Biomarker measurement in non-invasively sampled colorectal mucus as a novel approach to colorectal cancer detection: screening and triage implications.

BACKGROUND: Faecal tests are widely applied for colorectal cancer (CRC) screening and considered for triaging symptomatic patients with suspected CRC. However, faecal tests can be inconvenient, complex and expensive. Colorectal mucus (CM) sampled using our new patient-friendly non-invasive technique is rich in CRC biomarkers. This study aimed to evaluate diagnostic accuracy of CRC detection by measuring protein biomarkers in CM. METHODS: Colorectal mucus samples were provided by 35 healthy controls, 62 CRC-free symptomatic patients and 40 CRC patients. Biomarkers were quantified by ELISA. Diagnostic performances of haemoglobin, C-reactive protein, tissue inhibitor of metalloproteinases-1, M2-pyruvate kinase, matrix metalloproteinase-9, peptidyl arginine deiminase-4, epidermal growth factor receptor, calprotectin and eosinophil-derived neurotoxin were assessed using receiver operating characteristic (ROC) curve analysis. RESULTS: Colorectal mucus haemoglobin was superior compared to other biomarkers. For haemoglobin, the areas under the curve for discriminating between CRC and healthy groups ('screening') and between CRC and symptomatic patients ('triage') were 0.921 and 0.854 respectively. The sensitivity of 80.0% and specificities of 94.3% and 85.5% for the two settings respectively were obtained. CONCLUSIONS: Haemoglobin quantification in CM reliably detects CRC. This patient-friendly approach presents an attractive alternative to faecal immunochemical test; however, the two methods need to be directly compared in larger studies.

Colorectal cancer detection by biomarker quantification in noninvasively collected colorectal mucus: preliminary comparison of 24 protein biomarkers.

OBJECTIVES: Noninvasive colorectal cancer detection and screening remain global diagnostic challenges because the existing stool tests either lack sensitivity or are complex and expensive. Moreover, colorectal cancer screening uptake is low due to stool sampling inconvenience. We have developed a simple and patient-friendly noninvasive technique for collecting highly informative colorectal mucus. In this study, we aimed to comparatively assess a range of candidate biomarkers in colorectal mucus samples for colorectal cancer detection. METHODS: The study included 17 patients with colorectal cancer and 35 healthy controls, who provided noninvasively collected colorectal mucus samples. Protein biomarker quantification in these samples by enzyme-linked immunosorbent assays allowed comparing diagnostic performances of 24 candidate biomarkers that comprised haemoglobin, D-dimer, M2-pyruvate kinase, carcinoembryonic antigen, C-reactive protein, calprotectin, eosinophil-derived neurotoxin, protein S100A12, tumour necrosis factor α, clusterin, soluble cytokeratin 18, caspase-cleaved cytokeratin 18, citrullinated histone H3, peptidyl arginine deiminase 4, epidermal growth factor, epidermal growth factor receptor, matrix metalloproteinase 9, tissue inhibitor of metalloproteinase 1, periostin, vascular endothelial growth factor A, vascular endothelial growth factor receptor 1, vascular cell adhesion molecule 1, intercellular adhesion molecule 1 and mucin 2. Tested biomarkers were ranked for colorectal cancer detection efficiency using receiver operating characteristic curve analysis. RESULTS: High area under the curve values between 0.943 and 0.768 were observed for haemoglobin, tissue inhibitor of metalloproteinase 1, M2-pyruvate kinase, peptidyl arginine deiminase 4, C-reactive protein, matrix metalloproteinase 9, epidermal growth factor receptor, eosinophil-derived neurotoxin and calprotectin. CONCLUSION: Quantification of protein biomarkers in noninvasively collected samples of colorectal mucus certainly allows detecting colorectal cancer. Further clinical evaluation of the optimal biomarkers identified by this study is needed.

Systemic and skin-targeting beneficial effects of lycopene-enriched ice cream: A pilot study.

