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- Material and methods
In the course and prognosis of colorectal cancer (CRC), early detection and treatment are essential factors. Fecal immunochemical tests (FITs) are currently the most commonly used non-invasive screening tests for CRC and premalignant (advanced) adenomas, however, with restricted sensitivity. We hypothesized that fecal volatile organic compounds (VOCs) may serve as a diagnostic biomarker of CRC and adenomas. In this proof of concept study, we aimed to assess disease-specific VOC smellprints in fecal gas to distinguish patients with CRC and advanced adenomas from healthy controls. Fecal samples of patients who were scheduled to undergo an elective colonoscopy were collected. An electronic nose (Cyranose 320®) was used to measure VOC patterns in fecal gas from patients with histopathologically proven CRC, with advanced adenomas and from controls (no abnormalities seen at colonoscopy). Receiver operator characteristic curves and corresponding sensitivity and specificity for detection of CRC and advanced adenomas were calculated. A total of 157 stool samples (40 patients with CRC, 60 patients with advanced adenomas, and 57 healthy controls) were analyzed by electronic nose. Fecal VOC profiles of patients with CRC differed significantly from controls (area under curve ± 95%CI, p-value, sensitivity, specificity; 0.92 ± 0.03, <0.001, 85%, 87%). Also VOC profiles of patients with advanced adenomas could be discriminated from controls (0.79 ± 0.04, <0.001, 62%, 86%). The results of this proof of concept study suggest that fecal gas analysis by an electronic nose seems to hold promise as a novel screening tool for the (early) detection of advanced neoplasia and CRC.
Colorectal cancer (CRC) is one of the predominant cancers, contributing to a high burden of morbidity and mortality in the United States of America and Europe.[1, 2] Early detection and treatment are critical factors in the course and prognosis of CRC, and screening programs have proven to be an important means to reduce both mortality and secondary economic burden.[3-5] Colonoscopy is considered the gold standard for CRC and advanced adenoma screening. Fecal immunochemical tests (FIT) are currently the most commonly used non-invasive fecal screening tests. However, sensitivity of FIT for CRC is between 66–88%[6-10] depending on the cut-off values used, whereas sensitivity for advanced adenomas is disturbingly low (27–41%).[6, 8, 11, 12] As CRC prevention programs should primarily focus on early detection of premalignant advanced adenomas, the search for novel, more accurate non-invasive screening methods remains warranted.
Analysis of volatile organic compounds (VOCs) in exhaled breath has been reported as a potential non-invasive diagnostic biomarker test for lung cancer, breast cancer, malignant melanomas and CRC.[13-15] VOCs are gaseous carbon-based chemicals resulting from biochemical processes in the body, which are discharged by exhaled air, sweat, urine and feces. VOCs in fecal gases are mainly produced by the intestinal microbiota in the colon during fermentation processes and also derive from metabolic processes within microorganisms.[17-20] Molecular compounds of VOCs in exhaled air or from feces can be individually detected by gas chromatography and mass spectometry (GC-MS). However, this technique is expensive and requires time-consuming off-line analysis, making its widespread application in clinical practice unfeasible.
Electronic noses enable real-time, high-throughput analysis of the complete spectrum of VOCs in complex gas mixtures. The electronic nose (e-nose) technology is based on an array of nanosensors, each reacting to different fractions of the VOC mixture by a sensor-specific change in resistance. Combination of individual sensor measurements create a specific smellprint (fingerprint), which can subsequently be analyzed by means of pattern recognition algorithms.[21, 22] No studies on the usage of the e-nose in fecal gas analysis are currently available in literature.
We hypothesized that e-nose may discriminate fecal samples from patients with CRC from those with advanced adenomas and from (healthy) controls, by disease-specific pattern recognition of VOC smellprints. This hypothesis was tested in a cross-sectional proof of principle study analyzing VOC in the headspace of fecal samples, obtained from patients with CRC, patients with advanced adenomas, and controls. As secondary aim, accuracy of fecal VOC-analysis with the FIT was compared by performing a FIT on fecal samples from patients with CRC, patients with advanced adenomas, and controls.
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- Material and methods
In this proof of concept study, fecal VOC patterns of patients with CRC and advanced adenomas were compared with controls by means of e-nose. We have shown that fecal VOC analysis by e-nose is a feasible technique with high-throughput capability. Fecal samples from patients with colorectal carcinoma and advanced adenomas could firmly be distinguished from controls. Moreover, an e-nose could differentiate between advanced adenomas and CRC by fecal gas analysis.
Information on fecal VOC and their potential as diagnostic biomarkers in gastrointestinal diseases is limited, since most studies were performed with exhaled breath analysis. In few studies, differences in VOC profiles from patients with various gastrointestinal diseases, compared with controls have been reported, all of these measured by GC-MS.[18, 19, 29, 30] Since fecal VOC are mainly produced by the intestinal microbiome, it is not surprising that specific differences were detected in that set of diseases that are linked to alterations in the intestinal microbiota composition (e.g., inflammatory bowel disease (IBD), necrotizing enterocolitis (NEC), diarrhea-predominant IBS and bacterial gastro-enteritis.[18, 19, 29, 30] Although these studies were all pilot-studies with limited numbers of patients, fecal VOC seemed to contain biomarkers with clear diagnostic and disease activity monitoring potential, a finding we reproduced in our study aiming at (early) detection of advanced adenomas and CRC.
