined with washing of the enriched Salmonella cells was found crucial in eliminating amplification inhibitors originating from the faecal matrices. A total of 90 samples were tested that included 60 samples for sensitivity study and 30 samples for relative level of detection (RLOD, a level of detection in comparison to ISO 6579:1 reference method). Overall, the VETPOD had a sensitivity of 90%, 84.62% and 81.82% for boot sock, faecal and dust samples, respectively. The RLOD was 2.23 CFU/25 g which was found to be 1.33 times higher than the ISO 6579:1. Performing with an excellent agreement with ISO 6579:1, the VETPOD proved as a promising alternative to detect Salmonella spp. in primary production and animal husbandry samples.

Validation of Point-of-Care Device for Rapid Detection of Salmonella enterica in Meat Products.

Accurate and rapid detection of pathogens in foods of animal origin has been a critical part of the One Health Action Plan of the European Union (EU). Biosensors have the potential in bringing required technologies to accomplish this on the field, wherein loop-mediated isothermal amplification (LAMP) and lab-on-a-chip have proven to be ideal. We have developed a LAMP-based point-of-care (POC) device, the VETPOD, as a solution to the contemporary challenges in the rapid detection of Salmonella spp. The core technology in the VETPOD is a ready-to-use cartridge that included an injection-molded polymer chip with pyramid-shaped optical structures embedded within the chip. These pyramid-shaped optical structures direct the incident light, due to total internal reflection (TIR), through the reaction chambers to the phototransistor. The VETPOD was validated against the ISO 6579-1 reference method. A total of 310 samples were tested that included 180 Salmonella spiked samples in 6 different meat categories and 130 strains to determine the specificity. The overall results were satisfactory, wherein the VETPOD had an acceptable sensitivity (96.51%) compared to the reference (98.81%) and near perfect agreement with ISO 6579-1 with an overall Cohen's kappa of 0.94. The relative level of detection (RLOD) for the VETPOD was 1.38 CFU/25 g that was found to be 1.17 times higher than the reference. The VETPOD showed 98% precision for inclusivity and 100% precision for the exclusivity samples. The VETPOD proved as a useful alternative to detect Salmonella spp. that can be adaptable to a broader spectrum of pathogens in future.

Quality of life and symptom burden after rectal cancer surgery: a randomised controlled trial comparing patient-led versus standard follow-up.

PURPOSE: After curatively intended rectal cancer (RC) surgery, new follow-up strategies are warranted, seeking more individualised care and targeting health-related quality of life (HRQoL) and functional outcomes. The FURCA trial aimed to investigate the effect of patient-led follow-up on HRQoL and symptom burden 3 years after surgery. METHODS: RC patients from four Danish centres were randomised 1:1 to intervention (patient-led follow-up with patient education and self-referral to a specialist nurse) or control (standard follow-up with five routine doctor visits). Patients in both groups had a computed tomography (CT) at 1 and 3 years. The primary outcome (HRQoL) was assessed by the Functional Assessment of Cancer Therapy - colorectal (FACT-C) score (Ward et al. in Qual Life Res. 8(3):181-95, 18). Secondary outcomes were functional measures, patient involvement and satisfaction and cancer recurrence at 3 years. RESULTS: From Feb 2016 to Aug 2018, 336 patients were included of whom 248 completed 3 years of follow-up. Between-group differences were found neither for the primary endpoint, nor for functional outcomes. The recurrence rate did not differ between the groups. Patient involvement and satisfaction were higher in the intervention group with statistical significance in almost half of the items. CONCLUSIONS: We found no effect on HRQoL and symptom burden from patient-led follow-up, although it may improve patient-perceived involvement and satisfaction. IMPLICATIONS FOR CANCER SURVIVORS: The findings in this study suggest that patient-led follow-up is a more tailored approach to meet cancer survivors' needs and might improve their ability to cope with survivorship. GOV IDENTIFIER: R97-A6511-14-S23.

Optimizing Screening for Colorectal Cancer: An Algorithm Combining Fecal Immunochemical Test, Blood-Based Cancer-Associated Proteins and Demographics to Reduce Colonoscopy Burden.

