Serum haptoglobin concentrations in feline inflammatory bowel disease and small-cell alimentary lymphoma: a potential biomarker for feline chronic enteropathies

Abstract

Objectives

The aim of this study was to evaluate serum haptoglobin as a biomarker to differentiate between small-cell alimentary lymphoma and inflammatory bowel disease in cats.

Methods

Client-owned domestic cats with and without chronic gastrointestinal signs were enrolled in the study. Serum was collected from each patient and serum haptoglobin levels were measured using ELISA. In cats with gastrointestinal signs, histopathologic evaluation of endoscopic biopsies harvested from the intestinal tract was used to separate them into inflammatory bowel disease and small-cell lymphoma cohorts. Serum haptoglobin levels were statistically analyzed and compared among the three groups: healthy cats; cats with inflammatory bowel disease; and cats with small-cell alimentary lymphoma.

Results

Sixty-two cats were enrolled in the study, including 20 clinically normal cats, 14 cats with small-cell alimentary lymphoma and 28 cats with inflammatory bowel disease. The mean ± SD serum haptoglobin was 73.2 ± 39.1 mg/dl in normal cats, 115.3 ± 72.8 mg/dl in cats with inflammatory bowel disease and 133.1 ± 86.1 mg/dl in cats with small-cell alimentary lymphoma. Cats with inflammatory bowel disease and lymphoma had significantly higher serum haptoglobin than controls, with P values of 0.0382 and 0.0138, respectively. There was no statistical difference between the inflammatory bowel disease and lymphoma cohorts (P = 0.4235). For every one unit increase in serum haptoglobin, the odds of gastrointestinal inflammatory disease (inflammatory bowel disease or small-cell alimentary lymphoma) increased by 1.41% (P = 0.0165).

Conclusions and relevance

Serum haptoglobin is a useful biomarker for distinguishing between normal cats and those with gastrointestinal inflammatory disease, but it could not significantly differentiate between inflammatory bowel disease and lymphoma. Additional studies may be beneficial in determining the prognostic significance of serum haptoglobin as it may relate to the severity of gastrointestinal inflammation.

Keywords

Lymphoma inflammatory bowel disease biomarker haptoglobin non-invasive enteropathy

Introduction

Chronic gastrointestinal (GI) signs are a common presenting problem of cats to a veterinary practice. Two common causes of feline chronic enteropathy (CE) include inflammatory bowel disease (IBD) and small-cell alimentary lymphoma (SCALA).¹ These two disease entities present a diagnostic challenge, with accurate diagnosis typically requiring general anesthesia, invasive biopsies and specialized testing, such as immunohistochemistry (IHC) and PCR tests.^2–8

Numerous biomarkers have been proposed and evaluated in cats with CE, in an attempt to find a non-invasive means of differentiating between IBD and SCALA. These biomarkers include albumin, cobalamin, folate, total protein, lactate dehydrogenase, thymidine kinase type 1, serum amyloid A and fecal calprotectin, among others.^2,7,9–11 None has been able to replace the need for histopathologic evaluation of biopsied tissue.

Inflammation may play a significant role in malignancy. In fact, chronic inflammation has been associated with approximately 25% of human cancers.^12,13 It has been suggested that IBD and SCALA may represent a continuum of disease, with untreated IBD undergoing malignant transformation into small-cell lymphoma.^8,14–16 In 2018, Garraway et al found an increase in mucosal-associated bacteria in cats with SCALA vs those with IBD. This suggests that the chronic inflammatory state of lymphoplasmacytic enteritis seen in most cases of IBD may progress to the low-grade malignancy of SCALA.¹⁴

Haptoglobin (HPT) is a moderate, positive acute-phase protein with immunomodulating properties found in a variety of species, including humans and cats.^17–23 Its concentration decreases slowly in serum, compared with major acute-phase proteins,^16,19 making it a more accurate predictor of chronic or ongoing inflammatory disease processes. In humans, HPT has been found to be increased in a variety of inflammatory processes, including IBD,²³ and it has also been found to be elevated in a variety of malignancies, including lymphomas and leukemias.^22,24,25 Stiller et al validated the use of the sandwich ELISA for the measurement of HPT in cats for clinical use.¹⁸ Several studies have confirmed that serum HPT (sHPT) is elevated in ‘sick’ cats with a variety of inflammatory disease processes;^{19–21,26–29} however, none has directly evaluated sHPT in cats with chronic enteropathies or neoplasia.

The objective of this study was to evaluate sHPT as a potential biomarker for the differentiation of IBD and SCALA in cats, by evaluating sHPT levels in a healthy cohort and comparing them with those with IBD and SCALA.

