Biochemical Biomarkers of Mucosal Healing for Inflammatory Bowel Disease in Adults

Mucosal healing (MH) is the key therapeutic target of inflammatory bowel disease (IBD). The evaluation of MH remains challenging, with endoscopy being the golden standard. We performed a comprehensive overview of the performance of fecal-, serum-, and urine-based biochemical markers in colonic IBD to find out whether we are ready to replace endoscopy with a non-invasive but equally accurate instrument. A Pubmed, Web of Knowledge, and Scopus search of original articles as potential MH markers in adults, published between January 2009 and March 2020, was conducted. Finally, 84 eligible studies were identified. The most frequently studied fecal marker was calprotectin (44 studies), with areas under the curves (AUCs) ranging from 0.70 to 0.99 in ulcerative colitis (UC) and from 0.70 to 0.94 in Crohn`s disease (CD), followed by lactoferrin (4 studies), matrix metalloproteinase-9 (3 studies), and lipocalin-2 (3 studies). The most frequently studied serum marker was C-reactive protein (30 studies), with AUCs ranging from 0.60 to 0.96 in UC and from 0.64 to 0.93 in CD. Fecal calprotectin is an accurate MH marker in IBD in adults; however, it cannot replace endoscopy and the application of calprotectin is hampered by the lack of standardization concerning the cut-off value. Other markers are either not sufficiently accurate or have not been studied extensively enough.


Introduction
Inflammatory bowel disease (IBD), encompassing ulcerative colitis (UC) and Crohn's disease (CD), are lifelong, potentially devastating conditions of the gastrointestinal tract, and are characterized by a relapsing-remitting course and heterogeneous clinical presentation with extraintestinal manifestations. Although their pathogenesis remains not fully elucidated, a crucial role seems to be played by an interaction of genetic, epigenetic, immunological, and environmental factors. The uncertainty and complexity of IBD pathogenesis result in the absence of a single diagnostic tool, and what is the most vital, the absence of an effective causative therapy [1,2]. This, in turn, drives the search for better therapeutic goals, and in parallel, the dynamic development of new therapeutic agents. In this respect, mucosal healing (MH) in IBD is gaining increasing attention. MH seems to be a better prognostic indicator of the disease outcome compared to clinical scores. In UC, it is associated with a lower risk of clinical relapse, hospitalization, need for immunosuppression and colectomy, and colitis-associated neoplasia [3][4][5][6][7][8]. In CD, MH has been significantly associated with less severe inflammation after In contrast to UC, inflammatory changes in CD can also appear proximal to the terminal ileum. Moreover, a deeper layer of the gut wall can be involved with the formation of fistulas. The evaluation of the upper digestive tract, small intestine, and non-luminal CD can be performed with upper endoscopy, imaging techniques (magnetic resonance/computed tomography), or with capsule endoscopy. It must be highlighted, that non-invasive and widely available ultrasonography, namely simple intestinal ultrasonography or ultrasonography with contrast enhancement, is a valuable diagnostic tool for evaluating the disease activity and extent [18]. A standard ileocolonoscopy allows for the evaluation of injuries in the luminal mucosa and the detection of potential stenosis. Endoscopic activity may be reliably scored using either the Crohn's Disease Endoscopic Index of Severity (CDEIS) [23] or the Simple Endoscopic Score for Crohn's Disease (SES-CD) [24]. Both scores have been prospectively validated and are highly reproducible with excellent inter-observer agreement. A CDEIS score of < 3 is defined as mucosal remission [25]. The calculation of the score is complicated and time-consuming, which impedes its application in daily practice. SES-CD is a simplified version of CDEIS, making it easier to calculate. It has been validated and compared to CDEIS, with which it has shown a strong

