Investigation of potential early Histologic markers of pediatric inflammatory bowel disease

Diagnosing pediatric inflammatory bowel disease (IBD) may be challenging at times as a broad spectrum of gastrointestinal and extra-intestinal symptoms may complicate the clinical presentation. When IBD is suspected, endoscopy is performed to examine the mucosa and obtain tissue as gross and histologic findings are the gold standard for diagnosing IBD. Unfortunately, histologic confirmation cannot always be obtained early in the course of the disease, thus delaying the diagnosis and treatment, and ultimately the optimal growth potential and quality of life for these patients.

While the quality of the histopathologic diagnosis relies heavily on the clinician to provide helpful clinical information and multiple biopsy specimens from different sites of the gastrointestinal tract, there are well established pathologic criteria for the diagnosis of IBD [1‐7]. The typical histologic features of IBD are those of a chronic active colitis, encompassing chronicity features of crypt architectural distortion and basal plasmacytosis. Disease activity is determined by cryptitis, crypt abscesses, and ulcerations. A variable number of eosinophils may be present [1]. Ulcerative colitis (UC) demonstrates a gradation of activity indices depending on the acuity of disease [2, 3]. The inflammatory activity of UC is confined to the mucosa and submucosa; with extension of disease continuously and proximally from the rectum. Mucus depletion from goblet cells, cryptitis with crypt abscesses, and crypt distortion are findings not unique to UC, but may also be seen in Crohn’s colitis [4]. In Crohn’s disease (CD), the inflammation may involve any portion of the gastrointestinal tract from the mouth to the anus. The inflammatory activity is usually discontinuous with skip lesions and shows a transmural involvement [5]. Granulomas are the hallmark lesion of CD but are found in only 40–60 % of resection specimens and much less frequently (15–36 %) in mucosal samples [6, 7].

Importantly, 8–31 % of adult patients with UC and approximately one third of pediatric patients with UC have presented with absent or atypical findings on initial biopsies [2, 8, 9]. There is relative paucity of data addressing this matter specifically in pediatric CD. Studies regarding the diagnostic lag in children with IBD are also lacking. Heikenen et al. retrospectively evaluated 91 children diagnosed with IBD, noting that the average lag for diagnosis was 7.1 months in CD and 6.7 months in UC [10]. Children who presented with growth failure had the longest diagnostic lag. A recent study by Kappelman et al. noted that healthcare utilization by younger IBD patients was disproportionately increased and costs for IBD patients younger than 20 were significantly higher than those for adults, suggesting that effective-management strategies in this population could yield cost-effective benefit [11, 12].

Several cytokines and matrix proteins have been implicated in the pathogenesis of IBD. Tumor necrosis factor-alpha (TNF-α) immunoreactive cells have been noted in increased frequency in the lamina propria of surgically resected specimens of patients with CD and UC [13]. Matrix metalloproteinase-9 (MMP-9) has been suggested as a mediator of mucosal breakdown in IBD and has been shown to be markedly upregulated in intestinal fistulae specimens of patients with CD [14, 15]. However, expression of either TNF-α or MMP-9 is not routinely assessed in the diagnosis of IBD and has not been studied in the early stages of pediatric IBD.

In our pediatric practice, which includes both a large volume of patients with IBD and patients with functional abdominal pain, we have observed that initial biopsies from children with abdominal pain who are later diagnosed with IBD may reveal normal or only subtle non-diagnostic pathologic changes. We hypothesized that there would be a significant difference in the presence of certain histologic findings and inflammatory cell types apparent on review of initial endoscopic biopsies for a subset of diagnostically delayed pediatric IBD patients in comparison to a group of pediatric abdominal pain patients. We further hypothesized that IHC staining for TNF-α and MMP-9 would be helpful in distinguishing the two groups.

Our results demonstrated that initial biopsies in children with an eventual diagnosis of IBD in comparison to biopsies of children with functional abdominal pain have significantly greater frequency of inflammation in the stomach, crypt distortion in the colon, and eosinophilia in the rectosigmoid area. This study is novel in that the comparison group is not an adult population but a pediatric subset of abdominal pain patients. Additionally, the IBD group has been expanded to include patients with CD, as prior studies to our knowledge have been limited to findings in UC [16, 17].

This is one of the first studies to attempt to understand the histologic features associated with the development of pediatric IBD. Few studies have addressed the concern that the initial biopsies of IBD patients may be negative or inconclusive and these studies have largely been confined to children with UC [16, 17]. The pediatric IBD population differs from the adult IBD population as children with IBD may present with atypical or normal histologic findings. Markowitz et al. found that 5 of 12 (42 %) children with ulcerative colitis who ultimately required colectomy had rectal sparing or mild patchy inflammation of the rectum and sigmoid areas on initial biopsy [16]. Another study evaluated 73 pediatric patients and 38 adult patients, all with newly diagnosed UC, showing that 30 % of children in comparison with only 3 % of adults had less severe inflammation in the rectum compared with more proximal areas [17]. 21 % of children showed patchy inflammation, which was significantly greater than the adults who had none. Washington et al. demonstrated that chronicity features are lacking more often on initial rectal biopsies in children with ulcerative colitis when compared to adults, finding that the initial biopsies from children were less likely to show diffuse architectural abnormalities when compared to adults [9]. In the current study, we demonstrated that children who will eventually be diagnosed with IBD demonstrate crypt distortion early in their course more frequently than children with abdominal pain not associated with evolving IBD. The presence of crypt distortion should heighten the clinician’s suspicion of IBD even in non-diagnostic biopsies.

