Fecal S100A12 concentrations in cats with chronic enteropathies

Abstract

Objectives

The aim of this study was to compare fecal S100A12 concentrations in cats diagnosed with chronic enteropathy (CE) with healthy control cats.

Methods

This was a prospective, cross-sectional study. Forty-nine cats that had gastrointestinal signs for >3 weeks and a complete diagnostic work-up, including bloodwork, abdominal ultrasound and upper and/or lower gastrointestinal endoscopic biopsies, were enrolled into the CE group. Nineteen cats from the CE group were diagnosed with inflammatory bowel disease (IBD) or chronic inflammatory enteropathy (CIE) and 30 with alimentary lymphoma (LSA), based on histopathology results and additional testing with immunohistochemistry or molecular clonality testing with PCR if indicated. Nineteen apparently healthy control cats were included in the study. One fecal sample was collected from each cat and S100A12 concentrations were quantified by an analytically validated in-house ELISA.

Results

Fecal S100A12 concentrations differed between cats with LSA (median 110 ng/g; interquartile range [IQR] 18-548) and control cats (median 4 ng/g; IQR 2-25 [P <0.001]) and between cats with IBD (median 34 ng/g; IQR 15–973) and control cats (P <0.003). S100A12 concentrations in CE cats (median 94 ng/g; IQR 16–548) were statistically significantly higher compared with control cats (P <0.001). The area under the receiver operating characteristic curve (AUROC) to separate healthy cats from CE cats was 0.81 (95% confidence interval [CI] 0.70–0.92) and was statistically significant (P <0.001). The AUROC to separate cats with IBD from cats with LSA was 0.51 (95% CI 0.34–0.68) and was not statistically significant (P = 0.9).

Conclusions and relevance

Fecal S100A12 concentrations at the time of diagnostic investigation were higher in cats with CIE and LSA than in healthy controls but did not differ between cats with LSA and those with CIE/IBD. This study is an initial step toward evaluating a novel non-invasive marker of feline CIE. Further studies are needed to determine the diagnostic utility of fecal S100A12 concentrations in cats with CE, including comparing cats with IBD/CIE and LSA, and to compare them with cats with extra-gastrointestinal disease.

Keywords

Biomarker gastrointestinal inflammation inflammatory bowel disease lymphoma

Introduction

Chronic enteropathies (CE) are common in cats. Cats often present with clinical signs such as vomiting, diarrhea, anorexia, hyporexia and/or weight loss.^1–5 The diagnostic work-up for these patients involves a systematic evaluation to exclude secondary causes of gastrointestinal (GI) signs, and documentation of mucosal inflammation based on histopathology after biopsy.^3–5 Histopathology aims to distinguish between inflammatory bowel disease (IBD) or chronic inflammatory enteropathy (CIE) and alimentary lymphoma (LSA); however, changes in mucosal architecture are not always definitive. Additional testing with immunohistochemistry (IHC) and molecular clonality testing with PCR are often used to help distinguish between IBD and LSA.^6–8

The pathophysiology of inflammatory enteropathies is not fully understood, but has been hypothesized to involve interactions between environmental, genetic and immune factors, leading to chronic inflammation.^3–5,9 In humans and companion animals, mutations in innate immune receptors, upregulated epithelial major histocompatibility complex (MHC) class II molecule expression, microbiota alterations and increased antibody reactivity to antigens of commensal microbiota have been linked to CE.^9–15 Additionally, dietary factors are hypothesized to play a role, based on a clinical response to dietary trials in both dogs and cats.^2,16–19

S100A12 (calgranulin C) is a damage-associated molecular pattern molecule of the innate immune response and calcium-binding protein of the S100/calgranulin family.^20,21 It is predominantly released from activated polymorphonuclear cells and targets several different receptor proteins, including receptors for activated glycation end products (RAGE).^21,22 The ligand–RAGE interaction activates nuclear factor kappa B, leading to the production of proinflammatory cytokines and chemokines.^22,23 S100A12 plays a central role in both innate and acquired immune responses and has several cellular regulatory functions.^20,21,24 It is a sensitive and specific marker of localized inflammatory processes and has been associated with acute inflammation, chronic inflammation and malignancy in humans.^25–33 S100A12 accumulates at sites of inflammation and is stable in feces.^29,34 Fecal S100A12 is a biomarker of intestinal inflammation in dogs with CE and has been associated with the severity of endoscopic lesions and response to treatment.^35–38

