key: cord-0015119-1gkonujz
authors: Chow, Betty; Hill, Steve L.; Richter, Keith P.; Marsilio, Sina; Ackermann, Mark R.; Lidbury, Jonathan A.; Suchodolski, Jan S.; Cocker, Sarah; Steiner, Jörg M.
title: Comprehensive comparison of upper and lower endoscopic small intestinal biopsy in cats with chronic enteropathy
date: 2020-12-21
journal: J Vet Intern Med
DOI: 10.1111/jvim.16000
sha: 3d7cb3eddb9d413e05c709597622d9039f84c48b
doc_id: 15119
cord_uid: 1gkonujz

BACKGROUND: Integrating immunohistochemistry (IHC) and clonality testing with histopathology may improve the ability to differentiate inflammatory bowel disease (IBD) and alimentary small cell lymphoma (LSA) in cats. HYPOTHESIS/OBJECTIVES: To evaluate the utility of histopathology, IHC, and clonality testing to differentiate between IBD and LSA and agreement of diagnostic results for endoscopic biopsy (EB) samples from the upper (USI) and lower small intestine (LSI). ANIMALS: Fifty‐seven cats with IBD or LSA. METHODS: All cases were categorized as definitive IBD (DefIBD), possible LSA (PossLSA), probable LSA (ProbLSA), or definitive LSA (DefLSA) based on histopathology alone. Results from IHC and clonality testing were integrated. RESULTS: Based on histopathology alone, 24/57 (42.1%), 15/57 (26.3%), and 18/57 (31.6%) cats were diagnosed with DefIBD, PossLSA or ProbLSA, and DefLSA, respectively. After integrating IHC and clonality testing, 11/24 cases (45.8%) and 15/15 cases (100%) previously categorized as DefIBD and PossLSA or ProbLSA, respectively, were reclassified as LSA. A final diagnosis of IBD and LSA was reported in 13/57 (22.8%) and 44/57 (77.2%) cats, respectively. Agreement between USI and LSI samples was moderate based on histopathology alone (κ = 0.66) and after integrating IHC and clonality testing (κ = 0.70). However, only 1/44 (2.3%) of the LSA cases was diagnosed based on LSI biopsy alone. CONCLUSIONS AND CLINICAL IMPORTANCE: Integrating IHC and clonality testing increased the number of cases diagnosed with LSA, but the consequence for patient outcome is unclear. There was moderate agreement between USI and LSI samples. Samples from the LSI rarely changed the diagnosis.

IHC and clonality testing (κ = 0.70). However, only 1/44 (2.3%) of the LSA cases was diagnosed based on LSI biopsy alone.

Conclusions and Clinical Importance: Integrating IHC and clonality testing increased the number of cases diagnosed with LSA, but the consequence for patient outcome is unclear. There was moderate agreement between USI and LSI samples. Samples from the LSI rarely changed the diagnosis. decreased appetite, vomiting, diarrhea, or some combination of these. 1 Differentiating IBD and LSA can be challenging. Immunohistochemistry (IHC) and clonality testing by polymerase chain reaction for antigen receptor rearrangements (PARR) in conjunction with histopathological assessment may improve the diagnostic accuracy of endoscopic biopsy (EB) samples when differentiating IBD from LSA. [2] [3] [4] [5] [6] [7] Based on several studies, 15.6% to 26% of cases initially diagnosed as IBD based on histopathology subsequently were reclassified as LSA, or vice versa, after considering IHC. 3, 4, 8, 9 In addition, 4% to 53% cases initially diagnosed as IBD based on histopathology subsequently were reclassified as LSA, or vice versa, after considering clonality testing with or without IHC. 4, 9 However, a recent study in clinically healthy cats suggested that, similar to results in humans, PARR may lack specificity. Thus, reclassification of cases based on PARR may not be appropriate. 10 A previous study found poor agreement between duodenal and ileal EBs based largely on histopathology, with 44% of LSA cases being diagnosed only by evaluation of ileal but not duodenal biopsies. Results suggested that for accurate diagnosis, duodenal and ileal biopsy samples should be collected from cats with clinical signs of chronic GI disease. However, IHC and clonality testing were not performed in all cases. 11 Performing both lower (LGE) and upper gastrointestinal endoscopy (UGE) increases anesthesia time, patient risk, and procedure costs.

Our aim was to reevaluate the diagnostic utility of procuring both upper small intestine (USI) and lower small intestine (LSI) biopsy samples when results of histopathology are integrated with IHC and clonality testing for the diagnosis of IBD and LSA in cats.

Our study consisted of prospective and retrospective arms and was conducted at the Veterinary Specialty Hospital (VSH) in San Diego, California. The study protocol was approved by the VSH Research Advisory Committee, and written owner informed consent was obtained for each cat before enrollment into the prospective arm. 

