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Cancer Genetics/Canine Cancer Projects

Cancer is the leading cause of death in humans under the age of 85, as well as the leading cause of disease-related death in dogs. As such, it has gained exceptional importance in our society. Both genetic and environmental factors have major effects on the temporal occurrence of cancer, and there is thus a new emphasis to learn more about how these factors influence cellular and molecular changes in cancer. Dogs and people are susceptible to many of the same types of cancer and the natural history (incidence, age of onset, location, progression, outcome) of many cancer types is similar in both species. Our pet dogs share our environment closely, allowing us to examine not only the heritable risk factors, but also those associated with the environment. Moreover, when compared to humans, dogs have shorter generational life spans (as many as five or more related generations frequently co-exist), extended pedigrees with detailed family histories, and more homogeneous genetic backgrounds, which provide unique opportunities to address questions about the origin and behavior of cancer. The answers we obtain studying cancers of dogs will contribute to our ultimate goals to design strategies for prevention and treatment of cancer in both dogs and people.

To understand the implications of cancer, one must first realize that cancer is not a simple disease. Rather, the term cancer describes a large number of diseases whose only common feature is uncontrolled cell growth and proliferation. A very important concept that is now universally accepted is that “cancer is a genetic disease, although it is not always heritable.” Tumors arise from cells that accumulate mutations which eliminate normal constraints of proliferation and genetic integrity. These mutations provide cells a selective growth advantage within their environment. This is essentially the same evolutionary phenomenon that we call “natural selection”, albeit on a microscopic scale. Various theories have been proposed to explain the genetic basis of cancer. One explanation invokes stochastic (random) events – the inherent error rate of enzymes that control DNA replication during each division introduces about 1 in 1,000,000 to 1 in 10,000,000 mutations for each base that is replicated during each round of replication. The genome consists of many millions of base pairs, so each daughter cell is likely to carry at least a few mutations in its DNA. Most of these mutations are silent; that is, they do not present any problems to the cell’s ability to function. However, others can disable tumor suppressor genes or activate proto-oncogenes that respectively inhibit or promote cell division and survival. An alternative hypothesis is that mutations are not stochastic, but rather “directed” due to the presence of a “mutator phenotype,” where the factors that control DNA replication and repair are inherently prone to more errors than would be expected by simple stochastic events in particular individuals. This leads to different cancer predispositions, which would be higher than the mean in such individuals, and might explain why not all people (or dogs) exposed to similar environmental carcinogens develop the same forms cancer at the same rate. There is strong evidence to support both mechanisms (stochastic and directed) in people and animals.

In both cases, loss of function of tumor suppressor genes and gain of function of oncogenes appear to contribute disproportionately to the origin of tumors. Tumor suppressor genes encode proteins that constrain cell division, promote cell death, or are essential to maintain the integrity of DNA. These genes can even help eliminate renegade cells that have initiated the path to cancer; thus, mutations that disable tumor suppressor genes contribute to the development and progression of tumors. In a broadly oversimplified approach, tumor suppressor genes can be grouped in three categories. One that includes p53 and ATM, among others, is responsible for controlling DNA repair. Cells can undergo spontaneous mutations, and these tumor suppressor genes must ensure that the mutant cells do not divide until the errors in their DNA sequence are repaired. Another that includes various cyclin-dependent kinase inhibitors such as INK4 and even some proto-oncogenes such as Ras (see below) controls cellular aging. Each cell in the body has the potential for a finite number of divisions, and these genes prevent further replication when that number has been reached. A third serves to counteract the function of growth-promoting genes and survival genes. Among these are RB and PTEN. Inactivation of tumor suppressor genes such as those listed above increases the risk of cellular transformation that can result in various types of cancer. Moreover, cancers that arise due to other mutations but that retain the function of these tumor suppressor genes may respond more favorably to therapy, making these promising targets for genetic therapy of cancer. Proto-oncogenes are the polar opposites of tumor suppressor genes. They encode proteins that promote cell growth and survival. In most cases, these genes are “turned on” and “turned off” as needed to maintain an adequate balance of cell division. However, when these genes are targets of mutation, they may gain independent function that cannot be “turned off”, leading to the development of cancer. It is very imoprtant to note that, even though there are numerous prototypical tumor suppressor genes and oncogenes (or cellular proto-oncogenes), many different genes can function in one or the other category (and sometimes both), depending on the context in which they are expressed!

