Phenotypic properties of HSA cells can be used to distinguish them from normal (or benign) endothelial cells in tissue biopsies.
As expected from their malignant nature, HSA cells are incompletely differentiated hemangioblastic cells that express markers
associated with immature hematopoietic precursors, such as CD117 (c-Kit). Hence, a unique set of markers to distinguish HSA
cells from other committed hematopoietic cell lineages and from differentiated bone-marrow derived cells can be developed.
Specifically, these cells should express proteins that belie their hematopoietic origin with incomplete differentiation (e.g.,
CD117, CD34, CD133, CD45) along with proteins that document their commitment to the endothelial lineage (e.g., CD51/CD61 or
v beta 3 integrin, CD105, CD146). Similarly, these cells would lack expression of proteins normally found in hematopoietic
cells that are committed to leukocyte lineages, such as CD18, CD11b, CD3 and CD21. Moreover, the intimal contact of HSA cells
with the circulation provides malignant cells easy access to the circulation. It is now widely accepted that most tumors shed
cells into the microenvironment, and the process of metastasis may be more closely associated with an anti-apoptotic phenotype
than with an invasive phenotype. Thus, the frequency of primitive endothelial cells (or EPC-like cells) should be increased
naturally in dogs with HSA.
The number of EPC in the circulation has been reported to increase in a variety of conditions in humans and mice. These conditions
include stem-cell mobilization using cytokines, physical training and vascular injury (including myocardial infarcts and early
congestive heart failure). Other conditions, such as exposure to nicotine, are reported to both increase and decrease circulating
EPC suggesting that even under some pathological conditions the frequency of these cells in the circulation is fickle and
generally remains well below 1 percent. HSA lesions should shed EPC-like cells into the circulation continuously, meaning
that a diagnostic procedure to detect EPC-like cells in excess of a normal reference range would be a sensitive means to diagnose
canine HSA, even in early stages. In addition, these EPC-like cells would possess clonal abnormalities that would allow both
their characterization as malignant cells and their exploitation to develop new strategies for targeted therapies.
The development of such a method to provide early detection of canine HSA using multi-parameter flow cytometry has currently
been pursued. Flow cytometry has proven to be a robust technology to detect circulating cells present at extremely low frequencies.
In addition, it also provides the opportunity to isolate and purify HSA cells (by sorting) for confirmatory tests and therapeutic
development. Preliminary tests in dogs with biopsy-confirmed visceral hemangiosarcoma (N=7) show that these dogs harbor greater
than 1 percent EPC-like cells in the peripheral circulation. This assay requires less than 3 ml of anticoagulated blood, making
it a practical, non-invasive means to confirm a suspected diagnosis of HSA. The ultimate goal, however, is to refine the sensitivity
and specificity of this assay to detect the presence of HSA lesions in dogs at risk before they pose a clinical problem. Early
detection is likely to offer the highest probability of successful treatment outcomes.
The treatment options presently for HSA, even if detected early, are limited. The possible success of chemotherapy in asymptomatic
dogs would have to be balanced against the potential toxicity associated with treatment. However, customized experimental
treatments for various tumor types have reached (or are close to) clinical trials, suggesting that tailored cancer therapy
treatments are on the horizon. Until recently, such tailored therapies were not practical. In particular, immunologic approaches
(the gold standard for customized therapy) using tumor vaccines specific for an individual patient's tumor were laborious
and slow, and small molecules are only effective when the correct molecular target is present.
In the case of dogs where survival with standard-of-care is short-lived, the time required to generate tumor-specific vaccines
and the absence of defined molecular targets meant that customized treatments would not be available in a timely manner to
benefit the dog. Yet, canine HSA is clearly amenable to treatment with immunologic approaches. Rapid isolation of malignant
cells could be used to confirm the presence of targets that improve delivery of immunomodulators like interleukin (IL)-12.
Moreover, a recent technology that generates tailored vaccines in a matter of days has been shown to be practical for clinical
applications. In the case of HSA, this technology could be used to construct vaccines against mutant targets that regulate
production of vascular endothelial growth factor (VEGF), which is constitutively elevated in the tumors and found at increased
levels in blood samples from affected dogs.