The health-promoting dietary antioxidant lycopene has limited natural bioavailability, but lycopene-rich functional foods can improve its bioavailability. We assessed a new lycopene-enriched ice cream for systemic antioxidant effects and influence on morphological characteristics of facial skin surface in healthy volunteers. In a randomized crossover study, we used 4-wk dietary interventions with either control or lycopene-enriched ice cream. Samples of serum and residual skin surface components (RSSC) from facial skin were taken before interventions, at 2 wk, and at intervention end. Lycopene concentration, conventional blood biochemistry, and oxidative stress biomarkers comprising inflammatory oxidative damage and low-density lipoprotein peroxidase proteins were assessed in the serum. Lycopene-associated immunofluorescence, lipid droplet size, corneocyte desquamation, and microbial presence were measured in the RSSC. The results show that lycopene concentrations in the serum and skin steadily increased during lycopene-enriched ice cream consumption. Whereas we found no intervention-dependent changes in conventional biochemical parameters, both inflammatory oxidative damage and low-density lipoprotein peroxidase protein values significantly decreased by the end of intervention with lycopene-enriched ice cream, but remained unchanged during control ice cream consumption. Control ice cream significantly increased corneocyte desquamation and bacterial presence in the RSSC. These adverse effects, which could potentially predispose consumers to acne development, were absent when volunteers consumed lycopene-enriched ice cream. We concluded that lycopene-enriched ice cream is a new functional food with clear antioxidant properties. In addition, enrichment with lycopene may alleviate proinflammatory action of ice cream at the level of facial skin, thus decreasing diet-associated acne development risk in young consumers.

Continuous Dark Chocolate Consumption Affects Human Facial Skin Surface by Stimulating Corneocyte Desquamation and Promoting Bacterial Colonization.

Background: Nutrition can influence skin health. Dark chocolate possesses health promoting properties, but its consumption can exacerbate acne vulgaris in young people. Objective: We evaluated effects of continuous dark chocolate intake on morphological characteristics of the residual skin surface components (RSSCs) collected from the facial skin of young and middle-aged men. Methods: RSSC samples were taken from 17 young and 16 middle-aged men before and after a four-week consumption period of dark chocolate (10g per day). Lipid droplet size, corneocyte desquamation, and microbial presence levels were measured in the collected RSSC. The project was registered as ISRCTN89815519 in the ISRCTN registry (https://www.isrctn.com/). Results: Chocolate consumption caused a significant increase in corneocyte desquamation only in the group of young men, whereas Gram-positive microorganism presence significantly increased in both the young and middle-aged men, though this effect was noticeably stronger in the young men. Conclusion: Dark chocolate consumption appears to affect the facial skin of young men by enhancing corneocyte desquamation and promoting bacterial colonization of the RSSC. These changes might potentially contribute to acne development.

Continuous astaxanthin intake reduces oxidative stress and reverses age-related morphological changes of residual skin surface components in middle-aged volunteers.

Oxidative stress accelerates skin aging, and dietary supplementation with antioxidants may alleviate it. Morphological analysis of the residual skin surface components (RSSCs) allows detecting age-related changes in corneocyte desquamation, microbial presence, and lipid droplet size. We hypothesized that continuous ingestion of carotenoid antioxidant astaxanthin (4 mg/d) for 4 weeks could influence RSCC morphology and evaluated RSSC samples taken from middle-aged subjects before and after this dietary intervention. The study included 31 volunteers (17 men and 14 women) over the age of 40. RSSC samples were collected from the surface of the facial skin at the beginning (day 0) and end (day 29) of the study. In addition, blood samples were taken on days 0, 15, and 29 for measuring plasma levels of malondialdehyde that allowed assessing systemic oxidative stress. The results demonstrated that plasma malondialdehyde consistently decreased during astaxanthin consumption (by 11.2% on day 15 and by 21.7% on day 29). The analysis of RSSC samples has revealed significantly decreased levels of corneocyte desquamation (P=.0075) and microbial presence (P=.0367) at the end of the study. These phenomena as well as a significant (P=.0214) increase in lipid droplet size were more strongly manifested among obese (body mass index >30 kg/m2) subjects. All described RSSC changes correspond to a shift toward characteristics of skin associated with a younger age. The results confirm our hypothesis by demonstrating that continuous astaxanthin consumption produces a strong antioxidant effect resulting in facial skin rejuvenation which is especially pronounced in obese subjects.

Morphological Characteristics of Residual Skin Surface Components Collected from the Surface of Facial Skin in Women of Different Age.