Biomarkers of inflammation are considered valuable for the diagnosis and monitoring of IBD in humans.^25–32,39 S100A12 is linked to many human inflammatory GI diseases.^27–33 S100A12 and other minimally invasive biomarkers for CE diagnosis, disease monitoring, and monitoring response to therapy have been evaluated in dogs.^{35–38,40–42} Numerous biomarkers have been evaluated in cats with CE, including cobalamin, folate, albumin, serum amyloid A, serum haptoglobin, calprotectin and the diagnostic marker of ultrasonographic mucosal thickening, though histopathology remains the gold standard for a diagnosis of CIE.^43–47 There is a paucity of sensitive and specific non-invasive biomarkers of GI inflammation in cats, with a recent focus on the S100/calgranulin family of proteins.

To our knowledge, there have been no published reports of fecal S100A12 concentrations in cats diagnosed with CE. The aim of this initial study was to quantify and compare fecal S100A12 concentrations in cats diagnosed with CE and healthy control cats. We propose that cats with CIE/IBD and LSA have higher S100A12 concentrations than control cats.

Materials and methods

Ethics approval

The prospective, cross-sectional study design was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC number 11-25-20) at the Schwarzman Animal Medical Center. An informed consent form was provided to the owner of each cat prior to inclusion in the study.

Sampling population

Cats with CE aged >1 year (n = 56) presenting for routine diagnostic evaluation of chronic (⩾3 weeks’ duration) signs of GI disease (ie, vomiting, diarrhea, hyporexia or anorexia, abdominal pain and weight loss), including a planned endoscopic collection of GI tissue biopsies according to World Small Animal Veterinary Association (WSAVA) guidelines, were prospectively enrolled.⁴⁸ All cats underwent a routine diagnostic evaluation, including a complete blood count, serum chemistry profile, urinalysis, abdominal ultrasound imaging and additional diagnostics to rule out other causes of chronic GI signs (eg, fecal testing, serum trypsin-like immunoreactivity, feline pancreas-specific lipase, cobalamin and folate) at the discretion of the attending clinician at the Schwarzman Animal Medical Center. The severity of the cat’s clinical signs at presentation was quantified by a single investigator using the feline CE activity index (FCEAI) scoring system.⁴⁹ Treatment of each individual cat, including dietary management, antibiotic treatment, supplements, anti-inflammatory or immunosuppressant therapy, was also at the discretion of the attending clinician. Exclusion criteria applied retrospectively included an unclear histopathologic diagnosis of IBD vs LSA, normal histopathology or diagnosis of neoplasia other than LSA (Figure 1).

Figure 1

Diagnostic evaluation of eligible affected cats. Fifty-six cats with chronic gastrointestinal (GI) signs were prospectively enrolled. Seven cats were excluded due to unclear histopathologic diagnosis of inflammatory bowel disease (IBD) vs alimentary lymphoma (LSA; n = 5), normal histopathology (n = 1) or a diagnosis of neoplasia other than LSA (n = 1) for a final affected study population of 49 cats. The IBD group consisted of 19 cats: 14 cats diagnosed with IBD based on histopathology alone and five with PCR for antigen receptor rearrangement (PARR) for the T-cell receptor gamma (TRG) locus contribution. The LSA group consisted of 30 cats: 19 cats diagnosed with LSA based on histopathology alone, two with immunohistochemistry (IHC) contribution and nine with PARR contribution

Healthy control cats (n = 19) aged >1 year with no history of GI signs were included. Control cats were not on any medications for at least 30 days prior to sample collection. Dietary history was collected, and control cats were excluded if fed a raw diet.

Sample collection

Fresh fecal samples (~1 g) were collected from affected cats with gentle lubricated digital removal while under anesthesia and prior to bowel preparation at the time of endoscopy. Spontaneously voided fecal samples were provided by owners of healthy control cats in at-home litterboxes. Control fecal samples collected at home were refrigerated (at approximately 3°C) and provided within 24 h of defecation. Each fecal sample was immediately frozen (−80°C) until batch stool shipment to the Gastrointestinal Laboratory at Texas A&M University (TAMU).