Endoscopy was performed using a 103 cm endoscope with a 5.9 mm outside diameter and a 2.0 mm instrument channel (GIF-XP160, Olympus, Center Valley, Pennsylvania). Biopsy samples from the LSI were obtained either by cannulation of the ileocolic valve (direct visualization), or by passing a reusable or disposable 1.8 mm ellipsoid cup biopsy forceps, with or without spike, blindly through the ileocolic valve into the LSI for sample collection (blind technique). Histopathologic examinations of hematoxylin and eosin (H&E)-stained formalinfixed and paraffin-embedded (FFPE) tissue sections were reviewed by a single board-certified pathologist with a special interest in GI pathology (M.R. Ackermann) in all cases. The pathologist had access to information regarding history, clinical signs, and endoscopic findings.

Biopsy samples were evaluated for quantity and quality. The findings were reported descriptively and numerically scored according to the World Small Animal Veterinary Association (WSAVA) histopathologic scoring system. 5, 12 Briefly, morphological features (eg, surface epithelial injury, crypt hyperplasia, crypt dilatation or distortion, and fibrosis or atrophy) and inflammatory changes (eg, lamina propria lymphocytes, plasma cells, eosinophils, neutrophils, and macrophages) were assessed histologically and assigned a score (normal = 0, mild = 1, moderate = 2, or marked = 3).

Cases were categorized based on the histopathologic diagnosis as "definitive IBD" (DefIBD, the pathologist made a definitive diagnosis of IBD); "possible lymphoma" (PossLSA, the pathologist reported findings where LSA could not be ruled out but was deemed unlikely); "probable lymphoma" (ProbLSA, the pathologist was concerned about the presence of LSA and recommended IHC, clonality testing, or both for further evaluation); or "definitive LSA" (DefLSA, the pathologist made a definitive diagnosis of LSA). In cats with discordant results between USI and LSI, the diagnosis of LSA was determined when LSA was reported in either site. Association between morphological features and clinical features and diagnosis were evaluated.

Sections of FFPE tissue were sent to a single external laboratory for IHC and clonality testing (Leukocyte Antigen Biology Laboratory, University of California Davis, on a fee-for-service basis). The pathologist at the external laboratory was blinded to the H&E diagnosis. Sections were assessed by H&E staining, IHC, and clonality testing using a stepwise approach. Staining for T-, B-, and natural killer cell markers (ie, CD3, CD79a, granzyme B [GrB], respectively) were performed at the pathologist's discretion and based on histopathology results (ie, size and distribution of mucosal lymphocytes). Clonality testing was conducted on at least 2 FFPE tissue sections (each 25 μm in thickness) using PARR analysis. Total DNA content was measured before the procedure to ensure that sufficient tissue was present for accurate PARR testing. Results from the H&E-based histopathology, IHC, and clonality analysis were integrated and reported by the external pathologist.

All datasets were tested for normality using the Anderson-Darling test. Normally distributed numerical data were analyzed using the Student's t test and presented as means and standard deviations.

Non-normally distributed continuous data were analyzed using the Wilcoxon signed rank test and presented as medians and range.

Categorical data were analyzed using the Fisher's exact test or Wilcoxon rank-sum test. If a discrepancy was found between the USI and LSI diagnosis in a cat, the more malignant diagnosis category was used for statistical analysis. Agreement between diagnoses by intestinal location and by type of diagnostic testing (H&E staining alone, H&E with IHC and clonality testing) was assessed by calculating Cohen's kappa coefficient (κ). Kappa values 0-0.40 were characterized as poor, 0.41-0.75 as fair to good, and >0.75 as excellent agreement. 13 F I G U R E 1 Summary of case numbers and diagnoses. DefIBD, "definitive IBD"; DefLSA, "definitive LSA"; H&E, hematoxylin and eosin; IBD, inflammatory bowel disease; IHC, immunohistochemistry; LSA, lymphoma; PossLSA, "possible LSA"; ProbLSA, "probable LSA" Commercial software (Stata version 14.2) was used for statistical analysis. Statistical significance was set at P < .05.

Eighty cats that had undergone both UGE and LGE, with EBs obtained from the USI and LSI, between 2012 and 2017 were reviewed. Seventeen retrospective cases were excluded because of inadequate sample for IHC and clonality analysis (n = 4), poor sample quality (n = 6), insufficient DNA amplification from samples (n = 3), abdominal ultrasound examination was not performed (n = 1), diagnosis of gastric LSA with a normal small intestine on H&E (n = 1), diagnosis of feline GI eosinophilic sclerosing fibroplasia (n = 1), or diagnosis of histoplasmosis (n = 1). Six prospective cases were excluded because of inadequate sample for analysis (n = 2), poor sample quality (n = 3), or a diagnosis of gastric LSA with a normal small intestine on H&E (n = 1). Fiftyseven cases (17 retrospective and 40 prospective) were available for analyses ( Figure 1 ).