BACKGROUND: Problems of skin aging and its prevention currently attract increasing attention with the growth of human life expectancy. The morphology of the stratum corneum (SC) is well known, but investigation of age-related changes of its structure is difficult in the absence of non-invasive sampling methods. The residual skin surface components (RSSC) that overlay the SC can be easily collected non-invasively. OBJECTIVE: The aim of this study was to examine morphology of RSSC samples collected from the surface of facial skin of healthy female volunteers of different age. METHODS: RSSC samples were non-invasively collected from 53 adult female volunteers (22 aged in the range 18∼25 years and 31 aged in the range 50∼73 years). The samples were analysed microscopically. RESULTS: Distinct age-related changes were determined for lipid droplet size, corneocyte desquamation level and lipid crystal count. There was a significant (p=0.0006) decrease in lipid droplet size among older women. Similarly, significantly (p=0.0401) lower lipid crystal numbers were present in the older group. Conversely, corneocyte desquamation was significantly higher (p=0.0007) in older women. No age-related difference in microbial presence in the RSSC could be detected. Result patterns were generally similar to those previously found in male volunteers; however gender-related differences in the absolute values were revealed. CONCLUSION: Non-invasively collected RSSC samples allow identifying age-related changes on facial skin surface. The results of this study highlight gender-dependence of distinct elements of age-associated impairment of epidermal barrier and can be employed for developing new approaches to prevent changes associated with skin aging.

Inflammatory bowel disease detection and monitoring by measuring biomarkers in non-invasively collected colorectal mucus.

BACKGROUND AND AIM: Non-invasive detection and monitoring of inflammatory bowel disease (IBD) is an important clinical challenge. Stool calprotectin is the most popular among available options, but the necessity of stool collection limits its acceptability. This study aimed to evaluate biomarker measurement in non-invasively collected colorectal mucus as a new tool for IBD detection and activity monitoring. METHODS: Calprotectin, eosinophil-derived neurotoxin (EDN), and protein S100A12 were measured in colorectal mucus self-collected following defecation by 58 patients with IBD (before therapy), 50 patients with irritable bowel syndrome, and 33 healthy volunteers. Patients with IBD also collected samples at days 10, 20, and 30 of treatment for disease activity monitoring. RESULTS: Protein biomarker levels were significantly (P < 0.001) higher in IBD patients than in irritable bowel syndrome and control groups. Calprotectin and EDN effectively detected IBD with a respective sensitivity and specificity of 0.76 and 0.92 for calprotectin and 0.83 and 0.94 for EDN. S100A12 was less sensitive. Calprotectin and EDN results were combined in a new test (CALEDN) that had a sensitivity of 0.91 and a specificity of 0.89. Repeated biomarker measurement during IBD treatment demonstrated a steady decline of calprotectin and EDN levels as well as CALEDN values in patients responding to applied therapy and lack of this pattern in non-responders. CONCLUSIONS: Measuring calprotectin and EDN in non-invasively collected colorectal mucus presents a simple and efficient method for IBD detection and monitoring. Excellent performance of EDN for this purpose is reported for the first time. Combining calprotectin and EDN in one test improves IBD detection sensitivity.

Colorectal mucus non-invasively collected from patients with inflammatory bowel disease and its suitability for diagnostic cytology.

Colorectal mucus is a key component of the protective gut barrier which is altered in inflammatory bowel disease (IBD). We aimed to cytologically characterize colorectal mucus non-invasively collected from IBD patients using our new sampling technique. Colorectal mucus was self-collected by 58 IBD patients comprising 31 ulcerative colitis (UC) and 27 Crohn's disease (CD) cases. The samples were examined cytologically, and immunocytochemically. Large numbers of well-preserved granulocytes were typically detected (neutrophils undergoing degradation were observed as well). Plasma cells and erythrophagocytosis were present in 18.2% and 29.1% of cases, respectively, predominantly in patients with UC and distal CD. Immunocytochemical visualization of calprotectin in neutrophils, eosinophil-derived neurotoxin in eosinophils and tumour necrosis factor-α in macrophages was also achieved. Correct cytological diagnosis was made in 61.8% of analysed IBD cases. Our new method of colorectal mucus sampling provides highly informative material for cytology. Findings of the presence of plasmocytes and erythrophagocytosis in colorectal mucus are unique and may reflect previously unknown mechanisms of IBD pathogenesis. Immunocytochemical detection of inflammation biomarkers demonstrates the suitability of this material for biomarker quantification. These promising results suggest a potential role for colorectal mucus cytology in the non-invasive diagnosis of IBD.

