Comprehensive phenotypic and clonality analysis of intraepithelial lymphocytes of intestinal T-cell lymphoma and chronic enteropathy in dogs.

Human enteropathy-associated T-cell lymphoma (EATL) is considered to be derived from intraepithelial lymphocytes (IELs); however, the origin of canine intestinal T-cell lymphoma (ITCL) remains unclear. Histological, immunohistochemical, and clonality examinations were performed using endoscopically collected canine duodenum samples of mucosal lesions of chronic enteropathy (CE; 73 cases) and ITCL without transmural neoplastic mass lesions (64 cases). Histopathological examinations revealed the intraepithelial accumulation of lymphocytes (called "intraepithelial lymphocytosis") in 54/73 CE cases (74%) and the epitheliotropism of neoplastic lymphocytes in 63/64 ITCL cases (98%). Immunohistochemically, IELs in CE with intraepithelial lymphocytosis (IEL+CE) were diffusely immunopositive for CD3, with scattered immunopositivity for CD5, CD8, CD20, and granzyme B (GRB). The percentage of CD8+ in CD3+ IELs was significantly lower in IEL+CE than in CE without intraepithelial lymphocytosis (IEL-CE). Double-labeling immunohistochemistry revealed a high percentage of GRB expression in CD8- IEL among IEL+CE. Among 64 ITCL cases, CD3 was immunopositive in 64 (100%), CD5 in 22 (34%), CD8 in 8 (13%), CD20 in 12 (19%), CD30 in 13 (20%), and GRB in 49 (77%). In CD3+ cells, Ki67 immunopositivity was highest in ITCL, intermediate in IEL+CE, and lower in IEL-CE. A clonal TCR gene rearrangement was detected in 1/19 IEL-CE cases (5%), 15/54 IEL+CE (28%), and 38/58 ITCL (66%). These results indicate that the immunophenotype of canine ITCL (CD8-GRB+) is similar to that of the increased IELs in CE. The high proliferative activity and clonality of T cells in IEL+CE suggest that canine ITCL originates from these IELs, similar to human EATL.

Intestinal T-cell lymphoma is being more frequently diagnosed in dogs owing to the wide availability of endoscopy and clonality analysis in veterinary medicine. However, no epidemiological study on intestinal T-cell lymphoma has been previously performed, and hence, information about dog breed, age and sex distributions of intestinal T-cell lymphoma has largely remained unclear. In this study, breed predisposition to canine intestinal T-cell lymphoma was determined by calculating odds ratios and 95% confidential intervals. Of the 43 breeds identified, 7 appeared to have an increased risk of developing intestinal T-cell lymphoma, including Shiba dogs, German shepherds, Cairn terriers, Boston terriers, Papillons, Pugs and Maltese. Immunohistochemistry of representative Shiba cases revealed ubiquitous cytotoxic immunophenotype in both large and small cell lymphomas. Interestingly, CD20 co-expression was observed in 11% of cases. It could potentially be aberrant expression of CD20 or neoplastic transformation of a normal subset of CD20-positive T-cells. A comparison of mean age between representative breeds revealed that Shiba dogs were slightly younger than Miniature Dachshunds (P < .05). However, there was no difference in survival between the 2 breeds. As Shiba dogs are predisposed to chronic enteropathy, there may be underlying inflammatory process contributing to lymphomagenesis of intestinal T-cell lymphoma in this breed. Our findings provide insights into the underlying pathogenesis of breed-specific canine intestinal T-cell lymphoma.

Differentiating intestinal T-cell lymphoma from chronic enteropathy (CE) in endoscopic samples is often challenging. In the present study, automated machine learning systems were developed to distinguish between the two diseases, predict clonality, and detect prognostic factors of intestinal lymphoma in cats. Four models were created for four experimental conditions: experiment 1 to distinguish between intestinal T-cell lymphoma and CE; experiment 2 to distinguish large cell lymphoma, small cell lymphoma, and CE; experiment 3 to distinguish granzyme B+ lymphoma, granzyme B- lymphoma, and CE; and experiment 4 to distinguish between T-cell receptor (TCR) clonal population and TCR polyclonal population. After each experiment, a pathologist reviewed the test images and scored for lymphocytic infiltration, epitheliotropism, and epithelial injury. The models of experiments 1-4 achieved area under the receiver operating characteristic curve scores of 0.943 (precision, 87.59%; recall, 87.59%), 0.962 (precision, 86.30%; recall, 86.30%), 0.904 (precision, 82.86%; recall, 80%), and 0.904 (precision, 81.25%; recall, 81.25%), respectively. The images predicted as intestinal T-cell lymphoma showed significant infiltration of lymphocytes and epitheliotropism than CE. These models can provide evaluation tools to assist pathologists with differentiating between intestinal T-cell lymphoma and CE.