Endoscopic GI tissue biopsies were obtained from each cat with the extent of biopsy acquisition (upper vs upper and lower endoscopy with biopsies) left to the discretion of the attending clinician. Endoscopic evaluation was performed using either a 103 cm endoscope with an 8.6 mm outside diameter and a 2.8 mm working channel (GIF-160; Olympus), or a 140 cm endoscope with a 7.9 mm outside diameter and a 2.8 mm working channel (60714 NKSK; Karl Storz). All biopsies were obtained using 2.8 mm cup biopsy forceps. All tissue biopsies were immediately fixed in 10% formalin and transported to the laboratory for histopathologic evaluation.

Sample analyses

Histopathologic evaluation of GI tissue biopsies was performed by one of 10 board-certified veterinary pathologists at IDEXX Reference Laboratories. A diagnosis of IBD was based on a main histologic finding of diffuse lymphoplasmacytic inflammatory infiltrate, with severity of inflammation based on the reported WSAVA histopathological standards.⁵⁰ A diagnosis of lymphoma was based on an expanded monomorphic population of lymphocytes obscuring architecture with fusion of villous papillae, and the presence of intraepithelial lymphocytes forming discrete plaques, nests or clusters.⁵¹ In cases where histopathology was insufficient to differentiate IBD from LSA, additional diagnostic modalities were employed. Immunohistochemical staining was performed and interpreted by the same veterinary pathologist who interpreted the histopathology at IDEXX Reference Laboratories. PCR for antigen receptor rearrangement for the T-cell receptor gamma locus (PARR) was performed by a single board-certified veterinary pathologist at the Animal Health Laboratory at the University of Guelph, Ontario. IHC demonstrating strong cytoplasmic immunoreactivity with CD3 antigen, or PARR showing clonality combined with clinical, laboratory and histopathologic findings, was considered consistent with a diagnosis of lymphoma.^8,52

Fecal S100A12 concentrations were measured at the Gastrointestinal Laboratory at TAMU using an analytically validated in-house ELISA.^34,53–55 A canine S100A12-ELISA was first developed and shown to be accurate, reproducible, and sufficiently linear and precise for the measurement of S100A12 in cat serum and fecal samples.⁵⁵ A reference interval (RI) for feline fecal S100A12 was reported in a previous study.⁵⁵ However, since this RI has not been updated and the assay is only used infrequently, a statistical comparison between groups was performed rather than comparing results to the previously reported RI.

Data analyses

A commercially available statistical software package was used for all statistical analyses (SAS version 9.4, 2014). The normality of error residuals was analyzed by the Kolmogorov–Smirnov test and visual inspection. Descriptive statistics were used to analyze continuous variables and reported as median and interquartile range (IQR). Categorical data are presented as counts and percentages. A Kruskal–Wallis test was used to compare S100A12 concentrations between cats with IBD and healthy controls, cats with LSA and healthy controls, a combined CE group and healthy controls, and cats with IBD and LSA. Receiver operating characteristic (ROC) curve analysis was used to calculate the area under the ROC (AUROC) curve and served to calculate sensitivity and specificity at cutoff concentrations (determined by the Youden index) to differentiate (1) cats with CE from healthy cats and (2) cats with IBD from cats with LSA. Spearman’s rank-order correlation coefficient (r) was used to evaluate the correlation between S100A12 and other variables, including age and FCEAI score. A non-parametric Wilcoxon rank-sum test was used to compare S100A12 concentrations between cats with and without hyperthyroidism, and between cats with and without chronic kidney disease (CKD). Post-hoc Dunn’s multiple comparisons test was used to identify differences in S100A12 concentrations between healthy cats, CE cats with CKD and CE cats without CKD. Statistical significance was set at P <0.05.

Results

Study population

A total of 56 cats with CE were evaluated for enrollment into the study. Seven cats were excluded due to an unclear histopathologic diagnosis of IBD vs LSA (n = 5), normal histopathology (n = 1) or a diagnosis of neoplasia other than LSA (n = 1) for a final affected study population of 49 cats (median age 12 years [range 4–20]; 28 neutered males and 21 spayed females; median FCEAI score 6) (Figure 1). The most common presenting complaints included vomiting, weight loss, hyporexia, diarrhea, lethargy, hiding, hematochezia, regurgitation and constipation. Nineteen healthy control cats (median age 7 years [range 1–13]; 12 neutered males and 7 spayed females) were also enrolled into the study. The characteristics of each study population, including sex, age, breed, FCEAI score and most common presenting complaints are summarized in Table 1.

