The basis for histopathological diagnosis and characterization of tumours has for many years been the evaluation of sections stained by haematoxylin and eosin (HE) and examined by light microscopy. This procedure still remains the cornerstone for diagnosis and the starting stage for subsequent application of the techniques that will be described in this lecture. One major advance with routine HE microscopy has been the availability of slide digitization for computer-based analysis, which facilitates remote working by diagnostic pathologists and the ability to hold case discussions at a distance.

Immunohistochemistry

Immunohistochemistry (IHC) is defined as the use of monoclonal or polyclonal antibodies to detect and localize antigen within a tissue section. In contrast, immunocytochemistry (ICC) is the use of such reagents to detect and localize antigen within a cell monolayer. There are now numerous variations of procedures for IHC based on using different tissue samples (fresh frozen or formalin-fixed), pretreatment protocols (antigen retrieval and blocking steps) and detection reagents coupled to enzymes (for an enzyme-substrate reaction and colour change viewed by light microscopy) or fluorochromes (for emission of fluorescence when excited by light of particular wavelength under a fluorescence microscope). Details of these methodologies is beyond the scope of this presentation.

Immunohistochemistry (generally using immunoperoxidase enzyme-substrate based techniques) is now usually automated for high throughput and standardization between laboratories. Reagents are available for the detection of a wide range of structural or secreted molecules or the identification of infectious agents. The majority of reagents are broadly cross-reactive between animal species, which has allowed the application of numerous antibodies raised against human or rodent proteins to be used with tissue derived from companion animals. Testing laboratories now offer a wide menu of target antigens to the veterinary practitioner.

Immunohistochemistry for Tumour Phenotyping

The use of IHC in small companion animal tumour phenotyping began over 25 years ago with the first studies distinguishing between T- and B-cell lymphoma in the dog. Kaplan-Meier survival curves clearly showed that canine T-cell lymphoma had worse clinical prognosis compared with B-cell lymphoma. There are now numerous studies validating these concepts and further phenotyping the tumours by application of multiple markers.¹ More recent studies have extended immunophenotyping and correlation with survival to feline lymphoma.² One of the most useful applications of immunohistochemistry has been in helping to make the distinction between chronic lymphoplasmacytic enteritis and alimentary lymphoma—particularly in the cat.³ Different approaches have been taken to classification of feline alimentary lymphoma,^4,5 but consideration of low-grade and high-grade alimentary lymphoma and large granular lymphocytic lymphoma of the intestine is a useful framework. Characterization of feline alimentary diseases can be improved when IHC is used in combination with molecular analysis of lymphoid clonality (see below) and a useful diagnostic algorithm has been published.³ IHC has now been widely applied to many other tumour types and has revolutionized diagnostic histopathology.

One further example of a tumour type in which IHC has proven value is that of canine mast cell tumour. Evaluation of the Ki67 labelling index (an indication of mitotic activity) and assessment of membrane versus cytoplasmic expression of KIT are associated with survival and metastasis of these tumours. Mutations in the KIT gene are further linked to the potential of the tumours to respond to tyrosine kinase-inhibiting therapy.^6-8

Molecular Testing of Biopsy Samples

Analysis of rearrangements in genes encoding chains of T- and B-cell receptor molecules is now widely available for determining ‘clonality’ within a lymphocytic infiltration of tissue. A reactive or chronic inflammatory lesion involves a polyclonal infiltrate of lymphocytes of numerous different antigenic specificities; however, a neoplastic infiltrate is comprised of a clonal population with a restricted or monoclonal receptor type. It is recommended that clonality testing be performed only as an adjunct procedure following routine histopathological evaluation and IHC.⁹

The next advance in tumour diagnosis is larger scale molecular screening evaluating the expression of genes (i.e., mRNA production) encoding structural or metabolic molecules associated with particular types of tumour. One such technology applies the quantitative nuclease protection assay to analyse the small and fragmented mRNA that might typically be found in formalin-fixed and paraffin wax-embedded tissue samples. The same blocked tissue sample of tumour that was used for HE microscopy (and IHC) can be utilized for this technique that screens for a panel of tumour-associated genes. This method can not only phenotype the tumour (as for IHC), but can also provide prognostic and therapeutic information permitting customized therapeutic options for the patient.¹⁰

Serum Biomarkers

Although strictly not related to the subject of the present discussion (tissue-based diagnostics), there is active parallel research into serum biomarkers of animal tumours. For example, one study reports the evaluation of a diagnostic/prognostic algorithm based on clinical evaluation of canine lymphoma together with measurement of the serum concentrations of haptoglobin and C-reactive protein.¹¹

References

1.  Deravi N, Berke O, Woods JP, Bienzle D. Specific immunotypes of canine T cell lymphoma are associated with different outcomes. Vet Immunol Immunopathol. 2017;191:5–13.

2.  Wolfesberger B, Skor O, Hammer SE, et al. Does categorisation of lymphoma subtypes according to the World Health Organization classification predict clinical outcome in cats? J Feline Med Surg. 2017;19:897–906.

3.  Kiupel M, Smedley RC, Pfent C, et al. Diagnostic algorithm to differentiate lymphoma from inflammation in feline small intestinal biopsy samples. Vet Pathol. 2011;48:212–222.

4.  Moore PF, Rodriguez-Bertos A, Kass PH. Feline gastrointestinal lymphoma: mucosal architecture, immunophenotype, and molecular clonality. Vet Pathol. 2012;49:658–668.

5.  Barrs V, Beatty J. Feline alimentary lymphoma. 1. Classification, risk factors, clinical signs and noninvasive diagnostics. J Feline Med Surg. 2012;14:182–190.

6.  Scase TJ, Edwards D, Miller J, et al. Canine mast cell tumors: correlation of apoptosis and proliferation markers with prognosis. J Vet Intern Med. 2006;20:151–158.

7.  Webster JD, Yuzbasiyan-Gurkan V, Thamm DH, et al. Evaluation of prognostic markers for canine mast cell tumors treated with vinblastine and prednisone. BMC Vet Res. 2008;4:32.

8.  Horta RS, Lavalle GE, Monteiro LN, et al. Assessment of canine mast cell tumor mortality risk based on clinical, histologic, immunohistochemical, and molecular features. Vet Pathol. 2018;55:212–223.

9.  Keller SM, Vernau W, Moore PF. Clonality testing in veterinary medicine: a review with diagnostic guidelines. Vet Pathol. 2016;53:711–725.

10.  Davis B, Schwartz M, Duchemin D, et al. Validation of a multiplexed gene signature assay for diagnosis of canine cancers from formalin-fixed paraffin-embedded tissues. J Vet Intern Med. 2017;31:854–863.

11.  Alexandrakis I, Tuli R, Ratcliffe SC, et al. Utility of a multiple serum biomarker test to monitor remission status and relapse in dogs with lymphoma undergoing treatment with chemotherapy. Vet Comp Oncol. 2014;15:6–17.