Focus on Canine Cancer
For this program, we seek to define genetic lesions that underlie the pathogenesis of cancer in dogs. Mutations of specific genes that increase the probability, or risk, that an individual (actually a cell) will develop a tumor. In some cases, these mutations occur “sporadically”, that is, they alter the DNA of non-reproductive (somatic) cells. Generally, these mutations arise in susceptible individuals upon exposure to certain environmental insults; but the risk is not necessarily shared by relatives of the affected individual. Nevertheless, identifying the patterns of mutations associated with specific tumor types is likely to provide information to obtain a more accurate prognosis, and to develop more effective treatments. One example of this process has been extensively studied by Dr. Cheryl London and her colleagues, who described a common mutation of the c-Kit protein in canine mast cell tumors. Although the presence of this mutation is associated with a worse prognosis, it offers a potential target for treatments to improve the outcomes of dogs with this disease.(see, London et al, Exp Hematol. 1999 and Pryer et al, Clin Cancer Res. 2003)

On the other hand, when mutations occur in reproductive cells, they are passed on in the germ line. Identification of such mutations should help us predict relative cancer risk in individuals (or the likelihood of individuals to produce progeny with elevated cancer risks), allowing us to invest in practices to modify the environment that may reduce or eliminate the risk (cancer prevention). The investigation of cancer susceptibility in families or breeds of dogs is of critical importance to dog breeders and dog owners alike. Unlike other heritable conditions, genetic susceptibility to cancer may not manifest in disease until a dog has reached middle age, and long after it has achieved breeding potential. When present, this genetic susceptibility may be due to a process called loss of heterozygosity. Individuals inherit two copies of each gene upon conception, one from the sire, and one from the dam. Each of these gene copies is called an “allele.” A family or breed may have through the course of time, lost a functional allele of a “tumor suppressor gene” through mutation. The affected individuals are heterozygous (that is, they have two different alleles, and only one is functional). These individuals may not develop disease (cancer), unless the second, functional copy of the gene in question is mutated in a cell that retains the capacity to divide. Even in the best of circumstances, genetic analysis can only predict the probability or provide a relative risk, rather than a definitive assessment of whether or not the individual will in fact develop cancer. In an elegant series of research papers, Dr. Elaine Ostrander’s and Dr. Frode Lingaas’s groups recently reported an example of a heritable cancer syndrome (renal cystadenoma and nodular dermatofibrosis or RCND) in German Shepherd Dogs, where the gene defect was traced to a novel tumor suppressor gene called BHD (folliculin) (see, Lingaas et al, Hum Mol Genet. 2003 and Comstock et al, Mamm Genome 2004)

See the next section for information about our work regarding heritable traits that contribute to risk of lymphoma and bone cancer...

Another approach that has been successfully used to identify genes that contribute to cancer is the study of recurrent chromosomal abnormalities. Historically, this approach is responsible for the identification of the vast majority of tumor-associated genes (in humans). Until recently, major technical obstacles dampened the study of canine chromosomes, but many were overcome by the work of Dr. Matthew Breen and his colleagues, who developed reagents and adapted techniques to define a consensus karyotype for the dog.(see, Breen et al, Genome Res. 2001 and Thomas R, Fiegler et al, Cytogenet Genome Res. 2003 and Thomas R, Smith et al, Br J Cancer. 2003)

Selected Results from Our Laboratory
NOTE: If you wish to obtain information for ongoing studies, eligibility criteria, informed consent forms, or instructions for participation, please go to the STUDY INFO area of the site. If you require additional information, please feel free to contact us.