Assessment of cytology and mucin 2 in colorectal mucus collected from patients with inflammatory bowel disease: Results of a pilot trial.

BACKGROUND AND AIM: Non-invasive diagnosis of colorectal disease remains problematic, fecal biomarkers presenting the only current option. Colorectal mucus is the diagnostically informative element of stool samples, but its separation from stool is difficult. We aimed to: (i) test a novel method of non-invasive colorectal mucus sampling in a pilot clinical trial; (ii) evaluate sampling method acceptance by study participants; (iii) characterize the collected material cytologically; and (iv) assess feasibility of quantitative protein analysis in the samples. METHODS: A total of 141 patients with IBD (58), IBS (50), and healthy controls (33) participated in the study. Samples rich in colorectal mucus were self-collected by swabbing the anal area immediately following defecation. Collected samples were examined cytologically and subjected to quantitative analysis for total protein and mucin 2 (MUC2). RESULTS: The novel sampling technique was assessed as "good" or "adequate" by 96% of study participants. A total of 55% of the collected samples were free of fecal contamination. Cytology showed large numbers of well preserved inflammatory cells in IBD cases. Total protein values varied in all groups, being affected by fecal contamination. MUC2 levels were similar among all IBD-free individuals (control and IBS groups) and elevated in IBD patients (p < 0.001). MUC2 measurement applied as a test for IBD detection provided sensitivity = 72.4% and specificity = 86.7%. CONCLUSIONS: A novel non-invasive method for collecting human colorectal mucus has been successfully tested. The method was very well accepted by trial participants. The results have proven high quality of collected samples for both cytological investigation and diagnostic biomarker analysis.

Protein biomarkers in exfoliated cells collected from the human rectal mucosa: implications for colorectal disease detection and monitoring.

PURPOSE: Colorectal disease biomarkers in stool are actively explored, but instability of biomolecules in faeces constitutes a problem. Collection of exfoliated cells from the surface of the rectal mucosa provides an alternative to stool-based methods. We aimed to develop an original approach allowing preservation and quantification of protein biomarkers in exfoliated material and tested it in a pilot clinical study. METHODS: A novel method of cell and protein preservation in ammonium sulphate-rich buffers was developed using cultured human cells and applied to exfoliated cell samples collected from 139 faecal occult blood test (FOBT)-positive patients prior to colonoscopies. Protein biomarkers comprising calprotectin, eosinophil-derived neurotoxin (EDN), dimeric pyruvate kinase type M2 (M2PK), soluble cytokeratin-18, d-dimer and glyceraldehyde 3-phosphate dehydrogenase were quantified using enzyme-linked immunosorbent assays with parallel cytological and immunocytochemical analysis. RESULTS: Long-term preservation of cells and their protein constituents at ambient temperature was achieved using buffers containing saturated ammonium sulphate. Application of this approach to exfoliated cell samples allowed consistent protein quantification. Calprotectin, EDN, M2PK, soluble cytokeratin 18 and d-dimer showed dramatic increase in a few cases of inflammatory bowel disease (IBD) detected among trial participants. Cytological signs of inflammation were also present in these samples. CONCLUSIONS: Application of exfoliated cells collected from the surface of the rectal mucosa provides a reliable method for quantifying protein biomarkers of gastrointestinal diseases. Our preliminary results obtained in a limited number of cases indicate that the approach might be especially useful for IBD diagnosis and monitoring, but further studies are needed to assess its diagnostic value.

A case-control study of colorectal cancer detection by quantification of DNA isolated from directly collected exfoliated colonocytes.