Calprotectin
Although there is a wide range of biochemical, serological, immunological, genetic, epigenetic, and microbiological markers studied regarding IBD, fecal calprotectin (FC) is one of the few that was implemented in clinical practice and has been extensively used. Furthermore, it has a strong position in the international and local guidelines and can be used not only by gastroenterologists and general practitioners but also by patients at home.
Calprotectin, first described in 1980, is a 36 kDa zinc-and calcium-binding protein that belongs to the S100 family. The main source of calprotectin are neutrophils, and to a lesser extent, monocytes and macrophages. Calprotectin constitutes 60% of the soluble cytosolic proteins of neutrophils and it Diagnostics 2020, 10, 367 5 of 31 is used as a marker of neutrophil turnover. It can be detected in different biological fluids, such as sera, saliva, and urine, as well as in feces. The concentration of the calprotectin in stool is proportional to the neutrophils' migration to the gastrointestinal tract, and thus, calprotectin is the most broadly measured marker in feces [27].
In clinical practice, FC measurement is employed to differentiate between functional bowel disorders, mainly irritable bowel syndrome, and inflammatory bowel disease. In patients with IBD, it is applied as a valuable non-invasive tool to monitor the disease course in terms of evaluating disease activity and mucosal healing. It was repeatedly demonstrated that FC is a sensitive marker of mucosal inflammation in IBD and is related to the extent of inflammation in UC [28,29]. In Crohn's disease, FC (cut-off: ≥ 70 µg/g) performed more accurately in the identification of endoscopically active disease (overall accuracy: 87%) than elevated CRP, blood leukocytosis, and CDAI (overall accuracy: 66%, 54%, and 40%, respectively) [30]. Likewise, in UC, FC detected endoscopically active disease with a very good overall accuracy (89%), better than the Clinical Activity Index, elevated CRP, and blood leucocytosis (overall accuracy: 73%, 62%, and 60%, respectively) [31]. FC may additionally serve as a very good marker in the differentiation of acute severe colitis from mild to moderate colitis (cutoff value: 782 µg/g, sensitivity: 84%, specificity: 88%, AUC: 0.92) [32].
FC is a factor that predicts the treatment response [33], as well as the future course of the disease, namely relapse and postoperative recurrence. It can predict remission maintenance in CD (cutoff value: 327 µg/g, sensitivity: 92.3%, specificity: 82.4%, AUC: 0.924) [34], but also a flare in UC (cutoff value: 114 µg/g, sensitivity: 76%, specificity: 85%) [35]. In UC patients treated with infliximab, a reduction in the concentration of FC predicted the remission of disease [36].
FC has also become an integral part of the complex patient evaluation in clinical trials testing novel therapeutic agents. At the same time, home assays designed for use by patients have been developed. Smartphone-based home FC test was evaluated by 56% of patients as easy to perform and more than 90% were satisfied with it. Furthermore, there was a close correlation between laboratory and home tests [37]. Smartphone-based testing is an example of implementing an e-health strategy in real-life practice and can be a very useful alternative method of monitoring IBD patients [38].
The performance of FC in the evaluation of CD activity seems to be accurate irrespective of the disease location [39]. However, patients with ulcers limited to the ileum had significantly lower FC levels than patients with ulcerations in the colon [40]. In another study, FC correlated with the affected surface but not with the ulcerated surface and disease location [41].