BACKGROUND: Fecal Immunochemical Test (FIT) is widely used in population-based screening for colorectal cancer (CRC). This had led to major challenges regarding colonoscopy capacity. Methods to maintain high sensitivity without compromising the colonoscopy capacity are needed. This study investigates an algorithm that combines FIT result, blood-based biomarkers associated with CRC, and individual demographics, to triage subjects sent for colonoscopy among a FIT positive (FIT+) screening population and thereby reduce the colonoscopy burden. MATERIALS AND METHODS: From the Danish National Colorectal Cancer Screening Program, 4048 FIT+ (≥100 ng/mL Hemoglobin) subjects were included and analyzed for a panel of 9 cancer-associated biomarkers using the ARCHITECT i2000. Two algorithms were developed: 1) a predefined algorithm based on clinically available biomarkers: FIT, age, CEA, hsCRP and Ferritin; and 2) an exploratory algorithm adding additional biomarkers: TIMP-1, Pepsinogen-2, HE4, CyFra21-1, Galectin-3, B2M and sex to the predefined algorithm. The diagnostic performances for discriminating subjects with or without CRC in the 2 models were benchmarked against the FIT alone using logistic regression modeling. RESULTS: The discrimination of CRC showed an area under the curve (AUC) of 73.7 (70.5-76.9) for the predefined model, 75.3 (72.1-78.4) for the exploratory model, and 68.9 (65.5-72.2) for FIT alone. Both models performed significantly better (P < .001) than the FIT model. The models were benchmarked vs. FIT at cutoffs of 100, 200, 300, 400, and 500 ng/mL Hemoglobin using corresponding numbers of true positives and false positives. All performance metrics were improved at all cutoffs. CONCLUSION: A screening algorithm including a combination of FIT result, blood-based biomarkers and demographics outperforms FIT in discriminating subjects with or without CRC in a screening population with FIT results above 100 ng/mL Hemoglobin.

Adenoma and serrated lesion detection with distal attachment in screening colonoscopy: a randomized controlled trial.

BACKGROUND: Adenoma detection rate (ADR) is the single most important measure of quality in colonoscopy, but little is known about the detection rate of serrated lesions (SLDR). To improve ADR, Endocuff Vision (EV) can be used. Studies have shown differing results as to the effect on ADR; an effect on SLDR has not been shown. To investigate the effect of Endocuff Vision on ADR in a screening population, this randomized controlled open label trial with concealed allocation was performed. Randomization to trial group was carried out by the endoscopist using prepared numbered envelopes. METHODS: Patients referred as part of the national bowel screening program at Regional Hospital Herning, Denmark were recruited and allocated to one of two groups: Endocuff Vision colonoscopy (EVC) and standard colonoscopy (SC). Outcomes were ADR, mean number, site, and size of lesions per procedure. SLDR as outcome was added after inclusion had begun. RESULTS: A total of 1178 participants were included, with 1166 (EVC 583 and SC 583) available for analysis. There was no clinical relevant difference in ADR (59.2% [CI 55.1; 63.1] v 60.5% [CI 56.5; 64.4]) or SLDR (13.0% [CI 10.5; 16.0] v 10.3% [CI 8.0; 13.0]) between groups. More serrated lesions were found per procedure (MSP) (0.2 v 0.1, IRR 57% [CI 17; 109]. Removal rate of EV was similar in the two study groups. CONCLUSION: We found no significant effects of the use of Endocuff Vision on ADR, when compared to standard colonoscopy, but more serrated lesions were detected in the Endocuff group. TRIAL REGISTRATION: Clinical Trials NCT04651062.

Early detection of colorectal neoplasia: application of a blood-based serological protein test on subjects undergoing population-based screening.