Prognostic value of increased intraepithelial lymphocytes and lymphocytic clonality in dogs with chronic enteropathy or small-cell lymphoma

The accumulation of intraepithelial lymphocytes (IELs) is a histopathological feature of canine chronic enteropathy (CE), and IELs are considered the cells of origin of intestinal T-cell lymphoma (ITCL). However, the pathogenic mechanism of IEL activation in CE remains unclear. This study hypothesized that the expression of proinflammatory cytokines, associated with cytotoxic T/NK-cell activation, is upregulated in CE and ITCL, and examined the expression of IFN-γ, IL-2, IL-12p35, IL-12p40, IL-15, and IL-21 and the downstream signal transducers and activators of transcription (STAT) pathway in the duodenal mucosa of dogs without lesions (n = 11; NC), with IEL-CE (n = 19; CE without intraepithelial lymphocytosis), IEL+CE (n = 29; CE with intraepithelial lymphocytosis), and with ITCL (n = 60). Quantitative polymerase chain reaction (PCR) revealed that IFN-γ and IL-21 were higher in IEL+CE than in IEL-CE or NC. Western blot revealed upregulation of STAT1 and STAT3 in IEL+CE. Double-labeling immunohistochemistry revealed a positive correlation between the Ki67 index of CD3+ T-cells and IFN-γ expression levels. Immunohistochemistry revealed a higher ratio of p-STAT1-positive villi in IEL+CE and ITCL than IEL-CE and NC, which positively correlated with IFN-γ expression levels. Among the 60 ITCL cases, neoplastic lymphocytes were immunopositive for p-STAT1 in 28 cases and p-STAT3 in 29 cases. These results suggest that IFN-γ and IL-21 contribute to the pathogenesis of IEL+CE, and IFN-γ may be involved in T-cell activation and mucosal injury in CE. STAT1 and STAT3 activation in ITCL cells suggests a role for the upregulation of the STAT pathway in the pathogenesis of ITCL.

BackgroundA panel of IgA‐based serologic assays might aid in the diagnosis of chronic enteropathy (CE) in dogs, a syndrome encompassing conditions such as food‐responsive enteropathy, immunosuppressant‐responsive enteropathy, and inflammatory bowel disease (also referred to as chronic inflammatory enteropathy). However, it is unclear whether these biomarkers discriminate between CE and other types of primary intestinal disorders.ObjectivesTo evaluate a diagnostic panel that measures serum concentrations of IgA directed against OmpC (ACA), canine calprotectin (ACNA), and gliadin‐derived peptides (AGA) in dogs with well‐characterized intestinal diseases.AnimalsFifty‐five dogs with primary intestinal disease.MethodsSerum ACA, ACNA, and AGA concentrations were measured in 30 dogs with CE and 25 dogs with other intestinal diseases (non‐CE population), including histoplasmosis, parasitism, E. coli‐associated granulomatous colitis, and lymphoma. Serum IgA concentrations were compared among populations, and sensitivities and specificities were calculated using laboratory‐provided cut‐points.ResultsTwenty‐six of 30 (87%) CE dogs and 21 of 25 (84%) non‐CE dogs had abnormal concentrations (intermediate or high) of at least 2 markers; these proportions were not significantly different (P = .99). A serum ACA concentration ≥15 EU/mL was 86.7% (95% confidence interval [CI], 69.3%‐96.2%) sensitive and 24.0% (95% CI, 9.4%‐45.1%) specific for CE diagnosis. High AGA concentrations were observed in 16 of 25 (64%) non‐CE dogs.Conclusions and Clinical ImportanceThe evaluated serologic markers were poorly specific for CE diagnosis, which raises concerns that their use in clinical practice might lead to misdiagnoses and delayed or even detrimental treatments in dogs with non‐CE intestinal diseases.