It is very important to note that cancer can affect any dog of any breed at any age; however, the predisposition among breeds or families dogs to develop specific types of cancer underscores the importance of hereditary components in the development or progression of these diseases. We have worked to solidify the resemblance between naturally occurring tumors of dogs and people in the areas of lymphoma and leukemia, bone cancer, melanoma, and tumors of endothelial cells that line blood vessels (hemangiosarcomas). This, along with results from projects such as those exemplified above will have a visible and long-lasting impact on canine health by providing accurate and dependable information that can be used judiciously for breeding decisions and that will pave the way towards the development of advanced molecular therapies for canine cancer.
The information below briefly summarizes selected data from our laboratory on studies of three common types of cancer in dogs. For more extensive details please see our list of publications. You can also contact our laboratory staff if you have questions or requests for more specific information.

Canine Melanoma: among other work, we have characterized expression of antigens that can improve the diagnosis of canine malignant melanoma (see Table 1). We have also shown that, among other abnormalities, the INK4 product, p16, can be detected by immunostaining and it is inactivated in up to 85% of dogs with melanoma (see Figure 1).

Canine Lymphoma: we recently confirmed the aberrant expression of receptors for interleukin-2 and the expression of CD20 in canine lymphomas (see Figure 2) opening a door to develop therapeutic reagents that target these proteins. In addition, our results show for the first time that there are distinct patterns of phosphorylation of the retinoblastoma protein in canine lymphoma, which may have potential predictive value regarding tumor stage and grade, phenotype, response to therapy, and survival (see Figure 3).

An intriguing concept of lymphoma is that the risk among dogs is not uniform. Some breeds are known to be at greater risk, but the reasons for this, while presumed to be due to heritbable traits, are unclear. We also know that immunophenotypes ("T-cell" vs. "B-cell" origin) are prognostically significant for this disease, but we do not know how these may be affected by inherited genetic factors. In a manuscript published in the July 2005 issue of Cancer Research, the leading publication in the field, we tested the hypothesis that the prevalence of lymphoma immunophenotypes differs among dog breeds. Our results showed for the first time an association of risk with breed derivation - that is, that the oldest breeds, including Spitz dogs and Asian “lap dogs” that are most closely related to wolves shared a predisposition for excess T-cell lymphoma, suggesting they retain ancestrally inherited risk factors. In contrast, recently derived European breeds were predisposed to excess B-cell tumors. Golden Retrievers show approximately equal prevalence of B and T cell tumors, each with unique genetic characteristics, providing a unique opportunity to work "forwards" using standard linkage approaches and "backwards" from the affected genes to identify the first ever set of heritable factors that contribute to risk for specific subtypes of lymphoma.

Canine Hemangiosarcoma: we have used new molecular approaches to define genetic abnormalities in canine hemangiosarcomas. Our results show that the PTEN gene that encodes a dual protein phosphatase which dampens signaling through the phosphoinositide-3 kinase pathways is mutated in >70% of these tumors (see Figure 4). Our most recent work identifies this disease as the first characterized in dogs that may arise due to transformation of cells with properties of stem cells.

Other recent results are findings that extend the linkage of breed-specifc traits that may contribute to cancer risk. This work is being done in collaboration with Dr. Matthew Breen at North Carolina State University, Dr. Elaine Ostrander at the National Institutes of Health, Dr. Anne Avery at Colorado State University, Dr. Stuart Helfand at Oregon State University, and Dr. Cheryl London at The Ohio State University. Perhaps most significantly we have results from work done with Dr. Breen that indicate genetic lesions responsible for the development of various cancers are evolutionarily conserved between dogs and humans. What this means in practice is that we can now investigate precise mechanisms that predispose humans and dogs as individuals and as species to these specific tumors. In addition, many of these results are being translated into clinical applications. For example, our laboratory is working to develop a simple new blood test that will be useful to confirm a diagnosis of hemangiosarcoma, and that may provide a means to diagnose this disease before it is clinically evident, improving our ability to treat this disease. We also have begun testing gene therapy strategies to treat melanoma and osteosarcoma (see section on Immunotherapy).