BACKGROUND: Blood-based biomarkers used for colorectal cancer screening need to be developed and validated in appropriate screening populations. We aimed to develop a cancer-associated protein biomarker test for the detection of colorectal cancer in a screening population. METHODS: Participants from the Danish Colorectal Cancer Screening Program were recruited. Blood samples were collected prior to colonoscopy. The cohort was divided into training and validation sets. We present the results of model development using the training set. Age, sex, and the serological proteins CEA, hsCRP, TIMP-1, Pepsinogen-2, HE4, CyFra21-1, Galectin-3, ferritin and B2M were used to develop a signature test to discriminate between participants with colorectal cancer versus all other findings at colonoscopy. RESULTS: The training set included 4048 FIT-positive participants of whom 242 had a colorectal cancer. The final model for discriminating colorectal cancer versus all other findings at colonoscopy had an AUC of 0.70 (95% CI: 0.66-0.74) and included age, sex, CEA, hsCRP, HE4 and ferritin. CONCLUSION: The performance of the biomarker signature in this FIT-positive screening population did not reflect the positive performance of biomarker signatures seen in symptomatic populations. Additional biomarkers are needed if the serological biomarkers are to be used as a frontline screening test.

Targeting Germline- and Tumor-Associated Nucleotide Excision Repair Defects in Cancer.

PURPOSE: Nucleotide excision repair (NER) gene alterations constitute potential cancer therapeutic targets. We explored the prevalence of NER gene alterations across cancers and putative therapeutic strategies targeting these vulnerabilities. EXPERIMENTAL DESIGN: We interrogated our institutional dataset with mutational data from more than 40,000 patients with cancer to assess the frequency of putative deleterious alterations in four key NER genes. Gene-edited isogenic pairs of wild-type and mutant ERCC2 or ERCC3 cell lines were created and used to assess response to several candidate drugs. RESULTS: We found that putative damaging germline and somatic alterations in NER genes were present with frequencies up to 10% across multiple cancer types. Both in vitro and in vivo studies showed significantly enhanced sensitivity to the sesquiterpene irofulven in cells harboring specific clinically observed heterozygous mutations in ERCC2 or ERCC3. Sensitivity of NER mutants to irofulven was greater than to a current standard-of-care agent, cisplatin. Hypomorphic ERCC2/3-mutant cells had impaired ability to repair irofulven-induced DNA damage. Transcriptomic profiling of tumor tissues suggested codependencies between DNA repair pathways, indicating a potential benefit of combination therapies, which were confirmed by in vitro studies. CONCLUSIONS: These findings provide novel insights into a synthetic lethal relationship between clinically observed NER gene deficiencies and sensitivity to irofulven and its potential synergistic combination with other drugs.See related commentary by Jiang and Greenberg, p. 1833.

Transcriptomic and proteomic intra-tumor heterogeneity of colorectal cancer varies depending on tumor location within the colorectum.

BACKGROUND: Intra-tumor heterogeneity (ITH) of colorectal cancer (CRC) complicates molecular tumor classification, such as transcriptional subtyping. Differences in cellular states, biopsy cell composition, and tumor microenvironment may all lead to ITH. Here we analyze ITH at the transcriptomic and proteomic levels to ascertain whether subtype discordance between multiregional biopsies reflects relevant biological ITH or lack of classifier robustness. Further, we study the impact of tumor location on ITH. METHODS: Multiregional biopsies from stage II and III CRC tumors were analyzed by RNA sequencing (41 biopsies, 14 tumors) and multiplex immune protein analysis (89 biopsies, 29 tumors). CRC subtyping was performed using consensus molecular subtypes (CMS), CRC intrinsic subtypes (CRIS), and TUMOR types. ITH-scores and network maps were defined to determine the origin of heterogeneity. A validation cohort was used with one biopsy per tumor (162 tumors). RESULTS: Overall, inter-tumor transcriptional variation exceeded ITH, and subtyping calls were frequently concordant between multiregional biopsies. Still, some tumors had high transcriptional ITH and were classified discordantly. Subtyping of proximal MSS tumors were discordant for 50% of the tumors, this ITH was related to differences in the microenvironment. Subtyping of distal MSS tumors were less discordant, here the ITH was more cancer-cell related. The subtype discordancy reflected actual molecular ITH within the tumors. The relevance of the subtypes was reflected at protein level where several inflammation markers were significantly increased in immune related transcriptional subtypes, which was verified in an independent cohort (Wilcoxon rank sum test; p<0.05). Unsupervised hierarchical clustering of the protein data identified large ITH at protein level; as the multiregional biopsies clustered together for only 9 out of 29 tumors. CONCLUSION: Our transcriptomic and proteomic analyses show that the tumor location along the colorectum influence the ITH of CRC, which again influence the concordance of subtyping.

