Cancer testing: beyond the biopsy (Proceedings)


A standard and reliable approach to the diagnosis of a cancer case typically will lead the experienced veterinarian to an answer.

A standard and reliable approach to the diagnosis of a cancer case typically will lead the experienced veterinarian to an answer. Following a thorough physical examination and baseline bloodwork, fine needle aspiration of a mass will begin to distinguish a benign from malignant process. Cytology also can often categorize tumors as round/discrete cell, epithelial, or mesenchymal origin. Biopsy then usually confirms the diagnosis.

But what if this approach leads to a dead end at any one of these steps? What do you do when the dreaded "nondiagnostic sample" follows the word "results?" Sometimes the history, physical examination, cytology and biopsy all support a diagnosis, but the histologic changes are not definitive. Do you treat based on an educated guess? Sometimes. Or do you take one more step for that peace of mind in your diagnosis?

There are several advanced tests developed to confirm or diagnose cancer in animals. This talk will review several commercially available tests and introduce tests on the horizon.

Commercially available tests

ALP staining

Osteosarcoma (OSA) is a common tumor in dogs, with approximately 10,000 new cases diagnosed each year. Radiographic changes can be reliable when characteristic, sometimes more so than a biopsy. It can be difficult to confirm a diagnosis on histology because samples are small and reactive bone may predominate, or a chondroid or fibrous component make differentiating OSA from chondrosarcoma or fibrosarcoma, respectively. Alkaline phosphatase (ALP) is a hydrolytic enzyme present in multiple tissues and cell types, including osteoblasts. Neoplastic osseous tissue that is producing bone should stain positive for ALP. Cytology of the bone lesion can be strongly supportive of OSA, and if suspicious cells stain positively for ALP this can provide enough evidence of OSA to make treatment decisions. This test is available at many laboratories and is very sensitive for OSA, with a sensitivity of 100%, and specificity of 89%.


The Veterinary Bladder Tumor Antigen test is commercially available through Antech and other laboratories, or the kit can be ordered through Polymedco ( ). The basis for this test is antibody-mediated detection on a rapid latex agglutination dipstick for tumor-associated glycoproteins. These glycoproteins are of high molecular weight and consist predominantly of basement membrane proteins, released when invasive tumors degrade the basal lamina. This is as simple as a high school litmus test. Specificity and sensitivity for TCC are 41% and 88%, respectively. Though false positive tests can occur with hematuria, glucosuria, and pyuria, this test can serve as a simple interim step between inexpensive, noninvasive tests and more expensive or invasive tests because the negative predictive value is high. For older dogs with pollakiuria and with abnormal or suspicious epithelial cells and without hematuria, a positive VBTA should warrant an abdominal ultrasound and significantly increase the suspicion of a transitional cell carcinoma.


Petscreen, Inc. ( ), is a proteomics-based company out of the United Kingdom focused on novel biomarkers for a variety of disease conditions. Studies by the company found 19 serum protein peaks that are significantly different between dogs with (n=87) or without (n=92) lymphoma, 2 of which are attractive candidate markers and are currently being sequenced. This test can serve to confirm a suspected or elusive diagnosis, and may be useful in monitoring response to therapy. Studies are underway to clarify the role of this test in monitoring patients. This company also offers a chemosensitivity assay that uses a novel methodology compared to previously reported assays


Polymerase chain reaction for Antigen Receptor gene Rearrangement (PARR) for canine lymphoma has been developed at Colorado State University by Dr. Anne Avery. This test is based on the fact that the immune system produces a highly diverse set of antigen receptors in order to respond to as many antigens as possible. When clonal expansion of a lymphocyte occurs, as is the case when cancer develops from one renegade cell, the resulting population tends to produce only one type of receptor. This cell population can be amplified using PCR and the monotony of receptors is detected as a single band on a gel, whereas inflammatory conditions or normal lymphocytes produce a ladder effect. The test is available through Colorado State at . This test, as with the Petscreen test, can be used to confirm an elusive lymphoma diagnosis.

Flow cytometry

Immunophenotyping has traditionally been done on formalin-fixed biopsy samples using immunohistochemical markers. However, antibodies for this process can also be applied to cells in suspension, allowing immunophenotyping to be done on fine needle aspirate samples. This is convenient for dogs with lymphoma as it does not involve anesthesia and provides prognostic information with T-cell performing worse than B-cell for multicentric lymphoma. This service is available through many laboratories including Idexx, Veterinary Diagnostics (, UC Davis, North Carolina State University, and Colorado State University.

C-kit staining and mutation status

The role of the tyrosine kinase receptor c-kit in canine mast cell tumor has been elucidated and has lead to new advances in the treatment of this disease. It has been shown that dogs with mutated c-kit, most often in the juxtamembrane domain, have a higher response rate to novel receptor tyrosine kinase inhibitors (RTKIs) than do dogs with wild type c-kit (approximately 80% vs. 50%, respectively). Although c-kit staining is often included in "mast cell tumor panels" for proliferation markers, it is poorly predictive of outcome. Rather, it confirms the presence of c-kit as a target for therapy. Because wild type c-kit can respond to RTKIs, determination of mutation status has little clinical utility at this time. It may increase the hope that an RTKI will be effective but should not dictate whether it is tried. Staining for c-kit (immunohistochemistry) is available through several laboratories including:

1. University of Missouri ( 573-882-6811

2. Michigan State University ( )

3. The Animal Medical Center (, 212-329-8675

Determination of mutation status relies on rapid assessment of the size of the c-kit protein. If it is truncated or otherwise abnormal, it is assumed to be mutated.