1 M=male, F=female, C=castrated male, S= spayed female,
2 Y=yes, N=No, N/A=information not available
3 Vim=vimentin, Mel A=Melan A/MART-1
4 - neg, + weak, ++ weak to moderate, +++ moderate, ++++ strong
5 Cases 11 and 21 originated from the same dog
6 Cases 18 and 29 originated from the same dog

FROM Koenig et al, Vet Pathol. 2001 Jul;38(4):427-35. (PubMed)

Figure 1. Immunohistochemical assessment of the expression and accumulation patterns for p53, p21, Rb, p16, and PTEN in spontaneous cases of canine melanoma. (a) Negative control staining using an irrelevant primary antibody, (b) staining for p53 in case 2 (note the fine granular and focal staining in nuclei indicated by the white arrowheads), (c) staining for p21 in case 21 (note the intense staining in individual nuclei indicated by the white arrowheads), (d) staining for Rb in case 13, (e) staining for p16 in case 7 (note the increased intensity of staining in cytoplasmic Golgi regions indicated by the black arrows), (f) staining for PTEN in case 26 (staining occasionally extended to the nuclei in few cells, sot shown in this section). Bar = 5 µM. From Koenig et al, Vet Pathol. 2002 Jul;39(4):458-72. (PubMed)

Figure 2. CD20 expression in canine B cell lymphoma. High power photomicrographs from lymph node samples of dog 5 (canine T zone lymphoma) and dog 24 (diffuse large B cell lymphoma). Serial sections were stained with H&E, or immunostained with anti-CD3, anti-CD79a, and anti-CD20 using a modified streptavidin biotin complex procedure with Histomark Red as the chromogen and hematoxylin counterstain. Note expression of CD20 by malignant B cells from Dog 24. Bars = 10 µm. From Jubala et al, Vet Pathol. 2005 Jul;42(4):468-76. (PubMed)

Figure 3. Rb phosphorylation and Ki-67 expression in canine lymphoma. Expression of Ki-67 and phosphorylation of Rb at S249/T252 and T826 was examined by immunohistochemistry in paraffin embedded tumor blocks. The left panel shows examples from three tumors (RS, a,d,g,j,m; SD b,e,h,k,n; TT, c,f,i,l,o) stained with H&E (a-c), anti-CD3 (d), anti-CD79a (e,f), anti-Ki-67 (g-i) anti-RbpS249/T252 (j-l), and anti-RbpT826 (m-o). Expression was quantified as follows: 0-5% positive = 0; 6-20% positive = 1; 21-40% positive = 2; >41% positive = 3. Magnification for all photomicrographs was 400 X. Color development was done using Histomark Red. Scores (0-3) are shown in graphical format on the right panel. From Modiano et al, Proc Keystone Symposia: Molecular Targets of Cancer Therapy (E-1), abstract 232, 2003.

Figure 4. Localization of PTEN in canine vascular tumor cell lines. The presence and localization of PTEN was examined in three canine hemangiosarcoma cell lines (DD, DAL, DEN) by immunocytochemistry using a red chromogen for detection. The top left panel shows staining using an irrelevant antibody control. The top right panel shows staining of DAL cells with wild type PTEN. The bottom left panel shows staining of DD cells, which harbor a mutant PTEN gene that lacks a C-terminal phosphorylation site, resulting in constitutive localization to the plasma membrane. The bottom right panel shows staining for DEN cells, which harbor a mutant PTEN that appears to have reduced stability, albeit it retains the expected cytoplasmic localization. More information of the role of PTEN in vascular tumors is available in the published manuscript by Dickerson, et al. Vet Pathol. 2005 Sept;42:618-632. (PubMed)