Fecal Calprotectin as a Marker of Mucosal Healing
Switching the approach to the therapeutic goals regarding IBD from clinical remission to MH resulted in the extensive assessment of FC as a non-invasive mucosal healing marker. This issue is further complicated by the definition of mucosal healing, which is far from being precise. The question arises whether an endoscopic evaluation is enough to diagnose MH, and if so, which score is optimal. Other tactics require the histologic confirmation of MH or a combination of both methods. Nevertheless, endoscopy is currently the prerequisite to diagnose mucosal healing. An alternative MH predictor is sought for this procedure, which is invasive, costly, and poorly accepted by patients.
The FC as an MH marker was evaluated in 44 eligible articles. The reported AUCs, allowing for the evaluation of the overall accuracy of a marker independently from a selected cutoff, ranged from fair to excellent overall accuracy for UC (0.70-0.99) and from fair to very good for CD (0.74-0.94) (Tables 2 and 3, respectively). Unfortunately, a large variation in the selected optimal cutoff values was noted, ranging from 13.9 to 918 µg/g for UC and from 71 to 918 µg/g for CD. The analysis of FC's performance as an MH marker revealed that it displayed better sensitivity regarding CD than for UC, with respective median values of 83% against 79%, and better specificity for UC than for CD, with respective median values of 85% against 71%.  Summarizing, FC may be a useful tool to detect MH; however, consensus on the cutoff value is still missing, which can be explained, at least partly, by the inter-assay variability. Carlsen et al. [42] demonstrated that FC may serve as a good indicator of deep remission at a cutoff value at ≤ 25 mg/kg, where deep remission in this study was defined using combined endoscopic (MES < 1) and histologic (Goebes Score ≤ 1) indices. Additionally, the results of the study confirmed the utility of FC as a marker of endoscopically and histologically active IBD with a cutoff of > 230 mg/kg. In turn, when the concentrations of FC are at the level of 25-230 mg/kg, researchers have suggested that endoscopy should be performed if it is needed to optimize the therapy. The performance of other markers (hemoglobin, CRP, orosomucoid, erythrocyte sedimentation rate, albumins) or the Simple Clinical Colitis Activity Index (SCCAI) as predictors of deep remission and inflammatory activity was not accurate. Jha et al. proposed 158 mg/kg for the cut-off value of FC when the definition of mucosal healing was MES ≤ 1 [43]. Moreover, in a recently published study, Walsh et al. showed that FC correlated strongly with UC activity in terms of the endoscopic (UCEIS), histologic (Nancy score), and combined evaluations. Furthermore, they identified the optimal threshold of FC concentration for histologic remission to be 72 µg/g, and for the endoscopic and combined criteria, the cutoff was 187 µg/g [46]. Zittan et al. [52] also demonstrated that low levels of FC (< 100 µg/g) closely correlated not only with endoscopic indices of IBD activity but also with histological remission (Goebes score < 3.1). Lee et al. [49] showed that the concentration of FC correlated better with UCEIS than with the Mayo score in a group of 181 UC Korean patients. Besides the correlation with endoscopic indices, a strong correlation between FC and both clinical activity index and CRP was shown. The FC evaluation had similar performance characteristics to the fecal immunochemical test (FIT); however, its specificity for identifying mucosal healing could be improved by the combined assessment of FC, FIT, and clinical symptoms [79]. Patel et al. [50] showed that there is a correlation between the FC level and disease extent but also with the worsening of activity in terms of clinical, endoscopic, and histological indices. Furthermore, these authors demonstrated that patients with mucosal healing, defined as MES ≤ 1, had significantly elevated FC if their disease was histologically active (Nancy score ≥ 2). In another study, almost 12% of patients with endoscopic remission had histologically active disease, which was accompanied by significantly elevated FC [80]. In the same study, a baseline FC level of ≥ 321 mg/g was predictive of disease relapse at the 6-and 12-month follow-up [80]. Björkesten et al. [26] analyzed a group of 64 CD patients and found that FC, in contrast to CRP, CDAI, and the Harvey-Bradshaw Index (HBI), was a good marker of MH. However, 13% of patients with endoscopically active disease had a normal FC value. Hence, they attempted to create a score by incorporating all the aforementioned indices. Despite some advantages from the combination of HBI and FC, these parameters were not superior to the FC alone. As 24% of CD patients and 15% of UC patients have undefined disease activity according to their FC level (100-250 µg/g), it has been proposed that incorporating FC into the disease activity evaluation, together with clinical indices and CRP, would serve as a better marker of disease activity than FC alone [81].

Limitations of Fecal Calprotectin
FC may be elevated in many pathological conditions, such as gastrointestinal infections, gastric and colonic malignancies, eosinophilic colitis, lymphocytic colitis, and coeliac disease [82]. For instance, in a group of 870 outpatients referred for colonoscopy, an increased level of FC was demonstrated in 85% of patients with colorectal cancer and in 81% of patients with inflammatory conditions [83]. In turn, pregnancy does not affect the concentration of FC, and thus, its determination might be a useful non-invasive marker for monitoring the disease activity in pregnant IBD patients [84]. Multiple additional factors affect the level of FC and they all ought to be included when interpreting the result of the FC measurement.
Age, as well as concomitant medical treatment with proton pump inhibitors, nonsteroidal anti-inflammatory drugs, and acetylsalicylic acid, can all cause an increased concentration of FC. Correspondingly, Lundgren et al. [85] showed that around one-third of patients with a normal colonoscopy result had a slightly elevated FC concentration (>50 µg/g), which was associated with the patients' age and treatment with the above-mentioned drugs in the multivariate analysis.
Furthermore, wide daily variability of FC concentrations in stools has been observed [86]. Hence, this can be seen as a limitation for the FC application as a marker of the early response to induction therapy [87].