A Sensitive, Specific and Simple Loop Mediated Isothermal Amplification Method for Rapid Detection of Campylobacter spp. in Broiler Production.

Campylobacteriosis is one of the most common foodborne diseases worldwide. Two Campylobacter species - C. jejuni and C. coli in poultry and poultry products are considered to be the main source of human campylobacteriosis. Therefore, studying Campylobacter status in poultry flocks is needed to prevent transmission of disease and reduce human risk, health cost, and economic losses. In this study, we adapted and used a Loop-Mediated Isothermal Amplification (LAMP) assay for specific, sensitive, simple and cost-effective rapid detection of C. jejuni and C. coli in the poultry production chain. Amplified LAMP products were detected using a small, low-cost portable commercial blue LED transilluminator and a direct visual detection strategy was demonstrated. By using optimized conditions for amplification a limit of detection (LOD) of 50 CFU/ml was achieved for testing of C. jejuni and C. coli in spiked chicken feces without enrichment. The method took 60-70 min from receiving the samples to the final results (including 30 min for amplification). The optimized LAMP showed a relative accuracy of 98.4%, a specificity of 97.9%, and a sensitivity of 100% in comparison to real-time PCR method. Cohen's kappa index also showed an excellent agreement (0.94) between the two methods. The results showed that the method is specific, sensitive and is suitable to develop for rapid detection of Campylobacter spp. at poultry production.

Novel DNA methylation biomarkers show high sensitivity and specificity for blood-based detection of colorectal cancer-a clinical biomarker discovery and validation study.

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.

Classification of Multiple DNA Dyes Based on Inhibition Effects on Real-Time Loop-Mediated Isothermal Amplification (LAMP): Prospect for Point of Care Setting.

LAMP has received great interest and is widely utilized in life sciences for nucleic acid analysis. To monitor a real-time LAMP assay, a fluorescence DNA dye is an indispensable component and therefore the selection of a suitable dye for real-time LAMP is a need. To aid this selection, we investigated the inhibition effects of twenty-three DNA dyes on real-time LAMP. Threshold time (Tt) values of each real-time LAMP were determined and used as an indicator of the inhibition effect. Based on the inhibition effects, the dyes were classified into four groups: (1) non-inhibition effect, (2) medium inhibition effect, (3) high inhibition effect, and (4) very high inhibition effect. The signal to noise ratio (SNR) and the limit of detection (LOD) of the dyes in groups 1, 2, and 3 were further investigated, and possible inhibition mechanisms of the DNA dyes on the real-time LAMP are suggested and discussed. Furthermore, a comparison of SYTO 9 in different LAMP reactions and different systems is presented. Of the 23 dyes tested, SYTO 9, SYTO 82, SYTO 16, SYTO 13, and Miami Yellow were the best dyes with no inhibitory effect, low LOD and high SNR in the real-time LAMP reactions. The present classification of the dyes will simplify the selection of fluorescence dye for real-time LAMP assays in point of care setting.

Clean Colorectum at Diagnostic Colonoscopy: Subsequent Detection of Extracolonic Malignancies by Plasma Protein Biomarkers?