Proliferative indices

Various combinations of the tests below are offered in panels at laboratories including the Animal Medical Center and Michigan State University as shown above.


Agyrophilic nucleolar organizing regions are loops of proteins associated with the nucleoli that dissociate from the nucleoli in cancer cells, spreading throughout the nucleus, and increase in number and size. AgNORs can be performed on cytologic specimens and correlate well with grade and outcome. They have been the most predictive proliferation marker in dogs with mast cell tumors.


Ki-67 is a non-histone nuclear protein expressed throughout the cell cycle, only in cycling cells. Immunohistochemistry using the MIB-1 antibody can detect Ki-67 in tissues and this has been very helpful in determining the prognosis for tissues such as mast cell tumors and soft tissue sarcomas.


Proliferating Cell Nuclear Antigen is a non-histone protein that gradually increases in G1, peaks in S and decreases in the G2 phase of the cell cycle. Its role is to hold the DNA polymerase on to the DNA strand during transcription. PCNA has been the least useful of the markers of proliferation as it has correlated poorly with outcome and correlations have been inconsistent when present.


Microvessel density, or a measure of how many blood vessels are forming in a tumor and how close together they are, has been used as a surrogate endpoint for antiangiogenesis investigations and can correlate to tumor behavior, though inconsistently.

Mitotic index

As part of routine histopathology, mitotic index (number of mitotic figures per ten high power fields at 400x) is often reported. Recent studies have shown that a mitotic index greater than 5-7 in a canine mast cell tumor is associated with significantly shorter survival times.

Tests on the horizon

Thymidine kinase

Unlike the proliferation indices discussed above, thymidine kinase is a soluble biomarker of proliferation which means it can be measured in the bloodstream. This enzyme is part of the salvage pathway for DNA synthesis and can reflect a population of cells in the body that is proliferating and preliminary studies have shown great promise in cancers such as B-cell lymphoma and hemangiosarcoma. TK will most likely be used to confirm a diagnosis, assess the aggressiveness of a cancer as well as monitor response to treatment. Studies are in progress and the test will be commercially available by the time of the meeting. For more information, check the website at or visit their booth at CVC.

Hemangiosarcoma blood test

Research in the past few years has identified markers of hemangiosarcoma in circulating blood. Hemangiosarcoma cells were found to express both markers of stem cell origin (to identify them as neoplastic and as originating as multipotential, bone marrow-derived) and markers of lineage commitment (to identify them as hemangioblasts). These markers can be exploited to develop a blood test that may be used to confirm the diagnosis, improve our ability to diagnose cases early in the course of disease, monitor the efficacy of treatment, or monitor for relapse or metastasis. This is similar to the circulating tumor cell (CTC) test for carcinomas in people, as well as other screening or monitoring tests in people that are based on cluster of differentiation markers to identify the presence of abnormal cells (such as CD125 in ovarian cancer). This test is currently in development for commercial applications.

Genetic screening for cancer risk

Since the sequencing of the canine genome in 2005, researchers have been hard at work investigating the genetic basis of cancer, among other diseases. Cancers with a heritable component such as osteosarcoma will undoubtedly be better understood with these investigations and it is plausible that tests for certain genes associated with the development of this and other cancers will be developed. To contribute samples from purebred dogs with cancer to be a part of the big picture of finding a cure for cancer, go to .

Selected references

Ewing J. A review and classification of bone sarcomas. Archives of surgery 4:485-533, 1922.

Barger A, Graca R, Bailey K, et al. Use of alkaline phosphatase staining to differentiate canine osteosarcoma from other vimentin-positive tumors. Vet Pathol. March 2005;42(2):161-5.

Henry CJ, et al. Evaluation of a bladder tumor antigen test as a screening test for transitional cell carcinoma of the lower urinary tract in dogs. Am J Vet Res. August 2003;64(8):1017-20.

Romansik E, Reilly CM, Kass PH, et al. Mitotic index is predictive for survival for canine Cutaneous mast cell tumors. Vet Pathol 44(3):335-341, 2007.

Von Euler HP, Rivera P, Aronsson AC, et al. Monitoring therapy in canine malignant lymphoma and leukemia with serum thymidine kinase 1 activity- evaluation of a new, fully automated non-radiometric assay. International Journal of Oncology 34:505-510, 2008.

Lamerato-Kozicki AR, Helm KM, Jubala CM, Cutter GC, Modiano JF. Canine hemangiosarcoma originates from hematopoietic precursors with potential for endothelial differentiation. Exp Hematol 2006 Jul;34(7):870-8.

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