Most CD56 + Intestinal Lymphomas Are CD8 +CD5 − T-Cell Lymphomas of Monomorphic Small to Medium Size Histology

The expression of cytotoxic molecules in feline intestinal T-cell lymphoma cells was examined immunohistochemically using endoscopic samples of 50 cases. Cases included 14 large-cell lymphomas (LCLs) and 36 small-cell lymphomas (SCLs). Most LCL and some SCL exhibited marked erosion and villous atrophy. Clonal T-cell receptor (TCR) gene rearrangement was detected in 10/14 (71%) LCL cases and 33/36 (92%) SCL cases. No clonal immunoglobulin heavy chain (IgH) gene rearrangement was detected. Immunohistochemically, all cases were positive for CD3 and negative for CD79α, CD30, CD56, and Foxp3. LCLs were positive for CD8 in 13/14 cases (93%), T-cell intracellular antigen 1 (TIA1) in 14/14 cases (100%), and granzyme B in 6/14 cases (43%). SCLs were positive for CD8 in 28/36 cases (78%), TIA1 in 33/36 cases (92%), and granzyme B in 2/36 cases (6%). TIA1- and granzyme B-positive neoplastic lymphocytes were predominantly observed in the mucosal epithelium of 10/50 cases (20%) and 6/50 cases (12%), respectively. No significant differences in survival time were found based on cell size or epitheliotropism. However, cases with TIA1+ and/or granzyme B+ neoplastic lymphocytes predominantly in the mucosal epithelium had significantly shorter survival times (P < .05), suggesting that mucosal epithelium infiltration of neoplastic cells with a cytotoxic immunophenotype is a negative prognostic factor. Therefore, intraepithelial cytotoxic lymphocytes may be associated with mucosal injury and impaired intestinal function, leading to a poor prognosis in cats with intestinal T-cell lymphoma.

BackgroundSerum folate is considered a biomarker of chronic enteropathy (CE) in dogs, but few studies have examined associations with markers of CE.Hypothesis/ObjectivesTo evaluate serum folate concentrations in dogs with and without CE and associations with sample hemolysis and selected markers of CE. We hypothesized that hypofolatemia would be more common in dogs with CE and associated with hypocobalaminemia, higher CIBDAI, and hypoalbuminemia.AnimalsSix hundred seventy‐three dogs with available serum folate measurements performed at an academic veterinary hospital between January 2016 and December 2019.MethodsMedical records were retrospectively reviewed to categorize cases as CE or non‐CE and record clinical details and laboratory markers. Relationships between serum folate, cobalamin, and CE variables were assessed using chi‐square, Kruskal‐Wallis, or Spearman's correlation tests.ResultsOf the 673 dogs, 99 CE were compared to 95 non‐CE. In the overall cohort, serum folate concentration did not correlate with sample hemolysis (P = .75). In the CE subset, serum folate and cobalamin concentrations were positively associated (rho = 0.34, FDR = 0.02). However, serum folate concentrations (median [25th, 75th percentiles]) were higher (CE: 12.1 (8.9, 16.1), non‐CE: 10.4 (7.2, 15.5); P = .04) and cobalamin concentrations were lower (CE: 343 (240, 597), non‐CE: 550 (329, 749); P = .001) in the CE vs non‐CE group. Serum folate was not associated with markers of CE, but serum cobalamin was associated with albumin (P = .04) and cholesterol (P = .03).Conclusions and Clinical ImportanceHypofolatemia is an inferior biomarker of CE compared to hypocobalaminemia.