Table 1

Summary of the study populations

	Total CE population (n = 49)	IBD (n = 19)	LSA (n = 30)	Control (n = 19)
Sex	28 MN + 21 FS	11 MN + 8 FS	17 MN + 13 FS	12 MN + 7 FS
Median (IQR) age (years)	12 (8.25–14.5)	10 (7–14)	12 (10–15)	7 (4–10)
Breed (n)	DSH (34); DLH (3); Abyssinian (2); Ragdoll (2); American Shorthair (1); Bengal (1); Birman (1); Burmese (1); DMH (1); Oriental Shorthair (1); Siamese (1); Tonkinese (1)	DSH (13); DMH (1); DLH (1); Birman (1); Ragdoll (1); Siamese (1); Tonkinese (1)	DSH (21); DLH (2); Abyssinian (2); American Shorthair (1); Bengal (1); Burmese (1); Oriental Shorthair (1); Ragdoll (1)	DSH (18); DMH (1)
Median (IQR) FCEAI score	6 (5–7)	5.5 (5–7)	6 (5–8)	NA
Most common presenting complaint, n (%)	Vomiting, 32 (65);weight loss, 29 (59); hyporexia, 20 (41); diarrhea, 16 (32); lethargy/hiding, 7 (14); hematochezia, 4 (8); regurgitation, 1 (2); constipation, 1 (2)	Vomiting, 9 (47); weight loss, 10 (52); hyporexia, 6 (32); diarrhea, 6 (32); lethargy/hiding, 5 (26); hematochezia, 2 (11); regurgitation, 0 (0); constipation, 1 (5)	Vomiting, 23 (77); weight loss, 19 (63); hyporexia, 14 (47); diarrhea, 10 (33); lethargy/hiding, 2 (7); hematochezia, 2 (7); regurgitation, 1 (3); constipation, 0 (0)	NA

A feline CE activity index (FCEAI) score could not be calculated for one cat with chronic enteropathy (CE) in the inflammatory bowel disease (IBD) group due to lack of applicable history

LSA = alimentary lymphoma; MN = male neutered; FS = female spayed; IQR = interquartile range; DSH = domestic shorthair; DLH = domestic longhair; DMH = domestic mediumhair; NA = not applicable

Of the 49 cats with CE, 21 had biopsies performed of the stomach/duodenum alone, 10 had biopsies performed of the stomach/duodenum and colon, 16 had biopsies performed of the stomach/duodenum, ileum and colon, and two had biopsies performed of the colon only. Thirty cats were diagnosed with LSA (median age 12 years; 17 male neutered and 13 female spayed; median FCEAI score 6) and 19 were diagnosed with IBD (median age 10 years; 11 male neutered and eight female spayed; median FCEAI score 5.5) (Figure 1). Nineteen cats were diagnosed with LSA based on histopathology alone, nine with PARR contribution to diagnosis and two with IHC confirmation. Twenty-eight of 30 cats were diagnosed with epitheliotropic small-cell lymphoma. One of the cats with small-cell lymphoma was diagnosed concurrently with B-cell lymphoma of the stomach. Of the two cats whose histopathology was not small-cell lymphoma, one was diagnosed with intermediate-to-large-cell lymphoma without epitheliotropism, and the other had a neoplastic population of small-to-intermediate-sized lymphocytes, raising concern for large granular lymphoma. Of the 19 cats diagnosed with IBD, PARR was performed in five cases due to features consistent with LSA, but the PARR results were polyclonal and most consistent with inflammation. A total of three cats with IBD had ileal biopsies performed. Blind biopsies were acquired in two cats, and one cat had successful ileal intubation for biopsy acquisition. Thirteen LSA cats had ileal biopsies performed; blind biopsies were acquired in six cats, and seven cats had visualized ileal biopsies after successful intubation. Ten of these 13 cats had evidence of LSA in the ileum; however, all 13 cats had evidence of LSA on duodenal histopathology. All colonic histopathology showed inflammatory changes consistent with IBD. The location of biopsy acquisition and severity of lymphoplasmacytic histopathologic inflammation in the LSA and IBD populations is summarized in Table 2.