In the pathogenesis of CRC, changes in intestinal microbiome composition have been reported to be present, but a cancer-related microbiotal signature has not yet been identified.[31, 32] Recently, Altomare and colleagues identified disease-specific VOC in exhaled breath from 37 patients with CRC by means of GC-MS analysis, compared with 41 controls. On the basis of the differences in chemical compound composition, CRC could be distinguished from controls with an accuracy of 76%. Furthermore, it was shown that a specially trained Labrador dog was able to distinguish stool samples from CRC patients from controls by detection of canine scent (sensitivity 97%, specificity 99%). However, patients with advanced adenomas were not included in both studies. Whether the observed differences in VOCs or smell were because of specific changes in the intestinal microbiotal composition or due to alterations in (local) metabolism secondary to cancer remain to be clarified.
The e-nose technique is a non-invasive, high-throughput method based on pattern recognition of response to vapors. Real-time analysis of the complete spectrum of VOCs is possible, reinforcing its potential as clinical tool. Previous studies in exhaled breath have shown adequate repeatability and reproducibility.[21, 22, 34, 35] In this study, reproducibility of the e-nose technique in fecal gas analysis was reassuringly high. Because no previous studies on VOC analysis in fecal gases with this e-nose technique have been described, information on the optimal circumstances, such as temperature, amount of substrate, pressure and water content of the fecal material, was not available. To test our hypothesis, we used fecal samples instead of exhaled breath samples, because disease-specific VOCs are produced presumably by an altered (specific) intestinal microbiota composition due to the presence of premalignant or colorectal tumor cells. In addition, collection, storage and analysis of fecal samples can be controlled more vigorously compared with exhaled breath samples. Confirmation of our hypothesis as measured in fecal samples at body temperature would reinforce e-nose as a potential bedside CRC screening instrument.
This study has shown that e-nose could distinguish CRC from controls by detection of disease-specific fecal VOC profiles with a sensitivity of 85% and a specificity of 87%. We selected this cut-off point in the ROC curve to optimize corresponding specificity for CRC. To limit Type 1 errors, a different cut-off point in the ROC curve may be selected improving sensitivity for CRC to 93%, but at the expense of specificity. Reported historical FIT data show a sensitivity for CRC of 66–88% and a specificity of 87–96%.[6-10] More importantly, e-nose enabled differentiation between patients with advanced adenomas and controls, with a clinically relevant sensitivity (sensitivity 62%, specificity 86%) compared with reported FIT characteristics (sensitivity FIT for advanced adenomas 27–41%, specificity 91–97%).[6-10] Again, by selecting a different cut-off point on the ROC curve, sensitivity of e-nose for advanced adenomas may even be improved to 81% (with a corresponding decrease of specificity to 58%)
In this study, we compared the e-nose characteristics with FIT results obtained from the same fecal samples. Sensitivity and specificity of FIT for both CRC and advanced adenomas differed largely from historical FIT data. A potential explanation for these differences could be that all fecal samples had been stored at −20°C for a period of time, before FIT was performed. In the setting of this study, these circumstances may be detrimental for the condition of the stored samples, for example by a decrease in haemoglobin concentration. Another limitation of this study is the limited availability of demographic data of the included individuals. Information on use of medication potentially influencing VOC composition (like antibiotics), and dietary intake during collection of the fecal samples was not obtained. However, in a previous landmark study on fecal VOCs in healthy subjects, it was shown that the majority of fecal VOCs remain relatively constant in health, with only limited changes because of day-to-day dietary habits. To assess whether the limited demographic dataset might have influenced our e-nose results, we performed a stringent additional statistical analysis, revealing that the probability of a Type 1 error (false positive outcome) was very low for all of our diagnostic algorithms (p < 0,001). Future studies comparing and combining e-nose and GC-MS with FIT analysis, performed on fresh fecal samples, should include complete demographic data to allow clinical delineation of these non-invasive tests in CRC screening. Such an approach may help to identify biomarkers both specific and sensitive for CRC. Future tailoring of sensors toward such biomarkers may further increase the accuracy of the e-nose technique in the detection of CRC and advanced adenomas, enhancing its potential value as screening tool. In summary, the reported results showed that an e-nose can be used to distinguish CRC from healthy controls by analysis of fecal VOC profiles in the headspace of stool samples. Interestingly, advanced adenomas could be distinguished from controls with a higher sensitivity compared with historical FIT results. Therefore, analysis of fecal gas by e-nose seems to hold promise as a novel screening tool for the (early) detection of advanced neoplasia and CRC.