Effect of disease duration on fecal biomarkers in ulcerative colitis: a prospective cohort study

Ulcerative colitis (UC) is a refractory disease characterized by symptoms such as diarrhea, bloody stools, and abdominal pain with repeated relapses and remissions [1]. Evaluation of UC activity is important, and endoscopic evaluations of mucosal inflammation can facilitate subsequent prognostication [2, 3]. On the basis of these considerations, achievement of mucosal healing is important in inflammatory bowel disease (IBD), and evaluation of IBD using endoscopy is extremely important. However, endoscopic examinations are associated with problems such as physical burden to the patient, cost, and risk of complications.

Thus, to avoid the need for frequent endoscopy, noninvasive biomarkers that accurately reflect the endoscopic activity of UC have emerged, and fecal calprotectin (FC) and the fecal immunochemical occult blood test (FIT) are being widely used in clinical practice [4‐9]. FC is a calcium-binding protein found in neutrophils, monocytes, and macrophages, accounting for approximately 60% of the cytoplasm [10]. When inflammation occurs locally in the intestinal tract, leukocytes, including neutrophils, migrate into the lumen through the intestinal wall; thus, the inflammatory state of the intestinal tract can be determined by measuring the amount of calprotectin in feces [11]. The FIT was originally developed for colorectal cancer screening, and in UC, it reflects bleeding caused by inflammation of the mucosa and has been reported to correlate with the endoscopic score [6]. FC and FIT have been reported to not only reflect endoscopic activity but also predict subsequent relapse in patients with UC in clinical remission [12‐15]. In addition to these fecal biomarkers, serum leucine-rich alpha 2 glycoprotein (LRG) has been reported to be a useful marker that reflects endoscopic activity in UC and has been used in clinical practice [16‐18].

We had previously compared FIT with the urinary biomarker prostaglandin E-major urinary metabolite (PGE-MUM) [19]. The results of that study showed that disease duration affected the correlation between FIT concentrations and endoscopic scores; thus, the shorter the disease duration, the stronger the correlation. In contrast, PGE-MUM showed a high correlation with endoscopic activity during long-term disease. Thus, UC disease duration may affect the accuracy of each biomarker, and these effects may differ across biomarkers. However, though there are many reports investigating the relationship between endoscopic score and FC and FIT, the effect of disease duration on FC, which is a biomarker often used in clinical practice, has not yet been reported [5, 7, 8, 12, 13].

Therefore, this study examined the effect of UC disease duration on FC in comparison with FIT, which has already been shown to be particularly useful for evaluating short-term disease.

Mucosal healing is an important therapeutic goal in the management of UC, and biomarkers for the evaluation of mucosal healing have been widely used in clinical practice [26]. Endoscopic severity has been reported to predict the prognosis of UC, and the achievement of mucosal healing has been recognized to be important [2, 3]. For this reason, although biomarkers have been recognized to predict the prognosis of UC because they reflect endoscopic activity, recent studies have suggested that biomarker values themselves may also have prognostic significance [12‐15]. Furthermore, the prognosis of UC has been reported to be improved by strengthening treatment in cases with elevated biomarker levels; therefore, measurement of biomarkers can also contribute to improvements in the clinical management of UC [27, 28]. Thus, perceptions of biomarker measurements have changed progressively with accumulating evidence describing various aspects of biomarkers. While fecal biomarkers such as FC and FIT were initially thought to be the primary biomarkers, LRG, a serum biomarker, has recently gained more importance in clinical practice, and the role of biomarker measurement has expanded in the management of UC [16‐18]. C-reactive protein (CRP), a marker that reflects acute inflammation in various diseases, has also been shown to reflect disease activity in IBD. In addition, various other markers that are not widely used in current clinical practice, including fecal lactoferrin, PGE-MUM, and miRNA, have been investigated [29‐32]. Each biomarker has its own characteristics, and an understanding of these characteristics is important when making measurements.

For example, from the viewpoint of sample collection, while blood and urine can be collected in a relatively homogenous state, the collection of fecal samples and the accuracy of the tests using these samples can be influenced by the collection site and the condition of the stool. Additionally, patient hesitation is another factor to consider when a stool sample has to be collected and brought to the hospital. In terms of biomarker accuracy, FC has been reported to more accurately reflect endoscopic severity than CRP [33], and low CRP levels do not necessarily reflect the absence of endoscopic activity [34]. However, our previous report, which evaluated the relationships between biomarkers and the endoscopic score of the entire colon, showed that CRP might strongly reflect the activity of UC in cases showing endoscopic activity [35]. Thus, considering the variety of biomarkers for UC and the individual characteristics of each marker, as well as the effects of different factors on their accuracy, we aimed to investigate the effect of disease duration on FC and FIT, which are two commonly used biomarkers.