Materials and methods

sHPT assay

sHPT was evaluated by VDI Laboratory (Simi Valley, CA, USA) using a commercially available ELISA. The assay is a feline-specific sandwich ELISA for the quantitative determination of HPT in feline serum and has been previously validated by others for use in cats.¹⁸ The assay has low intra- and inter-assay precisions of 3.3% and 8.6%, respectively. Dilution linearity ranged from 90.4% to 102.9%, and spike recovery ranged from 98.4% to 103.8%.

Clinically healthy cats

Healthy control cats were client owned, with no prior history of GI signs. These cats had normal physical examinations and demonstrated no significant abnormalities on serum biochemistry, hematology, or feline immunodeficiency (FIV) and feline leukemia virus testing (FeLV). Approval from the Institutional Animal Care and Use Committee (IACUC) was obtained for all study procedures and owner consent was obtained prior to sample collection. Serum from 20 clinically healthy cats was collected and stored at –80°C at the Animal Medical Center in New York. Samples were shipped in batches to VDI Laboratory for analysis.

Clinical cases

IACUC approval was obtained for all study procedures and owner consent was obtained prior to sample collection. All 42 clinical cases were prospectively enrolled, client-owned cats being evaluated at The Animal Medical Center, New York. These cats were presented for chronic GI signs and had not received immunosuppressive therapy in the 4 weeks prior to sample collection. Owners independently elected to have their cats endoscopically evaluated, with biopsy samples obtained and submitted for histopathology. All biopsies were submitted through IDEXX Laboratories and all histopathologic diagnoses were made by a board-certified anatomic pathologist.

Enrolled cases met the following inclusion criterion: chronic GI signs of over 4 weeks’ duration. They could not have received immunomodulatory treatment within the 4-week period prior to receiving endoscopic evaluation with biopsies submitted for histopathology. Cases were excluded if they were currently on or recently had immunosuppressive therapy, were positive for FIV or FeLV, had a history of neoplastic disease or had another concurrent major illness that required medical management, with the exception of well-controlled hyperthyroidism and mild chronic renal disease (stage <3, according to International Renal Interest Society guidelines). Cases were not required to have had an exclusion diet trial performed to rule out food intolerance as a cause of chronic GI signs.

Serum from enrolled patients was collected and stored at –80°C. Samples were shipped in batches to the VDI Laboratory for analysis.

Cases were stratified into the IBD and SCALA groups based on histopathologic diagnosis. In cases where the diagnosis was unable to be made via histopathology alone, IHC and/or PCR for antigen receptor rearrangement (PARR) were submitted per the recommendation of the pathologist.

Statistical analysis

Baseline descriptive statistics are reported as mean ± SD for normally distributed variables and median and interquartile range for non-normally distributed variables. Between-groups analyses of baseline variables were performed using ANOVA as error residuals were normally distributed. Interaction of categorical clinical covariates was carried out by ANOVA generating the least square means for analysis. The distribution of error residuals was assessed by visual inspection followed by the Kolmogorov–Smirnoff test and deemed normal. Analyses for proportions of categorical variables were evaluated with a χ² test or Fisher’s exact analysis, as appropriate. Ordinal continuous parameters for HPT were regressed against dependent variables control vs IBD, control vs SCALA, control vs IBD + SCALA and IBD vs SCALA by way of a generalized linear logistic model to generate odds ratios, sensitivity and specificity. All analyses were performed with statistical software (SAS 2016) and P <0.05 was deemed to be statistically significant.

Results

Signalment

Clinically healthy cats

This group consisted of 20 cats, including nine castrated males and 11 spayed females. The median age of cats in this group was 106.5 months (range 48–174 months). The majority were domestic shorthair cats (n = 16; 80%). There were two domestic longhair cats (10%), one Balinese cat and one Siamese mix.

Clinical cases

Forty-two cats met the inclusion criteria for clinical cases, with 28 being diagnosed with IBD and 14 having SCALA. No clinical cases were normal on histopathologic diagnosis. In a single case, a diagnosis was unable to be made via histopathology alone. PARR was recommended by the pathologist and subsequently performed. This case was found to be polyclonal and was stratified into the IBD cohort.

IBD group

Twenty-eight cats were diagnosed with IBD via endoscopic biopsy. There was an even distribution of neutered males and females, with 14 in each group. Median age was 112.5 months (range 11–198 months). The majority were non-pedigree with 18 domestic shorthairs (64.3%), one domestic longhair (3.6%), one domestic mediumhair (3.6%) and one each of Russian Blue, Ragdoll mix, Himalayan, Maine Coon, Siamese, Turkish Angora, Devon Rex and Siberian.

