Introduction

A tumor biomarker is a biochemical entity used to measure the progress of a neoplastic disease or the effects of treatment on clinical outcome. Detecting this biomarker, most often a protein, in the patient's blood can be very useful in the clinic. Blood tumor biomarkers are used widely in human oncology. A similar interest in biomarkers is now emerging in veterinary medicine, where there is enormous potential for their development and application. However, their use is still hampered by lack of good validation, as they have been reported mostly in small retrospective studies and only a very few long-term follow-up studies in larger patient groups.

Tumor biomarkers can be diagnostic, predictive or prognostic. It is important to understand how to properly use the individual biomarker and the expected specificity/sensitivity. A diagnostic marker would be very useful in cancer-screening programs. Predictive markers are used to inform the treating veterinarian/client if a proposed treatment will be beneficial or not for the specific patient. Prognostic markers are helpful in deciding if at all, and when, a tumor treatment should be started. Today, there are very few useful tumor markers that meet these criteria in veterinary practice. As in human oncology, development of and algorithms for marker panels will likely be introduced in the near future as the diagnostic accuracy then often increases.

Refined diagnostics are, however, still needed in veterinary oncology. But a balance is also needed, where painful, time-consuming, and costly examinations are generally avoided out of concern for the animal's wellbeing and the cost of veterinary medicine for the pet owner. If better tumor markers can aid in selecting cases with unspecific symptoms that may be tumor-related, the ability to perform more advanced diagnostics in a lower number of cases, with a greater chance of making a cancer diagnosis, would be of great ethical and economic value. If tumor markers could help in prognosticating a treatment outcome at an early stage of treatment planning, the probable result would again be positive effects on quality of life and economy. Finally, if a tumor marker proves to have the capacity to detect microscopic disease before relapse is clinically evident, rescue therapy might be commenced earlier and, at least in theory, have a better chance of success, as a smaller tumor burden with a more rapid growth rate is likely to be more sensitive to, e.g., chemotherapy.

There are, however, controversies, potential biases, and considerations relative to the clinical application of blood biomarker assays for cancer screening and management and it is important to address these every time a new tumor marker is to be used in clinical practice. Below, a handful of tumor markers currently available will be discussed.

Biomarkers Used in Pathology

Mitotic Index

Mitotic index is a universal tool used in predicting treatment outcome and prognosis in most tumors. A high mitotic index mostly confirms a high-grade tumor. This information can also be predictive of how the individual patient responds to chemotherapy and radiotherapy as these treatment modalities are often used to target cell division. Examples of available biomarkers measuring cell proliferation in pathology are argyrophilic technique staining the nucleolus organizer region (AgNOR), proliferating cell nuclear antigen (PCNA), and Ki-67.

c-kit

Tyrosine kinases (TKs) have many functions in the body. Many TKs are associated with cancer formation. Hence, a large class of drugs has been developed to inhibit these functions. Examples of TK pathways are the TK enzyme Bcr-Abl found in chronic myelogenous leukemia, vascular endothelial growth factor (VEGF)-stimulating angiogenesis, which is very important for tumor growth, and, finally, c-kit which plays a role in cell survival, proliferation, and differentiation. In one third of canine mastocytomas, c-kit is mutated and endogenously activated, leading to uncontrolled proliferation. Recently, two drugs, masitinib/Masivet®/Kinavet®¹ and toceranib/Palladia®,² were registered that selectively block the mutated c-kit receptor function and thereby reduce the activity of the tumor. c-kit mutation can be analyzed on tissue samples of the tumor and predict activity of the described drugs, as the efficacy is 50% better in mutated forms of c-kit than in the wild type form. c-kit-mutated mastocytomas have been reported to carry a worse prognosis overall.

p-53

The tumor suppressor gene p-53 is involved in deoxyribonucleic acid (DNA) repair. If mutated, the function is impaired and damaged DNA risks being allowed to continue in the cell cycle. The risk for tumor development then increases. p-53 mutations have been reported in many canine, feline, and also human tumors. Overexpression of mutated p-53 can be analyzed using immunohistochemistry.