Technical Considerations
Currently, several commercial assays for FC measurement are available. Studies comparing the assays demonstrate similar performance when differentiating organic and functional bowel diseases [88]. Still, there are substantial inter-assay differences between FC tests from different manufacturers, hampering the establishment of a universal cutoff value [89]. New assays for simultaneous evaluation of FC and FIT (a latex agglutination turbidimetric immunoassay system) demonstrated similar performance to a standard ELISA assay [51].
Recently, a rapid test for FC evaluation using immunochromatographic methods has been developed as a point of care test (POCT). Although semiquantitative, rapid tests yield results in 30-40 min, allowing for an immediate adjustment of the therapy if needed. As such, they offer a useful alternative to ELISA, which is the gold standard for FC evaluation but is also time-consuming and requires laboratory infrastructure. The comparison of rapid tests with ELISA demonstrated a high level of agreement between both methods [90].

Other Fecal Markers
Among fecal markers other than calprotectin, the most intensively studied during the last decade were lactoferrin, another neutrophil-derived protein, which is responsible for limiting bacterial growth by lowering iron availability; matrix metalloproteinase (MMP)-9; and lipocalin-2. As an MH marker, lactoferrin and MMP-9 were more accurate in UC than in CD. In turn, lipocalin-2 was a more accurate marker of MH in CD (see Tables 4 and 5). However, the number of studies is limited and the conclusion regarding the potential utility of those markers in clinical practice cannot be drawn. Moreover, the properties of the aforementioned fecal markers were not shown to be superior to FC.

C-Reactive Protein (CRP)
The most intensively evaluated serum-based marker was CRP, examined in 30 eligible publications, which was frequently done as a reference for novel potential markers. CRP positively correlated with the endoscopic activity of the disease, yielding correlation coefficients from 0.29 to 0.63 for UC (Table 6) and from 0.31 to 0.71 for CD (Table 7). Reported areas under the receiver operating characteristics (ROC) curves (AUCs), allowing for the evaluation of the overall accuracy of a marker independently from the selected cut-off, ranged from fair to very good overall accuracy for both UC (0.60-0.96) and CD (0.64-0.92) (Tables 6 and 7, respectively).  There was a very large variation in the selected optimal cut-off values, ranging from 0.4 to 28 mg/L. Differences of two orders of magnitude are unlikely for a standardized marker, such as CRP, and might imply errors in the reported units of concentration. The analysis of CRP performance as the MH marker revealed that it displayed better sensitivity than the specificity for CD, with respective median values of 79.5% against 61%, but better specificity than sensitivity for UC, with respective median values of 82% against 66%. For UC, CRP performed better when mucosal healing was defined as MES ≤ 1, or equivalent, than when restricted to MES = 0. Rosenberg et al. [106], Arai et al. [74], and Shinzaki et al. [108] compared the diagnostic power of CRP using both MH definitions. They showed CRP performance to be superior in terms of sensitivity, specificity, and/or the overall accuracy in the case of the less restrictive definition. Chen et al. [47], in turn, compared CRP as the MH marker in a cohort consisting exclusively of selected patients in clinical remission with a cohort of UC patients with clinically active or inactive disease, finding the marker's performance to be superior in terms of specificity in the latter cohort. Accordingly, some authors have demonstrated that CRP was better at detecting moderate-to-severe disease activity but failed to differentiate between inactivity and mild endoscopic activity [30,31,78,103,105,107]. As demonstrated by Yoon et al. [110], CRP displayed similar accuracy, sensitivity, and specificity as the MH marker regardless of the type of score used for MH evaluation. However, it has to be mentioned that some authors failed to observe a significant association between CRP and endoscopic scores [32,[112][113][114][115][116][117][118] and/or did not find CRP to be significantly superior to a chance marker in detecting MH in UC [42,114,119] or CD [32].