SCALA group

Fourteen cats were diagnosed with SCALA via endoscopic biopsy. All were neutered: five spayed females and nine castrated males. Median age was 153 months (range 108–192 months). All but one (n = 13) were domestic shorthair cats (92.9%), with the remaining cat being a Russian Blue.

sHPT activity

The mean sHPT level for the healthy control cats was 73.4 ± 39.1 mg/dl (median 65.4 mg/dl). The IBD cohort was found to have a mean sHPT of 115.3 ± 72.8 mg/dl (median 99.0 mg/dl). Lastly, the SCALA group had a mean sHPT of 133.1 ± 86.1 mg/dl (median 107.9 mg/dl) (Table 1).

Table 1

Serum haptoglobin activity in clinically healthy cats, cats with inflammatory bowel disease (IBD) and cats with small cell alimentary lymphoma (SCALA)

Group	Median (mg/dl)	Mean (mg/dl)	SD (mg/dl)	Range (mg/dl)
Clinically healthy (n = 20)	65.4	73.4	39.1	8.9–143.6
IBD (n = 28)	99.0	115.3	72.8	17.5–318.1
SCALA (n = 14)	107.9	133.1	86.1	16.6–357.7

Mean sHPT concentrations were found to be statistically different between the control group and IBD group (P = 0.0382). Mean sHPT concentrations were also statistically different between the control group and the SCALA group (P = 0.0138). There was no statistical difference between the IBD and SCALA groups (P = 0.4235).

Logistic regression was performed and demonstrated that for every one unit increase in sHPT, the odds of a cat having IBD or SCALA increased by 1.4% (P = 0.0165). When evaluated separately, the odds of being positive for IBD increased 1.3% (P = 0.0328) with every unit increase in sHPT and, similarly, the odds of being positive for SCALA increased by 1.9% with every unit increase in sHPT (P = 0.0279).

A receiver–operating characteristic curve was also performed for sHPT, distinguishing ‘diseased’ (ie, IBD or SCALA) vs ‘healthy’ cats (Figure 1). The optimal cut-off point for separating normal cats from those with either IBD or SCALA was found to be 145 mg/dl, with a sensitivity and specificity of 0.357 and 1.0, respectively.

Figure 1

Receiver–operating characteristic curve for serum haptoglobin, distinguishing ‘diseased’ (ie, inflammatory bowel disease [IBD] and small-cell alimentary lymphoma [SCALA]) vs ‘healthy’ cats with an optimal cut-off point for separating normal cats from those with either IBD or SCALA of 145 mg/dl

Discussion

Biomarkers represent a non-invasive means of differentiating between disease processes. They are considered standard practice for many diseases in veterinary medicine, including renal disease, pancreatitis and cardiac disease. The use of tumor biomarkers has become standard practice in human medicine and is increasingly common in veterinary medicine, as a means of diagnosis and providing prognostic and treatment-related information.

Feline CE is extremely common in clinical practice. Currently, the only reliable way to differentiate between IBD and SCALA – two of the most common causes of CE – is histopathology, which remains the diagnostic recommendation as per the American College of Veterinary Internal Medicine (ACVIM).³⁰ Advanced, specialized testing with PARR and/or IHC on obtained samples is often still necessary for definitive diagnosis in many cases.^2,3,5–8 Some argue that advanced testing should be standard practice for obtaining a reliable diagnosis. These testing modalities are both invasive to the patient and costly to the owner. This is the first study, to our knowledge, evaluating whether sHPT can be used as a biomarker for differentiation between IBD and SCALA in cats.

The present study demonstrates that sHPT was significantly higher in cats with CEs than in normal cats. This was expected, given that both IBD and SCALA are associated with chronic GI inflammation and GI dysbiosis.^{1,2,7,14–16,30}

Given that many cases of SCALA are thought to arise from a progression of untreated inflammation in the GI tract of cats with IBD, leading to progressive bacterial dysbiosis and neoplastic transformation into SCALA,^7,14,15 it was hypothesized that SCALA cats would have significantly higher sHPT levels compared with the IBD cohort. In this study, however, sHPT was unable to differentiate between these two groups. The mean sHPT was higher in the SCALA group, but this difference was statistically insignificant. The lack of significance may represent a type II error, given the low sample sizes, most notably in the SCALA group (n = 14). We recommend future studies, with increased sample sizes, to evaluate this further.