In a previous study of biomarkers in UC, we reported a strong correlation between FIT concentration and endoscopic scores, especially in short-term disease-duration groups, and showed that UC disease duration could affect the accuracy of FIT evaluation [19]. We suspected that FIT represents the amount of bleeding from the intestinal tract due to inflammation of UC and that the amount of bleeding decreases due to scarring over longer disease durations. In contrast, PGE-MUM, a urinary biomarker of UC, tended to show a strong correlation even in long-term disease. On the basis of the results of these FIT and PGE-MUM analyses, we suspected that each biomarker might be affected differently by disease duration. Although FC is widely used in clinical practice for UC as well as in clinical trials, the effects of disease duration on UC have not been investigated. In this study, participants were grouped by disease duration, and the correlation and ROC analyses were performed. For FIT, the correlation coefficient with the endoscopic score tended to be lower in the long-term disease-duration group than in all cases and higher in the short-term disease-duration group than in all cases. This result was similar to the findings of our previous report [19]. For FC, the correlation coefficients with the endoscopic score tended to be lower in the long-term disease-duration groups than in all cases and higher in the short-term disease-duration groups than in all cases. FC showed similar results as FIT, indicating that the shorter the disease duration, the more accurate it may be. Nonetheless, the results of these analyses do not provide the necessary clarity to precisely define the duration of a “long” or “short” disease course. These terms are based on the observed tendency of the data. For accurate disease duration, additional cases need to be investigated first.

Although the mechanism underlying the greater accuracy of FC in the short-term disease-duration group is unclear, we hypothesized that fibrosis of the intestinal tract caused by prolonged disease duration might impede the normal expression of neutrophils in the intestinal tract. As a result, long-term disease duration affects FC concentration, which reflects the presence of neutrophils in the intestinal tract. In fact, in the comparisons among the groups in Cohort 2, although the median FC concentration was not significantly different, it was lower in the long-term disease-duration group (Table 2). In addition, the risk of carcinogenesis is known to increase with prolonged UC duration; Hata et al. reported that the cumulative incidence of invasive cancer in UC was 0.5% at 10 years, 4.1% at 20 years, and 6.1% at 30 years [36]. As a mechanism of inflammatory carcinogenesis in such cases of UC, the oxidative stress associated with inflammation may cause damage to the DNA of the mucous membrane and induce carcinogenesis through dysplasia by activation of the carcinogenic gene and suppression of the carcinogenic suppressor gene [37, 38]. We suspect that DNA damage via this mechanism may also alter the inflammatory cytokine profile, like the apoptosis signal pathway due to DNA damage, in the intestinal tract and may affect FC, which reflects neutrophils in the intestinal tract [39].

Furthermore, in the ROC analysis for predicting mucosal healing, both FC and FIT showed high AUCs, especially in the group with a disease duration of 0–4 years. The other two groups showed lower AUCs and had similar AUCs for both FC and FIT concentrations, respectively. As described above, in the ROC analysis, the effects of disease duration were similar between FC and FIT concentrations, and this effect may contribute to a more accurate prediction of mucosal healing in shorter disease durations. To date, no reports have investigated the effect of disease duration in UC on FC. A previous study evaluated the relationship between FC and disease duration for CD by dividing patients into groups with disease duration less than 10 years and ten years or longer. The authors reported that disease duration did not affect the diagnostic usefulness of FC [40].

This study had several limitations. The first limitation is the small number of participants since this was a single-center study. Although the analysis was performed in groups representing each disease duration in Cohort 2, a larger sample size per group would result in more accurate analysis. Additionally, a larger overall sample size would facilitate more detailed evaluations of disease duration divisions by increasing the number of subgroups. Second, in Cohort 1, the sample size differed depending on the subgroup for each disease duration. If the correlation coefficients are to be compared, it is desirable that the sample sizes are the same; however, in this analysis, the number of patients to be analyzed varied from 39 to 122 and 26 to 123 in the long-and short-term disease-duration groups, respectively. However, regarding the analysis of the short-term disease-duration groups, the short-term duration groups had high correlation coefficients despite the small sample size, and this result further supported the possibility that the correlation coefficient was high in the short-term disease-duration group. Third, cases without a total colonoscopy were excluded. Endoscopic examination in patients with severe IBD is associated with the risk of serious complications such as perforation [41, 42]. In addition, the evaluation of severe UC may have been insufficient because it was evaluated by a short colonoscopy, and colonoscopic examination was omitted in consideration of the patient's burden.

As a biomarker in UC, FC may be affected by disease duration similar to FIT. It was considered particularly useful in UC patients with short disease duration.