Other Acute Phase Reactants (APRs)
CRP was not the only acute phase reactant (APR) that has been evaluated in terms of its association with the endoscopic activity of IBD. During the last decade, other positive APRs, such as procalcitonin, fibrinogen, orosomucoid, and serum amyloid A, as well as negative APRs, such as albumin and transferrin, have been appraised in this respect, although less extensively than CRP and with less success. Procalcitonin (PCT) increases in response to pro-inflammatory stimuli, particularly of bacterial origin, where the sensitivity and specificity of which as a bacterial infection marker is claimed to be superior to CRP. Accordingly, in acute UC (UCEIS ≥ 3), PCT concentrations reflected endoscopic activity more precisely than those of CRP and albumin (respectively, r = 0.46, r = 0.18, and r = −0.23), as reported by Wu et. al. [120], and contrary to CRP, differed significantly between MES = 2 and MES = 3, as demonstrated by Koido et al. [121]. Unfortunately, none of the authors evaluated the diagnostic power of procalcitonin as a potential MH marker. Moreover, others failed to confirm a significant association between procalcitonin and the endoscopic activity of UC [47,112,122] or CD [47,112]. Zezos et al. [123] showed that fibrinogen concentration positively correlates with endoscopic scores to a similar extent as CRP in UC (r = 0.49, p < 0.001 for both) and Eder et al. [124] in CD (respectively, r = 0.59 and r = 0.57, p < 0.0001 for both); however, these were not followed by the evaluation of its diagnostic power. As reported by Miranda-Garcia et al. [114], fibrinogen, with an AUC < 0.05, failed to detect MH in UC patients (Table 8) but it displayed a good overall accuracy as an MH marker, accompanied by an excellent sensitivity in one study on CD patients (Table 9). In the same study investigating fibrinogen, orosomucoid was evaluated and yielded similar overall accuracy and sensitivity but slightly lower specificity in CD, but also failed as an MH marker for UC [114]. Its unsuitability for MH detection in UC has been confirmed by Carlsen et al. [42]. Furthermore, in CD, serum amyloid A (SAA) was found to display identical good overall accuracy in two studies but superior sensitivity in one [115] and superior specificity in the other [123], probably owing to the different cutoff values applied (Table 9). For UC, SAA accuracy was found to be insufficient [42]. Albumin displayed a good overall accuracy as an MH marker for the general UC population in a study of Uchihara et al. [109] but not those of Carlsen et al. [42] or Jusué et al. [32]. For CD, albumin has been reported to inversely correlate with the SES-CD score (r = −0.62, p < 0.0001) [124] but others have found it to fail as an MH marker [32]. Moreover, even if significantly better than a chance marker, albumin seems to share similar drawbacks to CRP, i.e., being less efficient in detecting MH among patients with clinical remission [65] (Table 8). Concerning transferrin, only its correlation with MES was reported (r = −0.37, p = 0.001) [125]. Uchihara et al. [109] argued that the unsatisfactory performance of APRs in differentiating mucosal inflammation from healing results from the fact that during endoscopy, only fragments of mucosa with the most severe inflammation are considered when reporting activity scores. To support their thesis, the authors compared CRP (Table 6) and albumin (Table 8) performance as the MH markers using the traditional MES score and cumulative MES score, in which scores obtained for six different bowel regions were added. Indeed, both CRP and albumin displayed superior AUCs and specificities when the MH definition was based on the cumulative MES score. N, number of observations; MH, mucosal healing; ↑/↓, increased/decreased; r, correlation coefficient; p, p-value; CR, subgroup of patients in clinical remission; NT, not treated with infliximab; T, treated with infliximab; (m)MES, Mayo Endoscopic Subscore; c(MES), cumulative Mayo Endoscopic Score; UCEIS, Ulcerative Colitis Endoscopic Index of Severity; RI, Rachmilewitz Index; EAI, Endoscopic Activity Index; SES-CD, Simple Endoscopic Score for Crohn's Disease; VEGF-A, vascular endothelial growth factor-A; ADA, adenosine deaminase; ST2, decoy receptor for IL-33 (member of IL-1 receptor family); NGAL, neutrophil gelatinase B-associated lipocalin; NGAL-MMP9, neutrophil gelatinase B-associated lipocalin-matrix metalloproteinase 9; sIL2R, soluble IL-2 receptor; sTREM-1, soluble triggering receptor expressed on myeloid cells-1; IL, interleukin; TNFα, tumor necrosis factor α; GM-CSF, granulocyte-macrophage colony-stimulating factor; IFNγ, interferon-γ; Nampt, nicotinamide phosphoribosyltransferase; LRG, leucine-rich alpha-2 glycoprotein; TTF3, trefoil factor-3; LL-37, cathelicidin; ALB, albumins; AUC, area under receiver operating characteristics (ROC) curve; Sens., sensitivity; Spec., specificity; na, not available; *, counted from a scatterplot. Numbers in superscript indicate parameters corresponding with a given MH definition or cohort.

