Osteosarcoma

No one doubts that genetic factors can affect osteosarcoma (OSA) risk. And everyone agrees that dogs arguably offer the best opportunity for identifying such genetic factors for two reasons: first, because of the existence of distinct populations (breeds), some of which have a high rate of osteosarcoma and others in which osteosarcoma is rare or nonexistent; and second, because individual dogs of a given breed are genetically very similar, while dogs of different breeds are genetically quite different.

The classical approach has been to compare the genetic maps of dogs with and without tumors in a single osteosarcoma-prone breed to identify a “region” in which a genetic risk factor lies, and then to compare the genetic maps of this region among several osteosarcoma-prone breeds to locate the precise gene and mutation. But the classical approach has so far failed to identify a genetic factor for osteosarcoma risk in any breed, despite two decades of effort. Why? One possibility is that current genetic mapping techniques cannot “see” the genetic differences between individuals with and without tumors. Another possibility is that there is no single genetic difference that increases osteosarcoma risk; instead, multiple genetic differences act together to increase risk. Or perhaps there are several genes that individually cause osteosarcoma, either in different breeds or even the same breed.

At present, several researchers are using Deerhounds as one breed in which to investigate possible genetic factors that increase osteosarcoma risk. (Many thanks to Dr. Jerold Bell of Tufts University Vet School who helped us to sort through OSA research projects.)

Ohio State University

Drs. Guillermo Couto and Carlos Alvarez at The Ohio State University are identifying novel genetic variation in osteosarcoma-prone breeds and comparing that variation across related breeds with different levels of osteosarcoma risk. For example, by measuring DNA quantity across the genomes of affected and unaffected dogs, they will identify DNA copy number variation (CNV). CNVs, which can be gains or losses of 50 to millions of base pairs of genomic DNA, can be suggestive of having biological effects when they are in or near genes. For example, the trait of furnishings (moustache and eyebrows) that is present in Deerhounds and several other breeds is due to a 167-base-pair insertion in the RSPO2 gene; and muscular dystrophy in German Shorthaired Pointers is caused by a deletion of 2.4 million base pairs. Alternatively, newly discovered CNVs and smaller variants can be queried for association with a trait using conventional genetic analysis.

As osteosarcoma is common in Deerhounds, the Ohio State group would like to include them in their study. Dr. Couto’s group kicked off this effort with blood collection of approximately 90 Deerhounds volunteered by their kind owners at the 2012 national specialty. Some owners will be contacted for additional information to complete the study, and there may be a call for more volunteers in the near future. Notably, this precious DNA and health information is being coordinated among multiple groups studying different aspects of Deerhound health.

North Carolina State University

Dr. Marlene Hauck studies the category of tumors called sarcomas, of which osteosarcoma is one. The SDCA has been working with her since we first started to collect blood samples for N.C. State in the late 1990s. Dr. Hauck initially used the DNA from the blood samples to search for a genetic factor that might increase osteosarcoma risk, but in recent years she has switched to studying DNA and RNA isolated from tumors themselves.

Historically, genes have been studied individually. However, not all disease, including cancer, can be studied just by looking at individual genes. An alternative approach is to study a group of genes that act in concert to produce or regulate the expression of a phenotypic trait. Such a set of genes is termed a “pathway,” and much cancer research now looks at changes in pathways as well as changes of genes within these pathways.

Dr. Hauck does something called deep sequencing of the tumors themselves in order to identify key mutations within a tumor type, as well as additional common pathway dysregulations, gene expression alterations, and critical mutations. She looks for driver mutations by sequencing all the expressed genes in a set of tumors and identifying mutations in common between tumors.

Dr. Hauck sequences all of the RNA in a tumor. She writes:

Since only about 5% of the entire genome (at least in people) is transcribed, this becomes a much more reasonable amount of data, including all the expressed genes (mRNA) and also the miRNA (which are thought to be important in regulating which mRNAs are translated into proteins (expressed). This is what we are funded to do in the soft tissue sarcomas, and what I had hoped to do in the OSAs as well. Knowing what we know about the region likely to be involved in the Scottish Deerhound, we could look at several tumors for the mutations/alterations in common. Any single tumor will have many mutations (think of tumors as having a mutator phenotype as they have lost normal DNA repair/cell cycle control mechanisms), so looking at just one tumor is not helpful in terms of driver mutations (as opposed to passenger, or “accidental” vs. “causal” mutations). Driver mutations are those that are thought to be responsible for tumorigenesis. The other mutations may alter response to therapy, or may have no effect—it can get a bit complicated.