Demographic characteristics and clinical signs are shown in Table 1 .

Mean age was significantly higher for LSA (mean, 11.7 years; SD, 2.4) than IBD (mean, 7.6 years; SD, 3.6; P < .001) cats. No significant associations were found between the diagnosis based on H&E or after integrating IHC and clonality testing and clinical signs or patient weight, nor between WSAVA score and clinical signs or patient weight (P > .05).

Biopsy sample numbers were reported as adequate for all cases, with exact numbers recorded in 37/57 cases, and a median of 11 USI biopsy samples (range, 6-20) and 5 LSI biopsy samples (range, [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] 

Based on H&E assessment alone ( Figure 1 ; 

As previously stated, IHC was performed using a stepwise approach, starting with the T-cell marker CD3+, but additional stains were pursued if deemed necessary. All sections were positive for CD3+. Based on PARR testing ( Figure 1 ; Table 4 ). 

The most common features were increased IEL, lamina propria lymphocytes or plasma cells, crypt dilatation or distortion, and mucosal or A stepwise approach of histopathology, followed by immunophenotyping, and finally testing to determine the clonality of infiltrating T-and B-cells generally is recommended. 4, 17, 20 Immunohistochemistry and clonality results were considered separately rather than in conjunction in another study. 9 T-cell receptor rearrangement pattern differed between USI and LSI sections in 11 LSA cases. Identical neoplastic T-cell clones in different GI segments is more common, although the occurrence of 2 unrelated clones in topologically distinct sites in a single cat has been reported. 15, 19 Epitheliotropism, present in 38% of feline alimentary LSA cases, is an important diagnostic feature, and the presence of clusters of IELs in villous or crypt epithelium is readily recognizable. 15 Higher interobserver variability however occurs in diffuse villous epitheliotropism, which may be under-recognized in H&E-stained sections although it appears more abundant on IHC. 15 The high sensitivity may explain the large proportion of cases (69%) that were reclassified as LSA in our study and a previous study (53%). 4 Some reclassified LSA cases may have had neoplastic infiltrates elsewhere in the GI tract, and a phenomenon known as lymphocyte trafficking may have led to clonal TCRG rearrangements despite a lack of apparent histopathological changes. 24 Indolent lymphocyte clones were a possibility in our study cats, although this occurrence would be difficult to prove without long-term follow-up by serial biopsies.

The specificity of standardized T-cell clonality assays in a study of lymphoproliferative disease in humans was shown to be as low as 54%. 25 In a recent study, many middle-aged to older, clinically healthy cats were diagnosed with intestinal LSA based on histopathology and IHC and clonality testing. 10 A study of CE in cats found clonality assays to have a poor specificity of 33%. 26 Poor specificity of clonality testing also may explain our high reclassification rate. False-positive clonality results can occur because of benign clones, canonical rearrangements, or pseudoclonality. 17 Therefore, results of clonality assays should be interpreted along with clinical, histopathology, and IHC data, 4 and performed in the same laboratory, preferably by the same pathologist. 17, 20 Unfortunately, not all veterinary laboratories follow this recommendation. 17, 18 Meanwhile, false-negative results also can be a problem, mainly because of insufficient primer coverage. 17, 20 Identification of all TCRG gene segments only will be possible when the feline genome is assembled. 15 Further evaluation of the specificity of clonality assays in veterinary medicine is warranted. gists would allow for assessment of the degree of interobserver variability and the impact on the proportion of cases that become reclassified after applying IHC and clonality testing. Finally, exact biopsy sample numbers were available for only 65% of cases, which may or may or alter the association between sample number and quality.

Our results show that the addition of IHC and clonality testing to H&E assessment increases the number of cases diagnosed with LSA.

Although the sensitivity of clonality testing is considered to be high, further assessment of its specificity is warranted. Further research to determine if a change in diagnosis correlates with patient outcome is in progress. Additionally, moderate agreement was found for the diagnosis of IBD and LSA between samples collected from the USI or LSI upon histopathology alone or when integrating IHC and clonality testing. Importantly, in our study, samples from the LSI rarely led to a diagnosis of LSA that would otherwise have been diagnosed as IBD on USI samples alone. 

At the time of the study, authors Marsilio, Lidbury, Suchodolski, and

Steiner are or were employed by the Gastrointestinal Laboratory at Texas A&M University, which offers laboratory tests, including histopathology services, on a fee-for-service basis. The author Ackermann is affiliated with Gastrointestinal Laboratory at Texas A&M University.

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The authors Chow, Hill, Richter, and Cocker have nothing to disclose.

Authors declare no off-label use of antimicrobials.

The study protocol was approved by the Veterinary Specialty Hospital Research Advisory Committee, and written owner informed consent was obtained for each cat before enrollment into the prospective arm.