Table 2

Summary of endoscopic gastrointestinal tissue biopsy acquisition for cats with inflammatory bowel disease (IBD) and alimentary lymphoma (LSA)

	LSA (n = 30)	Severity	IBD (n = 19)	Severity	Total (n = 49)
Stomach/duodenum	13	Mild: 2; moderate: 9; severe: 2	8	Mild: 2; moderate: 6; severe: 0	21
Stomach/duodenum/colon	4	Mild: 1; moderate: 3; severe: 0	6	Mild: 0; moderate: 4; severe: 2	10
Stomach/duodenum/ileum/colon	13	Mild: 0; moderate: 8; severe: 5	3	Mild: 0; moderate: 3; severe: 0	16
Colon only	0	Not available	2	Mild: 0; moderate: 2; severe: 0	2

The extent of tissue acquisition was left to the discretion of the attending clinician. Overall histopathologic severity of lymphoplasmacytic inflammation per site is also summarized based on the microscopic description. In cases with mixed severity of inflammation, the most severe degree of inflammation noted in the histopathology report was assigned

Six CE cats received glucocorticoids prior to diagnostic evaluation (prednisolone, n = 5; inhaled, fluticasone, n = 1). A total of three cats were receiving oral metronidazole at the time of endoscopy. One cat had received a subcutaneous injection of a long-acting antibiotic (cefovecin) 5 days prior for a dental procedure. Eleven cats were trialed on a hydrolyzed or novel protein diet trial with persistence of GI signs. One CE cat was being fed a raw diet. CE cats were not excluded for other comorbid conditions, although 17 cats had CKD and five had hyperthyroidism. Of the cats with CKD, 16/17 were of International Renal Interest Society (IRIS) stage 1 or 2; 1/17 cats had IRIS stage 3 CKD. All cats with hyperthyroidism were well controlled on medical management at the time of investigation.

Fecal S100A12 concentrations

Fecal S100A12 concentrations ranged from <1 ng/g to >81,241 ng/g in individual fecal samples from all cats diagnosed with CE (median 94; IQR 16–548). In cats diagnosed with IBD, fecal S100A12 concentrations ranged from <1 ng/g to 57,987 ng/g (median; IQR 15–973). In cats diagnosed with LSA, fecal S100A12 concentrations ranged from 3 ng/g to >81,242 ng/g (median 110; IQR 18–548). Fecal S100A12 concentrations in the control group ranged from 1 ng/g to 223 ng/g (median; IQR 2–25) (Figure 2).

Figure 2

Fecal S100A12 concentrations (ng/g) in control cats, cats with inflammatory bowel disease (IBD) and cats with alimentary lymphoma (LSA). In the IBD group, fecal S100A12 concentrations ranged from <1 ng/g to 57,987 ng/g (median 34; interquartile range [IQR] 15–973). In the LSA group, fecal S100A12 concentrations ranged from 3 ng/g to >81,242 ng/g (median 110; IQR 18–548). Fecal S100A12 concentrations in the control group ranged from 1 ng/g to 223 ng/g (median 4; IQR: 2–25). Fecal S100A12 levels were statistically significantly higher in cats diagnosed with IBD compared with healthy controls (**P <0.003) and in cats diagnosed with LSA compared with healthy controls (***P <0.001). There was no statistically significant difference in fecal S100A12 concentrations in cats with LSA compared with those with IBD (P = 0.9; not significant [ns])

Fecal S100A12 concentrations were statistically significantly higher in cats diagnosed with CE (combined IBD/LSA, median 94 ng/g; IQR 16–548) compared with healthy control cats (median 4 ng/g; IQR 2–25 [P <0.001]). Fecal S100A12 concentrations were statistically significantly higher in cats diagnosed with IBD compared with healthy control cats (P <0.003) and in cats diagnosed with LSA compared with healthy control cats (P <0.001). There was no statistically significant difference in fecal S100A12 concentrations between cats with LSA and those with IBD (P = 0.9).