A couple of years ago, Dr. Hauck’s team received a tumor sample from a Deerhound named Ace. Normally, looking at one dog, you cannot tell which mutations are important, but you can get relative expression levels for all genes with deep sequencing, and Ace hugely over-expressed a particular pathway. When Dr. Hauck’s team inhibited this pathway in his cell line (early passage tumor cells in culture), they died. She developed a second hypothesis based on the pathways dysregulated in Ace’s tumor: that there is a particular pathway—the pathway they found in Ace’s tumor—that is important in OSA. Several additional OSA tumors were tested; some seemed to echo the findings in Ace, some didn’t. So they have expanded the group studied and are working on that right now.

The research team at Ohio State and Dr. Hauck at North Carolina State are collaborating on researching OSA in Scottish Deerhounds and are actively seeking tumor samples from Deerhounds. If you live near either of these institutions, please contact the researchers there to discuss how to provide them with tumor samples from your dog, particularly if you are considering amputation as part of your dog’s treatment. If you are euthanizing your dog, please provide tumor samples from your dog. Both institutions offer full surgical and oncological sevices, so treatment at either place is an option. If you choose to do that, please let the researcher know that your dog is coming.

Van Andel Institute

As you may remember, Roe Froman from the Van Andel Institute spoke at our 2010 national specialty. VAI received a large grant to study several different types of cancer that appeared in both dogs and people in the hope that by studying the diseases in dogs that gains could be made for the treatment of these cancers in both species. One of the cancers was osteosarcoma. VAI began to aggressively collect samples from breeds that had OSA, including Deerhounds. Ultimately, a total of over 18,000 samples were collected from dogs of many breeds that were susceptible to the cancers being studied, including 140 Deerhound samples, 23 from dogs with osteosarcoma.

Research is a numbers game: to find the genes involved for hereditary traits, you need a certain number of samples for that trait. But the samples have to be confirmed: so, for instance, to study osteosarcoma, you need samples from dogs where the tumor was confirmed by biopsy; you can’t use samples where the disease looked and acted like osteosarcoma, so it was just assumed that it was osteo. Because of these restrictions, it is difficult to amass enough useable samples. For instance, for a complex trait such as osteosarcoma appears to be, you need 70 samples from affected dogs. In all the years we’ve been collecting osteo samples from Deerhounds, we don’t have samples from 70 affected dogs in toto, never mind for any one study.

The breed where enough samples were collected for VAI to research osteo ended up being the Great Pyrenees. VAI has found one loci for osteosarcoma in that breed that they are confident about and another that they still need to prove. Once they have this done for Great Pyrenees, they will go back to all of their other osteosarcoma samples to see if these genes are involved in other breeds.

We don’t know whether Deerhounds will have the same genetic presentation of the disease. In Pyrs, dogs most commonly get osteo between the ages of four and six. Three out of four dogs get it in a forelimb, and most cases are resistant to treatment. However, there are a handful of dogs with the disease whose legs are amputated that live for another four to five years before dying, usually of metastatic cancer that stems from the original osteosarcoma. VAI is very interested in how those dogs are different, because they feel that these dogs might be a key to possible future cancer treatments.

This is quite different from how the disease presents itself in Deerhounds, so it will be interesting to see if the diseases are genetically similar or not.

So at this time, VAI doesn’t need any more samples from Deerhounds, but they would like to keep in touch with us about their progress on this project and possible future projects.

 Jeff Phillips, DVM, Ph.D.

Jeff, who has been collecting Deerhound samples on his own for his private research on osteo, has switched positions and is now at Lincoln Memorial University in Harrogate, Tennessee. We continue to keep in touch, and most recently he has been waiting to hear from the National Cancer Institute, which was confirming some data for him. So he’s in a holding pattern, and we’ll let you know if there are any developments in his research.

The osteosarcoma research needs samples.