INTRODUCTION: Most of the subjects undergoing diagnostic colonoscopy do not have neoplastic bowel lesions. Potentially, some of the symptoms may therefore be caused by extracolonic malignancy, and subjects with persisting symptoms may need subsequent examinations. Blood-based, cancer-associated biomarkers may aid in directing the examinations for other specific malignant diseases. METHODS: EDTA plasma samples available from a previous prospective study of subjects undergoing diagnostic colonoscopy were used for analysis of 18 protein biomarkers. The study population of 3732 subjects included 400 patients with colorectal cancer (CRC) and 177 patients with extracolonic malignancies. Univariable analysis of the association of specific biomarkers and extracolonic cancers included those with 10 or more cases. Subsequently, reduced models of 4 or 6 biomarkers, respectively, were established by choosing those with the highest likelihood; age and sex were included as well. RESULTS: Univariable analyses showed that CyFra21-1 had an area under curve (AUC) of 0.87 for lung cancers (n = 33), CA19-9 had an AUC of 0.85 for pancreatic cancer (n = 22), CA125 had an AUC of 0.95 for ovary cancer (n = 16), B2M had an AUC of 0.81 for non-Hodgkin lymphoma (n = 12), and total prostate-specific antigen had an AUC of 0.99 for prostate cancer (n = 10). The multivariable analysis of 4 or 6 biomarkers plus age and sex as explanatory variables showed AUCs of 0.82 to 0.85 both for extracolonic cancers and CRC. The 4 biomarkers included in the model for detection of extracolonic cancers were CA125, hsCRP, CA19-9, and CyFra21-1; the 2 additional for the 6 biomarkers model were CEA and Galectin-3. Similarly, the 4 biomarkers included in the model for detection of CRC were CEA, CyFra21-1, Ferritin, and HE4; the two additional for the 6 biomarkers model were hsCRP and Pepsinogen 2. CONCLUSIONS: Results of this study indicate that it may be possible to detect subjects that have an increased risk of extracolonic cancer following a colonoscopy without findings of neoplastic lesions. Combinations of various protein biomarkers may direct subsequent examination after colonoscopy with clean colorectum. The results, although preliminary, may form the basis for additional research directed both for primary examinations of subjects with symptoms of malignancy and subsequent examinations after colonoscopy.

Characterization of genetic intratumor heterogeneity in colorectal cancer and matching patient-derived spheroid cultures.

Patient-derived in vitro cultures of colorectal cancer (CRC) may help guide treatment strategies prior to patient treatment. However, most previous studies have been performed on a single biopsy per tumor. The purpose of this study was to analyze multiple spatially distinct biopsies from CRCs and see how well intratumor heterogeneity (ITH) was recapitulated in matching patient-derived spheroids. Three to five biopsies were collected from six CRC tumors. Each biopsy was split in two; one half was used for spheroid culturing, while the other half was used for DNA and RNA purification. For two patients, lymph node metastases were analyzed. Somatic mutations were called from whole exome sequencing data. Each tumor contained mutations shared across all biopsies and spheroids, including major CRC drivers such as APC, KRAS, and TP53. At the same time, all tumors exhibited ITH on both mutation and copy number level. The concordance between biopsies and spheroids ranged between 40 and 70% for coding mutations. For three patients, the biopsy and spheroid from matching areas clustered together, meaning that the spheroid resembled the area of origin more than the other areas. However, all biopsies and spheroids contained private mutations. Therefore, multiple cultures from spatially distinct sites of the tumor increase the insight into the genetic profile of the entire tumor. Molecular subtypes were called from RNA sequencing data. When based on transcripts from both cancer and noncancerous cells, the subtypes were largely independent of sampling site. In contrast, subtyping based on cancer cell transcripts alone was dependent on sample site and genetic ITH. In conclusion, all examined CRC tumors showed genetic ITH. Spheroid cultures partly reflected this ITH, and having multiple cultures from distinct tumor sites improved the representation of the genetic tumor subclones. This should be taken into account when establishing patient-derived models for drug screening.

Open versus laparoscopic rectal cancer resection and risk of subsequent incisional hernia repair and paracolostomy hernia repair: a nationwide population-based cohort study.