ROC analysis

The AUROC to separate healthy from CE cats was 0.81 (95% confidence interval [CI] 0.70–0.92) and was statistically significant (P <0.001; Figure 3a). When the cutoff value of S100A12 concentration between CE and healthy cats was set at 13.8 ng/g, the sensitivity was 79.6% (95% CI 66.3–88.5) and specificity was 68.4% (95% CI 46.0–84.6) for differentiating between the two groups. The AUROC to separate cats with IBD from cats with LSA was 0.51 (95% CI 0.34–0.68) and was not statistically significant (P = 0.9; Figure 3b). When the cutoff value of S100A12 concentration between IBD and LSA cats was set as 39.1 ng/g, the sensitivity was 63.3% (95% CI 45.5–78.1) and specificity was 52.6% (95% CI 31.7–72.7) for differentiating between the two groups.

Figure 3

(a) Receiver operating characteristic (ROC) curve of fecal S100A12 concentration in healthy vs cats with chronic enteropathy. The area under the ROC (AUROC) curve is 0.81 (95% confidence interval [CI] 0.70–0.92; P <0.001). (b) ROC curve of fecal S100A12 concentration for cats with inflammatory bowel disease vs those with alimentary lymphoma. The AUROC curve is 0.51 (95% CI 0.34–0.68; P = 0.9)

Other variables

There was no correlation between the age of all the cats and S100A12 concentration (r = 0.085, P = 0.49), or between the age of cats with CE and S100A2 concentration (r = –0.031, P = 0.83). There was also no correlation between FCEAI score and S100A12 concentration (r = –0.056, P = 0.71). There was no statistically significant difference in S100A12 concentration between cats with CE with hyperthyroidism (n = 5) and cats without hyperthyroidism (n = 63; P = 0.64). There was a statistically significant difference in S100A12 concentrations between CE cats with CKD (n = 17) and all cats without CKD (n = 51; P <0.003). However, while there was a statistically significant difference in S100A12 concentrations between control cats and CE cats without CKD (P <0.006) and control cats and CE cats with CKD (P <0.001), there was no statistically significant difference in S100A12 concentration between CE cats without CKD and CE cats with CKD (P = 0.34).

Discussion

The purpose of this study was to quantify and compare fecal S100A12 concentrations in cats with CE to healthy control cats. To our knowledge, this is the first study to evaluate fecal S100A12 in cats with CE. A diagnosis of IBD/CIE and lymphoma requires evaluation of biopsy specimens in combination with clinical signs, dietary trials and assessment of clinical response.⁴⁸ However, GI biopsy acquisition is not appropriate or feasible for every cat with chronic GI signs. Factors taken into consideration include anesthetic concerns, client expectations, financial constraints and the limitations of an endoscopic procedure vs the invasiveness of a surgical procedure. For cases in which endoscopy is not pursued, clinically useful non-invasive biomarkers indicating GI inflammation may be helpful to raise the index of suspicion for underlying GI disease and guide empiric therapy. S100A12 is an inflammatory biomarker that can be measured in fecal samples.^{27,29,31,32,35} Collection of feces for measurement of S100A12 is non-invasive and can be done at home by the owner, making this a user-friendly diagnostic tool.⁵⁶

This study demonstrates that fecal S100A12 was significantly higher at the time of diagnostic investigation in cats with CE compared with healthy controls, with significant discrimination based on AUROC. Though we studied cats and recognize differences in disease processes, the results are in alignment with former studies performed in dogs and humans that showed concentrations of fecal canine S100A12 to be significantly higher in dogs with IBD compared with healthy controls, and a sensitive marker of GI inflammation with exceptional specificity in distinguishing between children with IBD and children without IBD.^29,31,36 The present study represents a first step toward developing a novel non-invasive marker of feline CIE as part of a multimodal approach to diagnosis of GI disease.

A secondary aim of the study was to compare fecal S100A12 concentrations in cats with IBD and LSA. The etiopathogenesis of feline low-grade LSA is not yet fully elucidated, but some cases are thought to arise from chronic untreated GI inflammation and neoplastic transformation of IBD to LSA over time.^6,43 In the present study, fecal S100A12 concentrations did not significantly differ between cats with IBD and LSA. There was no discrimination between IBD and LSA groups based on AUROC, and possible cutoffs had low sensitivities and specificities. A recent study that evaluated IHC for S100A8/A9 and S100A12 in GI biopsies of cats with CE showed slightly higher GI mucosal S100A8/A9 and S100A12 cell counts than controls, but there was no significant difference between cats with IBD and LSA.⁵⁷ The lack of differentiation between IBD and LSA aligns with the current challenges in veterinary medicine in distinguishing between the two disease processes; as stated earlier, differentiation between IBD and LSA is not always definitive with a single diagnostic test such as histopathology alone. Similarly, fecal S100A12 concentrations did not conclusively distinguish between the two disease entities in this cohort of cats.

