Breen Lab @ NCSU

Many human cancers are associated with characteristic chromosome aberrations that have diagnostic and prognostic significance. The study of chromosomes (cytogenetics) and the identification of chromosome aberrations now plays a key role in defining many human cancers and in selecting appropriate therapy. The knowledge of such aberrations has identified areas of the human genome to be targeted for further research. In the dog the extent and identity of chromosome aberrations associated with specific cancers is still largely unknown. Historically, this has been a consequence of the difficulties in identifying reliably the chromosome comprising the dog karyotype. However, with an internationally recognized chromosome nomenclature for the dog, researchers now have the ability to maintain consistency in canine cytogenetic descriptions. A more recent tool for the classification of many human tumors has been provided by cellular genomics, in particular by molecular cytogenetic evaluation using fluorescence in situ hybridization (FISH) techniques. In the human, many cancers are actually now defined by their recurrent genetic abnormalities. These include; whole chromosome numerical abnormalities (e.g. trisomies and monosomies); partial chromosome numerical abnormalities (e.g. duplications and deletions); and structural abnormalities (e.g. translocations). Molecular cytogenetic evaluation of tumors using FISH has revolutionized the way in which we interrogate the cancer genome for recurrent changes. The FISH techniques widely used in cancer studies include metaphase FISH, interphase FISH and comparative genomic hybridization (CGH). All three techniques play an important role in the molecular cytogenetic evaluation of tumors.

It is estimated that almost 50% of all dogs over the age of 10 years will develop cancer and approx 1 in 4 of all dogs will at some stage in their life develop a cancer. Not all cancers kill dogs and the high level of veterinary care that is available in the 21st century is providing man’s best friend with a very high quality of medical care.

Our research activities are focused on exploring the cellular genomics of canine cancers and the investigation of any evolutionarily related genetic aetiology shared with human.

Our lab is focused:

1.  the comparative aspects of genetic changes in canine and human cancers,
2. the identification of cancer-related genes in dog and their counterparts in human cancer.

This work involves three major areas:

Direct molecular cytogenetics analysis of tumor cells. Tumor specific chromosome aberrations are widely documented in human malignancies and cytogenetics plays a key role in the diagnosis and prognosis of many forms of human cancer. To help with dog chromosome identification we have developed extensive panels of chromosome specific single locus probes (SLP) (numbering over 1,500 SLPs) and of whole chromosome paint probes (WCPP) that we use in multicolor fluorescence in situ hybridization (FISH) in order to characterize genetic aberrations in individual tumor cells. Use of a panel of 41 multicolored SLPs to identify each chromosome in the canine karyotype is shown below.

 

Indirect molecular cytogenetics analysis of tumor cells. Comparative genomic hybridization (CGH) is a whole genome screening approach that allows detection and localization of DNA sequence copy number variation anywhere in the tumor genome. The approach is based on competitive differential hybridization of labeled genomic DNA from a tumor, competed with DNA from normal cells. CGH has proved very helpful in defining the locations of previously unknown oncogenes and tumor suppressor genes. We have developed canine CGH and are now applying this technique to a variety of key tumor types. We have already begun to find associations between genetic changes detected by CGH analysis and the survival of dogs with certain cancers.

To improve the throughput, resolution and accessibility of canine CGH, we developed array-based CGH for the dog, first with a panel of 2,200 canine BAC clones ordered throughout the genome at 1Mb intervals, and now with a set of 1,000,000 oligonucleotide sequences spaced at 2.5kb intervals. The increase in resolution and throughput has led to array CGH replacing chromosome based CGH as the method of choice for genome wide assessment of DNA copy number aberrations.

The use of laser microdissection allows us to accurately micro-dissect tumor cells from archival sections. Using degenerate oligonucelotide primer (DOP) PCR, and fluorescent labeling, we are able to perform CGH using DNA obtained from just a few tumor cell nuclei. By characterizing the presence and location of chromosomal changes in the earliest identifiable changes in tumor cells, we are identifying possible targets for positional cloning and gene-based therapies.

Gene expression profiling. We are also developing array-based expression profiling to compare gene expression patterns in tumor cells compared to the pattern of expression of normal cells in adjacent tissue. A combination of the data obtained from genomic and expression arrays will allow us to 1) identity regions of the genome imbalance and then 2) define deregulated genes within these regions. In this way we are defining loci for mutation screening.