BackgroundThe optimal medical treatment for chronic enteropathy (CE) in dogs and cats is controversial. Sequential treatment using diet, antimicrobials, and immunosuppressive drugs is the most common strategy used by clinicians.ObjectivesTo review the evidence for the effectiveness of dietary, drug, and alternative health interventions for inducing clinical remission in dogs and cats with CE.AnimalsRetrospective study of dogs and cats with a diagnosis of chronic enteropathy.MethodsMEDLINE and Centre for Agriculture and Bioscience International (CABI) databases (1950 to March 2017) were searched for randomized controlled trials (RCTs), observational studies, and case series. The primary outcome was induction of clinical remission. All studies were evaluated using the quality of evidence grading guidelines (I‐IV), which assign a score defining the strength and quality of the evidence.ResultsTwenty‐two studies (11 RCTs in dogs and 2 in cats and 9 cohort studies or case series) met the inclusion criteria for inducing remission of gastrointestinal (GI) signs. Of the 13 RCTs achieving grade I scores, 10 studies (totaling 218 dogs and 65 cats) compared single treatment: diet (n = 3), immunosuppressives (n = 3), antimicrobials (n = 2), anti‐inflammatory drugs (n = 1), and probiotics (n = 1). Three case series (grade III) reported clinical remission using an elimination diet fed to 55 cats and use of enrofloxacin to induce remission in dogs with granulomatous colitis (2 studies totaling 16 dogs).Conclusions and Clinical ImportanceThe current evidence for treatment of CE is much greater in dogs than in cats. There is sufficient strong evidence to recommend the use of therapeutic GI diets, glucocorticoids, enrofloxacin, or some combination of these in dogs with CE. Therapeutic GI diets and glucocorticoids are most useful in cats with CE.

Chronic enteropathy in dogs represents a significant clinical challenge that often requires innovative therapeutic strategies for effective management. This case study investigated the simultaneous use of budesonide, cannabidiol (CBD) oil and probiotics. Budesonide represents a corticoid therapy primarily used in human medicine for autoimmune diseases. Cannabidiol oil is one of the cannabinoids that does not affect sensory perception and is known for its anti-inflammatory properties. Probiotics help maintain gut microbiota balance as a treatment modality for chronic enteropathy in dogs. This report details the clinical symptoms, diagnosis, and treatment plan, highlighting the positive response seen in the patient. The findings suggested that the combination of budesonide, CBD oil, and probiotics may be a beneficial strategy for the treatment of chronic enteropathy in dogs.

CD4-positive small T-cell lymphoma of the intestine presenting with severe bile-acid malabsorption: a supportive symptom control approach.

Chronic enteropathies (CEs) in dogs describe a group of idiopathic disorders characterized by chronic persistent or recurrent gastrointestinal (GI) signs. Three major subgroups of CE can be identified by their response to treatment: Food-responsive disease (FRD), antibiotic-responsive disease (ARD), and steroid-responsive disease (SRD). The clinical diagnosis of CE is made by exclusion of all other possible causes of chronic diarrhea and includes histologic assessment of intestinal biopsies. The process of diagnosing canine CE can therefore be very time-consuming and expensive, and in most cases, does not help to identify dogs that will respond to a specific treatment. The development of novel diagnostic tests for canine CE has therefore focused on the accuracy of such tests to predict treatment responses. In this article, several novel assays that have the potential to become commercially available will be discussed, such as genetic tests, perinuclear anti-neutrophil cytoplasmic antibodies (pANCA), antibodies against transglutaminase/gliadin, antibodies against E coli OmpC/flagellin, and micro RNAs.

Introduction: Recently, patients with refractory sprue have been shown to contain a clonal proliferation of phenotypically abnormal intraepithelial lymphocytes in their intestine. Whether this signifies early enteropathy-type intestinal T-cell lymphoma (EITCL) or a reactive condition is not clear. We report on a patient presenting with the findings of refractory sprue who subsequently developed overt EITCL. Material and Methods: Duodenal biopsies from 1997 (refractory sprue) and duodenal and jejunal biopsies from 1998 (intestinal T-cell lymphoma) were compared by immunohistochemistry and PCR for the detection of T-cell receptor (TCR)-γ gene rearrangements. Clonal PCR products were sequenced. Results: The duodenal biopsies from both 1997 and 1998 and the jejunal tumor biopsy showed villus atrophy and an increase of intraepithelial lymphocytes with an abnormal immunophenotype (CD3+, CD4–, CD8– and TCR-β–). In all duodenal specimens including the one from 1997, and the jenunal tumor biopsy, an identical clonal amplificate was detected by enzymatic amplification of the TCR-γ gene. Conclusion: These data suggest that refractory sprue containing a clonal proliferation of phenotypically abnormal intraepithelial lymphocytes may represent an early manifestation of EITCL. The detection of immunohistochemical negativity for several antigens normally found on intraepithelial lymphocytes such as CD8 or the TCR-β chain in combination with clonal T-cell populations by PCR may be helpful in identifying refractory sprue with a malignant transformation.

Association of celiac disease and intestinal lymphomas and other cancers