This pilot study aimed to assess an original test based on the analysis of exfoliated colonocytes as a new approach to colorectal cancer (CRC) detection. DNA was isolated from exfoliated cells collected from the surface of the rectal mucosa by a standardized minimally invasive procedure in a case-control trial involving 66 patients with CRC diagnosis and 110 healthy volunteers (age 50-70). PicoGreen staining and quantitative real-time PCR (QRTPCR) were used for DNA quantification. Mean DNA scores in microg/ml obtained for the control and cancer groups were 2.1 (95% CI 1.7-2.5) and 9.0 (CI 6.7-11.2) respectively (p < 0.001) for PicoGreen and 0.8 (CI 0.6-0.9) and 3.8 (CI 1.9-5.7) respectively (p = 0.003) for QRTPCR. The PicoGreen assay better detected CRC presence. At DNA score cut-off point of 2.5 microg/ml this assay gave sensitivities of 77.8% (CI 52.4-93.6) for proximal tumours, 91.4% (CI 76.9-98.2) for distal CRC and 86.8% (CI 74.7-94.5) for all CRC with specificity at 74.0% (CI 64.0-82.4). Increasing the cut-off point to 5.0 microg/ml resulted in sensitivities of 38.9% (CI 17.3-64.3) for proximal tumours, 71.4% (CI 53.7-85.4) for distal CRC and 60.4% (CI 46.0-73.5) for all CRC. Specificity for this cut-off point increased to 94.8% (CI 88.3-98.3). The new procedure of exfoliated cell collection from the surface of the rectal mucosa is a simple, safe and well-tolerated technique providing high quality cells. These early results suggest that exfoliated cell collection in combination with DNA quantification can potentially be employed as a tool for CRC early detection.

Colorectal cancer detection by measuring DNA from exfoliated colonocytes obtained by direct contact with rectal mucosa.

The purpose of the study was to explore the potential of direct exfoliated colonocyte collection from human rectal mucosa for colorectal cancer screening. A special device was designed for standardized collection of exfoliated cells from the surface of human rectal mucosa. Material was collected from 120 outpatients selected for colonoscopy and 36 patients with confirmed diagnosis of colorectal cancer or large polyps. Determination of total DNA amounts in the collected samples (DNA scores) by PicoGreen assay and real-time PCR was employed alongside cytological assessment. Well preserved cells with cytological patterns characteristic for different colorectal conditions (cancer, inflammatory bowel disease) were detected in the collected material. In the outpatient group DNA scores were higher in patients with cancer and inflammatory bowel disease compared to those with no abnormalities detected, diverticular disease and small polyps (P<0.001 for PicoGreen assay; P=0.002 for real-time PCR). The sensitivity and specificity of the quantitative DNA test (PicoGreen assay; cut-off point 3.0 microg/ml) for detecting serious colorectal conditions were 1.00 and 0.74, respectively. In the group with confirmed tumours, the PicoGreen assay performed better for distal colorectal cancer (sensitivity 0.83; specificity 0.76) compared with proximal colon malignancies (sensitivity 0.57; specificity 0.76). It can be concluded that the proposed technique of direct collection of exfoliated cells from the surface of human rectal mucosa provides abundant cellular material suitable for diagnostic and research applications. Further refinement of the quantitative DNA test may lead to a new approach for colorectal cancer early detection and screening.

Isolation of exfoliated colonocytes from human stool as a new technique for colonic cytology.

Cell exfoliation in the gut is an important cell renewal mechanism. To approach its investigation we applied a novel immunomagnetic technique for isolation of exfoliated cells from human stool. Exfoliated colonocytes were isolated from 168 stool samples. The cells were assessed microscopically using conventional stains and immunohistochemistry. The technique allowed us to obtain well-preserved colonocytes displaying characteristic features of well-differentiated colonic epithelium and positive immunostaining for cytokeratin 5/8. No mucin-producing cells were found. Exfoliated cells did not produce inducible nitric oxide synthase, albeit cultured colon carcinoma cells HT-29 analysed in parallel showed strong immunostaining. Analysis of exfoliated cell numbers in consecutive stool samples from the same subjects revealed considerable interindividual variation. Overall exfoliated colonocyte numbers were relatively low, isolation being unaffected by addition during the procedure of excessive amounts of HT-29 cells. Apoptosis was extremely rare among exfoliated colonocytes. Well-preserved exfoliated colonocytes can be consistently isolated from human faeces using a simple procedure. Our findings suggest that the actual process of cell exfoliation in the human colon may be much less intense than is generally accepted. Exfoliated cell isolation from human stool constitutes a convenient non-invasive approach that can be used for diagnostic and research purposes.