OBJECTIVE: To investigate the risk of incisional hernia repair (IHR) and paracolostomy hernia repair (PHR) following open and laparoscopic rectal cancer resection with curative intent. BACKGROUND: Laparoscopic rectal cancer resection has been implemented to varying degrees around the world. IHR and PHR following open and laparoscopic rectal cancer resection have only been sparingly evaluated. METHODS: Patients who underwent rectal cancer resection were identified in the Danish Colorectal Cancer Group's database. To identify IHR and PHR following rectal cancer resection, we linked data to the Danish Ventral Hernia Database. The absolute risk of IHR and PHR was estimated as cumulative incidence proportions, treating death as competing risk. We used Cox proportional hazard regression analysis with multivariable adjustment to compute hazard ratios (HRs) comparing open and laparoscopic approach. RESULTS: The 5-year risk of IHR was 4.1% among patients undergoing open resection (n = 3090) and 3.2% among those undergoing laparoscopic resection (n = 3099), corresponding to a risk difference of 0.9% (95% CI 0.0-2.0, P = 0.057). Laparoscopic rectal resection was not associated with lower risk of IHR (adjusted HR 0.94, 95% CI 0.67-1.31, P = 0.709). A total of 2577 patients had a colostomy at rectal cancer resection and the 5-year risk of PHR was 2.1% after open surgery compared with 6.7% after laparoscopic surgery, corresponding to a risk difference of -4.6% (95% CI -6.4 to -2.7, P < 0.001). Laparoscopic surgery was associated with increased risk of PHR (adjusted HR 2.56, 95% CI 1.53-4.29, P < 0.001). CONCLUSION: We observed no association between surgical approach of rectal cancer resection and subsequent IHR. Laparoscopic surgery was associated with increased risk of PHR.

Direct detection of early-stage cancers using circulating tumor DNA.

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.

Changes in incontinence after hysterectomy.

PURPOSE: Information about the perioperative incontinence following hysterectomy is limited. To advance the postoperative rehabilitation further we need more information about qualitative changes in incontinence, fatigue and physical function of patients undergoing hysterectomy. METHODS: 108 patients undergoing planned hysterectomy were compared pre- and postoperatively. In a sub-study of the prospective follow-up study the changes in incontinence, postoperative fatigue, quality of life, physical function, and body composition were evaluated preoperatively, 13 and 30 days postoperatively. Sample size calculation indicated that 102 women had to be included. The incontinence status was estimated by a Danish version of the ICIG questionnaire; further, visual analogue scale, dynamometer for hand grip, knee extension strength and balance were applied. Work capacity was measured ergometer cycle together with lean body mass by impedance. Quality of life was assessed using the SF-36 questionnaire. Patients were examined preoperatively and twice postoperatively. RESULTS: In total 41 women improved their incontinence after hysterectomy and 10 women reported deterioration. Preoperative stress incontinence correlated with BMI (r = 0.25, p < 0.01) and urge incontinence with age (r = 0.24, p < 0.02). Further, improvement after hysterectomy in stress incontinence was associated with younger age (r = 0.20, p < 0.04). Improvement in urge incontinence was positively associated with BMI (r = 0.22, p = 0.02). A slight but significant loss was seen in lean body mass 13 and 30 days postoperatively. CONCLUSIONS: Hysterectomy was not significantly associated with the risk of incontinence; in particular, when no further vaginal surgery is performed. Hysterectomy may even have a slightly positive effect on incontinence and de-novo cure.

Molecular-Subtype-Specific Biomarkers Improve Prediction of Prognosis in Colorectal Cancer.

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.

Follow-up after rectal cancer: developing and testing a novel patient-led follow-up program. Study protocol.

BACKGROUND: The main treatment for non-metastatic rectal cancer (RC) is surgical resection. Late adverse effects that are highly prevalent and negatively impact patients' symptom burden and quality of life are: bowel-, urological and sexual dysfunctions; psychological distress; fear of recurrence. Patients and clinicians have requested a more patient-centred follow-up, balancing the focus on detection of recurrence, and physiological and psychological late adverse effects. The current follow-up program primarily focuses on detection of recurrence, with less attention on late adverse effects. As a consequence, the randomized controlled trial Follow-up after Rectal Cancer (FURCA) has been launched, testing the effect of a new patient-led, follow-up program. The aim of this paper is to describe the methodology used in the FURCA study and to report results from the development of the patient-led, follow-up program. Adult patients, treated with curative intent for primary adenocarcinoma in the rectum are included from four Danish centers. MATERIAL AND METHODS: Patients are randomized into an intervention group, receiving standardized education and access to self-referral to an assigned project nurse, or a control group following the current follow-up program with routine medicals. The primary outcomes are symptom burden and quality of life, measured by the Functional Assessment of Cancer Therapy - Colorectal (FACT-C) questionnaire. Other outcome and demographic data are collected as patient-reported measures and register-based data. Results from developing the intervention: The education program is based on data from two focus group interviews and the feedback from experts. An algorithm is developed in order to qualify the research nurses' responses to patients' self-referral. Discussion and perspectives: The results of the FURCA study will strengthen the evidence base for RC follow-up, and qualify the ongoing transformation in cancer follow-up programs.