Cytokines, Their Receptors, and Growth Factors
CRP and other positive APRs are produced in response to cytokines, mainly interleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)-α, which also affect the concentrations of negative APRs [135]. Unsurprisingly, those cytokines, as well as many others, have also been of interest as potential MH markers, especially given that the imbalance between pro-and anti-inflammatory cytokines is a hallmark of IBD [136]. For UC, TNFα and IL-9 displayed an excellent overall accuracy, IL-6 and IL-17A displayed very good accuracy, granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon (IFN)γ displayed good accuracy, and soluble IL2 receptor (sIL2R) and IL-12(p70) displayed fair accuracy. Most of those cytokines had higher specificities than sensitivities (Table 8). However, TNFα, IL-6, IL17, and IFNγ in Rodriguez-Peralvarez et al.'s [132] study failed to detect MH and the only cytokine better than a chance marker was IL-8, displaying only a fair accuracy. IL-6 displayed an excellent overall accuracy as an MH marker for CD, IL-9 and IL-1β displayed good accuracy, and sIL2R displayed fair accuracy. The AUC for IL-17 was not calculated but the cytokine displayed good sensitivity and specificity (Table 9). While some authors reported significant correlations between the endoscopic activity of CD and IL-1, IL-6, IL-12, IFNγ, and TNFα (all r > 0.9, p < 0.0001) [137], others failed to observe significant associations for IL-6, TNFα, and IFNγ [78]. Algaba et al. [126] evaluated a panel of angiogenic factors as potential markers differentiating mucosal inflammation from healing, of which, only vascular endothelial growth factor (VEGF)-A for UC (Table 8) and angiopoietin-1 for CD (Table 9) performed significantly better than a chance marker, displaying fair overall accuracy and better sensitivities than specificities. Good and very good accuracies were reported for ST2 (interleukin 1 receptor-like 1) by two research groups for UC (Table 8) and fair accuracy by one group for CD (Table 9).

Other Serum-Based Markers
Most of the remaining serum-based markers evaluated during the last decade as potential MH markers are also associated with inflammatory and immune responses. Several authors have evaluated lipocalin-2 (neutrophil gelatinase-associated lipocalin (NGAL)), a glycoprotein that is physiologically present in serum at low concentrations but over-secreted in response to stimuli associated with epithelial damage, either alone or in a complex with matrix metalloproteinase 9 (MMP9). NGAL displayed good overall accuracy for UC (Table 8) and fair to good accuracy for CD (Table 9). Trifoil factor (TTF)-3, a protein involved in healing of the epithelium, protecting the mucosa from insults, and the stabilization of the mucus layer, positively correlated with UCEIS [141] and displayed a fair overall accuracy as an MH marker for UC (Table 8), but failed to reflect endoscopic activity [141] or indicate MH for CD [124].
In UC, individual studies have been dedicated to adenosine deaminase activity, a non-specific marker of T-cell activation, antimicrobial peptide LL-37, adipokine and immunomodulatory cytokine visfatin (Nampt), and lysine-rich α2 glycoprotein (LRG), a protein involved in inflammatory responses to bacterial infections as well as in angiogenesis; all of these have been characterized by good overall accuracy, sensitivity, and specificity (Table 8).
While visfatin performed well for UC patients, it failed to detect MH in a mixed cohort of UC and CD patients examined by Trejo-Vasquez et al. [134]. The authors analyzed a diabetic panel including, among others, plasminogen activator inhibitor (PAI)-1 and adipokines (leptin, resistin, and visfatin), of which, only leptin displayed a fair overall accuracy as an MH marker (Table 8). Jung et al. [103] evaluated a soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) in both UC and CD and found the marker to display fair sensitivity and good specificity for UC (Table 8), but with an estimated 29% sensitivity, it failed as an MH marker for CD. TREM-1 belongs to the immunoglobulin superfamily and is present on monocytes, neutrophils, macrophages, and endothelial cells, and its soluble form accompanies inflammatory and infectious conditions. Presepsin (a soluble cluster-of-differentiation 14 subtype (sCD14-ST)) is a truncated form of the co-receptor for toll-like receptor 4 (TLR4), which is expressed on phagocytic cells and shed into the circulation upon lipopolysaccharide binding in a manner proportional to the severity of the inflammatory response. Although correlated with endoscopic scores for both CD and UC (r = 0.277, p = 0.022 for UC), presepsin, in turn, differentiated mucosal inflammation and healing only for CD patients with good accuracy, sensitivity, and specificity (Table 9). Moreover, Hosomi et al. [112] demonstrated that the combined evaluation of presepsin with CRP substantially improves the sensitivity (100%) compared to individual assessments of CRP and presepsin. Similarly, regarding presepsin, the ratio of estrogen receptors (ER) β-to-α was a good MH indicator in CD (Table 9) but not UC. Solely for CD, ficolin-2, a liver-synthesized pattern recognition molecule involved in inflammation and immunity, was examined. Unlike most of the evaluated markers, its concentrations had already significantly increased in mild mucosal inflammation without a further elevation in moderate and severe inflammation, potentially making it a unique tool for differentiating mild inflammation from complete healing [77]. However, the reported overall accuracy and sensitivity have only been fair and the specificity of ficolin-2 was rather poor (Table 9). Yarur et al. [115,140] examined the performance of adhesion molecules and key proinflammatory cytokines, namely V-CAM, I-CAM, IL-1β, IL-6, IL-8, and TNFα, as potential MH markers in CD. They found that each marker's performance in a general population of CD patients was unsatisfactory. However, as a panel, they achieved good overall accuracy (AUC = 0.81) [115]. Moreover, IL-1β and I-CAM were good predictors of MH in a subpopulation of CD patients resistant to anti-TNFα therapy [140].