Upper endoscopy is considered an essential component of the initial evaluation of a child with possible IBD [18, 19]. In a prospective evaluation of the importance of EGD in the diagnosis of IBD, focally enhanced gastritis was noted in 11 of 21 cases [19]. Focally enhanced gastritis has been seen more commonly in children with CD than UC and has also been noted to be present in 76 % of adults with CD compared to 0.8 % of controls [20, 21]. An increased association of active gastritis in children with CD has also been noted [22]. The gastritis noted in our patients was not active or “focally enhanced” but more indicative of chronic inflammation with a diffuse increase of lymphoplasmacytic cells in the lamina propria with or without reactive epithelial changes. The frequency of gastritis did not differ between CD and UC patients within the IBD group. As our finding of gastritis is rather nonspecific, it invites further investigation regarding the different types of gastritis noted in early IBD in children.

In the current study, we demonstrated significantly higher eosinophil density in the rectosigmoid area of patients who would eventually be diagnosed with IBD. Eosinophil infiltration can be nonspecific and may be observed in conditions such as allergy (e.g. allergy-associated colitis in adults and allergic proctocolitis in infants), eosinophilic gastroenteritis, eosinophilic colitis, IBD, parasite infection, infectious colitis, neoplasm, celiac disease, and autoimmune diseases [23]. Increased eosinophil density has been reported in active inflammation in both CD and UC and is associated with upregulation of eosinophil chemoattractants, eotaxin and RANTES [24]. CD is associated with enhanced secretion and accumulation of eosinophil cationic protein released with eosinophil degranulation [25]. The diagnostic and prognostic values of tissue eosinophilia in IBD patients, however, remain unclear. Some studies have suggested that tissue eosinophilia in the rectum may be predictive of a more favorable prognosis in UC patients as a paucity of eosinophils has been noted to be associated with an increased risk of eventual colectomy [26, 27]. More recent studies, however, have reported a possible dual role for the eosinophil with noted involvement in tissue destruction and repair in the different stages of UC as eosinophils were actually higher in inactive UC than in the active phase of the disease [28, 29]. Shen et al. found that tissue eosinophilia in ileal pouch mucosa in IBD patients treated with restorative proctocolectomy was found to be more prominent than that in the afferent limb, suggesting that luminal factors in different areas of the bowel may contribute to eosinophil-mediated inflammation [30]. Our finding of eosinophilia in the rectosigmoid area of the patients later diagnosed with IBD in comparison to abdominal pain patients suggests that eosinophilia may be an early indicator of disease progression. It should be noted that we did not control for allergic disease which may have influenced the results. However, previous studies have found no association between colonic eosinophil densities and a history of atopy and no significant seasonal variation [31, 32]. Further investigation in a prospective manner with a larger sample size may be warranted to evaluate for the presence of eosinophils in other areas of the gastrointestinal tract.

Prior studies have shown increased expression of TNF-α and MMP-9 in IBD tissue specimens [13‐15]. The TNF-α immunohistochemistry by Murch et al. was performed on frozen tissue samples that were not fixed in formalin or paraffin embedded. We failed to detect a significant difference in expression of either marker between the IBD group and the comparison group of abdominal pain patients. This may be due to limited sample size, timing of disease progression, or technical limitations of IHC staining on mucosal specimens that are formalin-fixed and paraffin-embedded. We believe that IHC on formalin-fixed paraffin embedded tissue is less sensitive than western blotting and real time PCR for detecting cytokine expression, however the latter techniques require fresh or frozen tissue. Future studies evaluating TNF-α and MMP-9 on fresh or frozen tissue utilizing western blotting or real time PCR over the course of disease progression may be helpful in understanding the role of TNF-α and MMP-9 as potential early biomarkers for IBD.

There are several limitations to this study, most importantly, the retrospective nature and limited sample size. The initial biopsies of approximately 30 % of the IBD patients included only upper endoscopy specimens. The addition of colon and terminal ileum biopsies in those patients may have provided more diagnostic clues. A prospective design may be challenging as this patient population is difficult to identify in the early stages of disease. Although IHC staining for TNF-α and MMP-9 did not provide significant results in this study, further investigation into other potential IHC markers may be warranted.

Although typical changes consistent with IBD are present in most cases, occasionally mucosal biopsies will show subtle or nonspecific changes. The absence of chronic changes in the colorectal mucosa on initial biopsies of some children may delay the diagnosis of IBD and initiation of appropriate medical therapy, thus histopathologic criteria predicting disease progression in pediatric IBD may potentially improve patient prognosis and quality of life. Histopathologic findings of colonic crypt distortion and possibly rectosigmoid eosinophilia may be early features of IBD in children. In such cases, careful observation should ensue and consideration should be given to repeat endoscopy if concerning symptoms continue.