Characterization of Genetic Alterations in Feline Vaccine-Associated Sarcoma 
Using Whole Chromosome Painting Probes

E.A. Hoots. E.A. McNiel, S.M. LaRue.
Department of Radiological Health Sciences
Colorado State University
Fort Collins, Colorado

Feline vaccine-associated sarcoma poses clinical, ethical, legal and economic problems for the veterinary profession.  Prevention and treatment of this cancer requires an understanding of the underlying causes.  Cancer is a genetic disease and chromosomal aberrations have been shown to cause tumors in humans.  Characterization of such chromosomal aberrations in feline tumors will allow us to identify the genomic loci important in oncogenesis.  Fluorescence in situhybridization techniques such as fluorescent-labeled painting probes are state-of-the-art, powerful tools for evaluating cytogenetic abnormalities in cancer cells.  With painting probes it is possible to directly visualize and identify chromosomal aberrations such as translocations, insertions, amplifications, and deletions more rapidly and reliably than with traditional cytogenetic techniques.  These techniques have never been used to evaluate feline tumors.  Therefore, we have developed a technique to efficiently generate chromosome specific painting probes for each of the chromosomes of the domestic cat (Felis cattus n=38).

Metaphase chromosome spreads on coverslips were made from mitogenstimulated, cultured lymphocytes.  These coverslips were stained with 5% giemsa and viewed under 1000X magnification.  A glass needle controlled by a micromanipulator was used  to scrape 10 copies of a chromosome from the glass surface.  The chromosomes were transferred to a microfuge tube and the DNA is amplified using degenerate oligonucleotide primed polymerase chain reaction (DOP-PCR), a form of PCR that amplifies all genomic elements equally.  The amplified DNA fragments are then labeled via a second round of DOP-PCR amplification in the presence of a fluorescent nucleotide, which is incorporated into the newly synthesized DNA.  The fluorescent-labeled DNA (painting probe) is then hybridized onto a metaphase spread.  In the presence of blocking DNA to eliminate preferential binding to repetitive sequences (Feline Cot-1 DNA), the labeled painting probe will "light up" chromosome specific sequences when viewed with a fluorescent microscope.

Whole chromosome painting probes that specifically identify feline chromosomes were created and can be used to identify chromosomal abnormalities in feline tumors.  Our goal is to produce painting probes for each feline chromosome and to screen tumors from clinical cases.  Identification and characterization of discrete chromosomal abnormalities in feline tumor cells may be important in understanding the behavior, prognosis, and potential response to therapy of feline cancers.

Supported by grants from the Morris Animal Foundation and the National Cancer Institute.

Characterization of Chromosomal Aberrations in Feline Vaccine-Associated Sarcoma 
Using Comparative Genomic Hybridization

E.A. McNiel, E.A. Hoots, S.M. LaRue.
Department of Radiological Health Sciences
Colorado State University
Fort Collins, Colorado

Epidemiologic evidence strongly associates vaccination of cats for rabies and feline leukemia virus with the development of soft tissue sarcomas at the site of administration.  These tumors are histologically aggressive, locally invasive, and constitute a difficult legal, ethical, and clinical problem for the veterinary  profession.  Prevention and definitive treatment of feline vaccine-associated sarcoma requires an understanding of the pathogenesis of this cancer.  Cancer is a genetic disease.  The transformation of normal cells to tumor cells with increasingly malignant characteristics results from the accumulation of mutations.  Causative chromosome rearrangements are recognized in human tumors and many cancer-associated genes have been identified with cytogenetic study.  The location of recurrent chromosomal aberrations signals the presence of cancer associated genes at particular genomic locations.  Chromosomal studies of human cancers provide an important clinical diagnostic and prognostic tool.  We have initiated efforts to characterize the genomic alterations in feline vaccine-associated sarcoma through cytogenetic evaluation of tumors.

Over the last decade, the development of molecular cytogenetic techniques including chromosome and gene specific fluorescence in situ hybridization (FISH) has greatly enhanced the resolution, specificity, and efficiency of tumor cytogenetics.  These advancements are particularly important for the study of the complex karyotypic  rearrangements found in solid tumors.  Comparative genomic hybridization (CGH) is a state-of-the-art technique for detecting numerical chromosome changes, such as amplifications, deletions, aneuploidy, and unbalanced translocations in tumors.  CGH involves the simultaneous hybridization of tumor and normal DNA, labeled with 2 different fluorochromes, onto a normal metaphase chromosome spread.  Computer image analysis  allows for detection of differences in the fluorescent intensity of the two fluorochromes  indicating gains or losses in the tumor at particular chromosomal loci.

We have modified CGH for use in the study of feline tumors.  So far, five feline vaccine-associated sarcomas have been characterized using CGH and classical banding techniques.  These tumors have highly aberrant karyotypes.  Further evaluations will be necessary to characterize recurrent mutations.  Comparative genomic hybridization provides a rapid method for screening the genome  for mutations in cancer related genes.

Supported by the Morris Animal Foundation and the National Cancer Institute.

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