In this study, there was no correlation between clinical disease activity (FCEAI score) in the CE group and fecal S100A12 concentration. FCEAI score is calculated by tallying the presence of and severity of GI signs (ie, appetite, vomiting, diarrhea, weight loss and attitude/activity), the presence of endoscopic lesions and changes in biochemical parameters, including total protein, alanine transaminase/alkaline phosphatase and phosphorous.⁴⁸ Cats with CE can present in a variety of different ways, leading to a varying degree of severity of FECAI score. Some cats may only present with weight loss, despite a good appetite, energy level and normal biochemical parameters, leading to a low FCEAI score. In contrast, cats may have severe GI signs and biochemical changes, leading to high FCEAI scores. Lack of correlation between FCEAI score in the affected cats and fecal S100A12 concentration is likely explained by the diverse clinical presentation of cats with CE.

In dogs, cutoff values for fecal S100A12 have been shown to distinguish between dogs requiring anti-inflammatory or immunosuppressive treatment and dogs with food-responsive or antibiotic-responsive enteropathies (⩾490 ng/g; sensitivity 64%, specificity 77%).³⁷ A study in dogs also showed that fecal S100A12 concentrations >2700 ng/g distinguished between dogs with CE refractory to treatment and those with partial remission.³⁷ In our cohort of cats, a cutoff value of 13.8 ng/g distinguished between cats with CE and control cats with a sensitivity of 79.6% and specificity of 68.4%. Given that there was no significant differentiation between fecal S100A12 concentrations in IBD and LSA cats based on AUROC analysis, cutoff values in the two groups were not reliable and had a low sensitivity and specificity. For example, a cutoff of 39.1 ng/g distinguishes between cats with LSA and IBD with a sensitivity of 63.3% and a specificity of 52.6%. In this cohort of cats, we were unable to establish a statistically significant cutoff between IBD and LSA cats.

The median age of cats in the CE and control groups differed (12 years vs 7 years); however, a recent study did not find an association between S100A12 concentrations and age in healthy puppies and adult dogs.⁴² In our study, there was no correlation between age and S100A12 concentration in all cats, or between age and S100A12 concentration in CE cats. The presence of controlled hyperthyroidism did not significantly affect fecal S100A12 concentration. Interestingly, the presence of CKD resulted in a significantly higher fecal S100A12 concentration compared with cats without CKD. However, all cats with CKD were in the CE group. Cats with CE without CKD still had significantly higher fecal S100A12 concentrations compared with healthy controls, and there was no significant difference in fecal S100A12 concentration between CE cats with CKD and CE cats without CKD. Further studies are needed to evaluate for an individual effect of CKD on fecal S100A12 concentration.

There were a few outliers in the CE group, with fecal S100A12 concentrations >35,000 ng/g. The largest concentration was >81,241 ng/g in a cat in the LSA group. Two cats in the IBD group had fecal S100A12 concentrations of 57,986 ng/g and 35,215 ng/g, respectively. Two of these outliers had large bowel signs as part of their presenting complaints and had evidence of colitis on histopathology. The third outlier (fecal concentration 35,215 ng/g) did not have large bowel signs and the colon was not included in endoscopic evaluation. In the absence of other explanations for these outliers, it is possible that colitis may have a more profound effect on fecal S100A12 concentrations, though further studies are needed for investigation.