The current literature demonstrates great variability in HPT levels in normal feline populations.^19,28,29 Similarly, in our study sHPT levels were found to be variable within each cohort, including the control group. The SD increased from the normal to IBD to SCALA groups, likely representing varying degrees of inflammation within each patient. As the disease progresses, the potential for a marked inflammatory response increases, making variability between cats more likely, as exemplified by our data. Correlating sHPT with the degree of histopathologic inflammation and severity of clinical signs of each patient was beyond the scope of this study. It seems likely that those with increased clinical signs and increased inflammatory infiltrate on biopsy would have higher HPT levels and that variability in sHPT within each cohort represents varying degrees of disease severity. Should this be found to be true, sHPT may be a valuable prognostic tool and may also be used as a means of evaluating response to therapy.

There were several limitations to the present study. Low sample sizes, most notably in the SCALA group, likely contributed to a type II error and a lack of statistically significant difference in sHPT between the SCALA and IBD groups. The use of the ELISA method described has only been validated in cats by a single study.¹⁸ This could be viewed as a limitation of its potential accuracy. Also, samples obtained for this study were performed via endoscopy and were not full-thickness surgical biopsies. The adequacy of endoscopic biopsy specimen for the differentiation between IBD and SCALA can be controversial;^4,5,8 however, given the ACVIM 2010 consensus statement recommendation of endoscopic biopsy as ‘the preferred choice’, it was deemed appropriate and acceptable in this study.³¹ Advanced diagnostic testing with IHC and PARR was not uniformly performed on all samples. Sabattini et al⁸ demonstrated that IHC and clonality testing increase the likelihood of accurate differentiation between these two disease processes;⁸ therefore, it is possible that some of the included cases may have been misdiagnosed. Currently, as per the 2010 ACVIM consensus statement, advanced testing should be considered in situations where the pathologist is unable to make a definitive diagnosis with histopathology alone.³¹ Given the expense of these tests, in the current study, additional testing was only added at the recommendation of the pathologist. This occurred in a single case in which PARR was performed and the sample found to be polyclonal, thus stratifying it into the IBD cohort. Interestingly, the sHPT in this case was 97.2 mg/dl, which is very close to the median of the group (99.0 mg/dl), suggesting that the correct classification was made. The use of PARR in cats could also be seen as a limitation owing to its decreased sensitivity in cats vs dogs. While this is true, that is, PARR is less sensitive and specific than canine PARR, detection of the T cell receptor gene (TRG) by PCR has been found to be >70% sensitive and >90% specific in most studies.^32–34 While the sensitivity and specificity of PARR is lower in feline B-cell lymphoproliferative diseases,³⁴ SCALA is a T-cell lymphoma and thus PARR should be considered a reliable test, especially when found to be clonal. Lastly, our inclusion of cats into the control cohort based on a reported lack of clinical signs, patient history and bloodwork alone may be seen as a limitation. Abdominal ultrasound and GI biopsies were not performed in these patients, so it is possible that some were misclassified as ‘normal’ when they may have had subclinical GI disease. This could account for some of the sHPT outliers in the control population.

Future studies should include larger cohorts of patients and evaluate sHPT as a marker of disease severity and response to therapy. Inclusion of a large-cell GI lymphoma cohort could also provide valuable information on increased uses for sHPT in GI disease in cats.

Conclusions

This is the first study to directly compare serum haptoglobin levels in normal cats, cats with IBD and cats with SCALA. It confirmed that sHPT is a valuable biomarker of inflammation in cats, being able to differentiate between normal cats and those with CEs. It was unable to show a statistically significant difference in sHPT between the IBD and SCALA cohorts. Additional studies evaluating its ability to accurately quantify the degree of GI inflammation for use as both a prognostic tool and as a means of evaluating response to therapy in cats with CEs are warranted.

Footnotes

Acknowledgements

We would like to thank all enrolling clinicians, technicians, assistants and participating owners at The Animal Medical Center in New York. We would also like to thank all employees at VDI Laboratory for handling and running our samples with care.

Accepted: 30 December 2020

Conflict of interest

Randy Ringold is an employee at VDI Laboratory. He had no knowledge of the specific cases involved during enrollment, sample collection and data analysis. No conflict of interest exists for the remaining authors.

Funding

Financial support for hematology, serum biochemistry and histopathology submission for cases was provided by Caspary Research Institute of the Animal Medical Center. Serum samples were run free of charge through VDI Laboratory for serum haptoglobin levels.

Ethical approval

This work involved the use of non-experimental animals (owned or unowned) and procedures that differed from established internationally recognized high standards (‘best practice’) of veterinary clinical care for the individual patient. The study therefore had ethical approval from an established committee as stated in the manuscript.

Informed consent

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

ORCID iD

Edwina K Love

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