Interpretative Synthesis of Data: Urine-Based Markers
The only eligible study evaluating potential MH markers in urine was that of Arai et al. [142], who examined prostaglandin E and found it to be a good or even excellent marker, depending on the MH definition, which showed a stepwise increase along with MES (Table 10).

Review Limitations
Although all possible efforts were made to make the review complete, some potentially relevant publications could be overlooked due to the huge number of records to be screened at the stage of the title and abstract selection. Moreover, several studies reporting marker performance were excluded upon a full-text reading due to a lack of clarity concerning whether clinical or endoscopic remission was evaluated. Furthermore, it must be pointed out that most studies included in the final analysis were conducted on small cohorts and/or had an unbalanced design with a substantial disparity between patients with endoscopic remission and inflammation, and thus the reported accuracies, sensitivities, and specificities ought to be interpreted with caution.

Conclusions
At the beginning of the 21st century, we are facing a growing incidence of IBD, especially in regions where the previously reported incidence was low, i.e., in Asia and Eastern Europe [143]. The complex nature of these diseases makes IBD patients a heterogeneous group of subjects, which can benefit from an individualized approach [144]. Still, available diagnostic and therapeutic possibilities are limited. MH has recently received interest as a therapeutic goal, for the evaluation of which, endoscopy is mandatory. At the time being, biochemical biomarkers, although possessing many advantages, cannot substitute for an invasive, expensive, and time-consuming colonoscopy. Out of those used in clinical practice, fecal calprotectin remains the closest to "the perfect" MH marker. However, as long as the standardized cutoff value of FC is missing, it cannot be broadly applied as the surrogate marker of endoscopic remission. Other fecal or serum-based markers also seem to be promising. However, as they were not evaluated in replicated and cross-validated studies and were often conducted on small cohorts, further research is required before they could substitute or be used alongside fecal calprotectin.
Ongoing research on novel biochemical biomarkers of MH should focus on validation of the most promising indices in larger and better-defined cohorts. As indicated by this review, interleukins upstream of CRP in an inflammatory cascade, namely IL-1β and IL-6, have the greatest diagnostic potential for colonic CD. For UC, IL-6, as well as TTF3, IL-9, ST2, TNFα, and ADA, seem to be more powerful than other markers. A promising tactic for getting the most out of biochemical biomarkers would be to combine multiple markers into panels, especially these displaying a high sensitivity but lower specificity with those characterized by a high specificity combined with a lower sensitivity. Such an approach already yielded excellent results for CD prediction, where a panel of 51 serum antibodies and proteins, developed on a large cohort (n = 400), was demonstrated as being able to predict CD diagnosis within a year with an 87% accuracy [145].