This study had several limitations. Fecal S100A12 concentrations were quantified using a single fecal sample, whereas prior studies have suggested and used fecal samples collected on three consecutive days for analysis, owing to intra-individual variability.⁵³ Validation of the canine S100A12 for use in cats found a low level of short-term biologic variation in fecal samples.⁵⁵ Thus, using fecal samples from three consecutive days would be preferred but was determined not to be feasible for this study based on owner compliance to stool acquisition.⁵⁵ Collection of the stool samples in the CE group was performed by gentle lubricated digital removal under anesthesia prior to bowel preparation. As S100A12 concentrations are reflective of bowel granulocytes, the method of stool collection may have affected the results, though no studies have been performed to evaluate this in cats. Additionally, stool samples for all cats were stored in −80°C freezers and sent as a batch for analysis. CE group samples were immediately frozen, and healthy control samples were frozen within 24 h of collection. The length of storage may have affected S100A12 concentrations, thus lowering the values, but no study on the impact of feline fecal S100A12 concentrations has been done to date, although S100A12 has been demonstrated to be stable in fecal samples at various temperatures for 7 days in canine studies and up to 6 months in human studies.^29,34,36,56

Several cats with CE were receiving medications at the time of sample collection, including corticosteroids (n = 6) or antibiotics (n = 4). A previous study showed that corticosteroid treatment did not have a significant effect on canine serum S100A12 concentrations.²⁴ However, the effect of corticosteroid therapy on fecal S100A12 in cats and possible effects of antibiotics on fecal S100A12 concentrations are unknown. Ideally, all cats enrolled would have been free of any medications prior to endoscopy and stool collection; however, this cohort of cats represents a typical patient population. Additionally, the diet of enrolled cats was not standardized, and diet recommendations were clinician and owner dependent. We relied on observed health of control cats and provided owner and veterinary record history, rather than a full work-up to ensure systemic health. Lastly, the healthy control group sample size was smaller than the total affected population sample size, and controls were not breed or age matched to affected cats. This may have affected the results, though most cats in all groups were neutered/spayed, domestic shorthair, domestic mediumhair or domestic longhair, and there was no correlation between the age of all cats and S100A12 concentration.

Alimentary LSA may affect any section of the GI tract; however, the most common sites include the jejunum, followed by the ileum and duodenum.^51,58 Not every CE cat had a full endoscopic evaluation, including biopsy of the duodenum, jejunum, ileum and colon. While 13 cats with LSA had biopsies performed of the ileum, only three cats with IBD had ileal biopsies performed. While all LSA cats with ileal biopsies also had evidence of small-cell lymphoma in the duodenum, it is possible that some cats with IBD may have had undiagnosed LSA without full endoscopic evaluation. This could have contributed to the lack of statistically significant difference in fecal S100A12 concentrations between the two CE groups. Interestingly, all colonic histopathology was consistent with colitis, regardless of the presence of IBD or LSA in other biopsy sites. This aligns with a previous study demonstrating the presence of LSA in only 1/5 colonic biopsies.⁵⁸

This study provides important initial data supporting future studies evaluating fecal S100A12 as a novel non-invasive biomarker of feline inflammatory CE. Further research with the establishment of RIs, multiple daily stool samples, pre- and post-therapy measurements, and larger-scale studies comparing cats with IBD to LSA may help determine whether this biomarker might be useful for the diagnosis of disease or monitoring response to therapy.

Conclusions

Fecal S100A12 concentrations are increased in cats with CE, and should be further evaluated as a biomarker to identify cats with GI inflammation and its severity. However, in this population of cats, fecal S100A12 did not distinguish between cats with IBD or LSA.

Footnotes

Accepted: 1 March 2023

Author note

Part of this material was presented as an abstract at the 2021 ECVIM-CA congress via a virtual poster presentation.

Conflict of interest

JA Lidbury, JS Suchodolski and JM Steiner are employees of the Gastrointestinal Laboratory at Texas A&M University, which offers diagnostic testing, including fecal S100A12 measurement, on a fee-for-service basis.

Funding

The authors received no financial support for the research, authorship, and/or publication of this article.

Ethical approval

The work described in this manuscript involved the use of non-experimental (owned or unowned) animals. Established internationally recognised high standards (‘best practice’) of veterinary clinical care for the individual patient were always followed and/or this work involved the use of cadavers. Ethical approval from a committee was therefore not specifically required for publication in JFMS. Although not required, where ethical approval was still obtained, it is stated in the manuscript.

Informed consent

Informed consent (verbal or written) was obtained from the owner or legal custodian of all animal(s) described in this work (experimental or non-experimental animals, including cadavers) for all procedure(s) undertaken (prospective or retrospective studies). No animals or people are identifiable within this publication, and therefore additional informed consent for publication was not required.

ORCID iD

Kailee A Zornow

Jennifer E Slovak

Joerg M Steiner

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