Detection of colorectal neoplasia: Combination of eight blood-based, cancer-associated protein biomarkers.

Serological biomarkers may be an option for early detection of colorectal cancer (CRC). The present study assessed eight cancer-associated protein biomarkers in plasma from subjects undergoing first time ever colonoscopy due to symptoms attributable to colorectal neoplasia. Plasma AFP, CA19-9, CEA, hs-CRP, CyFra21-1, Ferritin, Galectin-3 and TIMP-1 were determined in EDTA-plasma using the Abbott ARCHITECT® automated immunoassay platform. Primary endpoints were detection of (i) CRC and high-risk adenoma and (ii) CRC. Logistic regression was performed. Final reduced models were constructed selecting the four biomarkers with the highest likelihood scores. Subjects (N = 4,698) were consecutively included during 2010-2012. Colonoscopy detected 512 CRC patients, 319 colonic cancer and 193 rectal cancer. Extra colonic malignancies were detected in 177 patients, 689 had adenomas of which 399 were high-risk, 1,342 had nonneoplastic bowell disease and 1,978 subjects had 'clean' colorectum. Univariable analysis demonstrated that all biomarkers were statistically significant. Multivariate logistic regression demonstrated that the blood-based biomarkers in combination significantly predicted the endpoints. The reduced model resulted in the selection of CEA, hs-CRP, CyFra21-1 and Ferritin for the two endpoints; AUCs were 0.76 and 0.84, respectively. The postive predictive value at 90% sensitivity was 25% for endpoint 1 and the negative predictive value was 93%. For endpoint 2, the postive predictive value was 18% and the negative predictive value was 97%. Combinations of serological protein biomarkers provided a significant identification of subjects with high risk of the presence of colorectal neoplasia. The present set of biomarkers could become important adjunct in early detection of CRC.

Protocol Outlines for Parts 1 and 2 of the Prospective Endoscopy III Study for the Early Detection of Colorectal Cancer: Validation of a Concept Based on Blood Biomarkers.

BACKGROUND: Programs for population screening of colorectal cancer (CRC) have been implemented in several countries with fecal immunochemical testing (FIT) as the preferred platform. However, the major obstacle for a feces-based testing method is the limited compliance that reduces the clinical sensitivity for detection of participants with non-symptomatic CRC. Therefore, research approaches have been initiated to develop screening concepts based on biomarkers in blood. Preliminary results show that protein, genetic, epigenetic, and metabolomic components may be valuable in blood-based screening concepts, particularly when combinations of the various components appear to lead to significant improvements. OBJECTIVES: The protocol described in this paper focuses on the validation of concepts based on biomarkers in blood in a major population screened by FIT. METHODS: In Part 1, participants will be identified and included through the Danish CRC Screening Program comprising initial FIT and subsequent colonoscopy to those with a positive result. Blood samples will be collected from 8000 FIT-positive participants, who are offered subsequent colonoscopy. Findings and interventions at colonoscopy together with personal data including co-morbidity will be recorded. Blood samples and data will also be collected from 6000 arbitrarily chosen participants with negative FIT. In Part 2, blood samples and data will be collected from 30,000 FIT-negative participants three times within 4 years. The blood samples will be analyzed using various in-house and commercially available manual and automated analysis platforms. RESULTS: We anticipate Part 1 to terminate late August 2016 and Part 2 to terminate late September 2022. The results from Parts 1 and 2 will be presented within 12 to 18 months from termination. CONCLUSIONS: The purpose of this study is to improve the efficacy of identifying participants with neoplastic bowel lesions, to identify false negative participants, to identify participants at risk of interval neoplastic lesions, to improve the compliance in screening sessions, and to establish guidelines for out-patient follow-up of at-risk participants based on combinations of blood-based biomarkers.