Serum Tumor Biomarkers

Hypercalcemia

Hypercalcemia is probably the best known surrogate marker for neoplasia in dogs and cats. Usually, it is caused by production of parathyroid hormone-related peptides that increase serum calcium. However, hypercalcemia can also be the result of any imbalance of the calcium homeostasis (involving, e.g., intestinal absorption, renal function, parathyroid and thyroid gland function, or bone as storage of calcium). Known tumors where hypercalcemia is reported are lymphoma in dogs and cats, apocrine gland anal sac adenocarcinoma in dogs, and thymoma and squamous cell carcinoma in cats. For analysis, measurement of ionized calcium is recommended as there are reports of cases where total calcium was normal, but ionized calcium was elevated.

Alkaline Phosphatase

Elevation of alkaline phosphatase (ALP) is best known for being a negative prognostic factor in osteosarcoma in canine patients. This has been reported, both for total ALP and for bone-specific isoenzyme of ALP.

Acute Phase Proteins

Many acute phase proteins are reported to be changed in neoplastic conditions. The best known of these are C-reactive protein (CRP) and haptoglobin. As CRP is elevated in many different conditions (e.g., inflammation, postsurgical trauma, liver disease, diabetes mellitus), its usefulness as a tumor biomarker is debated. As inflammation is a hallmark of cancer, however, it undoubtedly has a role in cancer diagnostics. In patients with a specific cancer diagnosis, measurement of CRP can be prognostic. Moreover, CRP is also included in a combination panel with thymidine kinase 1 (TK1) to create an algorithm called the Neoplasia Index.

Thymidine Kinase 1

The S phase-specific protein thymidine kinase 1 (TK1), which is closely correlated to cell proliferation, can be used in immunohistochemistry to detect ribonucleic acid (RNA)/protein expression in tissue specimens and also to detect activity or protein/peptide levels in serum from patients. Thymidine kinase 1 is part of the first step in the salvage pathway for deoxythymidine triphosphate (dTTP), serving as the DNA precursor necessary for replication and repair. To date, TK1 has been used mainly as a serum marker in hematologic malignancies in humans, but also been found beneficial in canine malignancies, such as lymphoma and hemangiosarcoma.^3,4 Since TK1 expression reflects cell proliferation, TK1 is used in a variety of ways in clinical oncology. Proven applications are early diagnosis of malignancy, prognosis of certain tumors, detection of response to therapy, and early detection of relapse ahead of clinical signs.⁵

Canine Lymphoma Blood Test

The canine lymphoma blood test (cLBT) is commercially available. Studies demonstrate that this test is able to reliably detect recurrence of lymphoma up to 2 months prior to signs such as lymphadenopathy. It is a proteomic-based assay developed from a study where initially 19 candidate proteins were found to be overexpressed in canine lymphomas.⁶ Later, the algorithm was broken down to only two proteins, combining clinical data about the case with these two (acute phase) proteins to generate a LBT score that can discriminate benign lymphadenopathy from malignant lymphoma. It has yet to be proven whether instituting rescue chemotherapy before overt clinical signs appear is beneficial for overall survival in dogs and cats.

References

1.  Hahn KA, Ogilvie G, Rusk T, Devauchelle P, Leblanc A, Legendre A, et al. Masitinib is safe and effective for the treatment of canine mast cell tumors. J Vet Intern Med. 2008;22(6):1301–1309.

2.  London CA, Malpas PB, Wood-Follis SL, Boucher JF, Rusk AW, Rosenberg MP, et al. Multi-center, placebo-controlled, double-blind, randomized study of oral toceranib phosphate (SU11654), a receptor tyrosine kinase inhibitor, for the treatment of dogs with recurrent (either local or distant) mast cell tumor following surgical excision. Clin Cancer Res. 2009;15(11):3856–3865.

3.  von Euler H, Einarsson R, Olsson U, Lagerstedt AS, Eriksson S. Serum thymidine kinase activity in dogs with malignant lymphoma: a potent marker for prognosis and monitoring the disease. J Vet Intern Med. 2004;18(5):696–702.

4.  Thamm DH, Kamstock DA, Sharp CR, Johnson SI, Mazzaferro E, Herold LV, et al. Elevated serum thymidine kinase activity in canine splenic hemangiosarcoma*. Vet Comp Oncol. 2012;10(4):292–302.

5.  von Euler H, Eriksson S. Comparative aspects of the proliferation marker thymidine kinase 1 in human and canine tumour diseases. Vet Comp Oncol. 2011;9(1):1–15.

6.  Ratcliffe L, Mian S, Slater K, King H, Napolitano M, Aucoin D, et al. Proteomic identification and profiling of canine lymphoma patients. Vet Comp Oncol. 2009;7(2):92–105.