Overview of the Presentation

 Define molecular cytogenetics and its application in human and veterinary medicine

 Describe key techniques and resources used for cytogenetic characterization of tumors

 Provide examples of molecular cytogenetic findings from ongoing feline studies

 Outline future goals and prospects for molecular cytogenetics of feline cancer

Overview of the Issue

Overview of Molecular Cytogenetics

The field of cytogenetics concerns the study of chromosomes within normal and abnormal cells, both within and between individuals. Following rapid advances in DNA-based technologies, the cytogenetics field has moved progressively from gross evaluation of chromosome structure and organization using conventional light microscopy towards increasingly more detailed analysis at the molecular level. Molecular cytogenetics analysis plays a key role in determining the organizational status of chromosomal DNA in normal individuals, and in understanding how disruptions to the normal status play a role in genetic disease, such as cancer.¹

Application of Molecular Cytogenetics in Human Cancer

Malignant cells from human cancer patients commonly exhibit a variety of chromosome abnormalities, reflecting dysregulation of the normal cell cycle. These may comprise structural abnormalities (aberrant breakages or fusions within and between chromosomes) or numerical abnormalities (gains or losses of entire or partial chromosomes), or, typically, a combination of both. These aberrations vary in frequency and distribution both within and between different tumors. The specific combination of chromosome aberrations in a given tumor can be defined using molecular cytogenetics techniques, and then compared to other patients with the same cancer to identify common and unique features.

The presence or absence of certain chromosome aberrations is not random, and among these are recurrent features that fall into two primary categories with extensive overlap:

1.  Chromosome aberrations that are encountered at high frequency in one or more specific forms of cancer but are rare or absent in others, and which therefore have diagnostic value

2.  Chromosome aberrations whose presence is consistent with the clinical behavior of the tumor, and which therefore have prognostic value

Molecular cytogenetics provides opportunities for developing novel therapeutic strategies targeted to specific, recurrent chromosome aberrations. A well-known example is the 'Philadelphia chromosome', an abnormal structural rearrangement ('translocation') between human chromosomes 9 and 22, present in approximately 90% of patients with chronic myelogenous leukemia, but also infrequently in other leukemia subtypes. The translocation results in the formation of an abnormal protein whose activity can be inhibited by therapy with a family of drugs (tyrosine kinase inhibitors).² This represents a highly successful example of advancement in clinical management through the application of molecular cytogenetics technology.

As a result of the extensive genomics tools that have been developed for the domestic dog, data regarding recurrent chromosome aberrations in human cancers can be extrapolated directly in terms of the corresponding DNA sequence on the equivalent dog chromosome. It now becomes evident that the same cancer subtype in humans and canine patients often involve defects of the same genes, and that the same diagnostic, prognostic and therapeutic findings may be applicable across different species.³

Progress in Feline Cancer Cytogenetics

The few prior reports of chromosome abnormalities in feline cancer have been typically limited both in terms of the number of cases studied and the level of detail with which they have been analyzed. These limitations are gradually being overcome as technologies advance and become more time- and cost-effective. Critical to future advancements is the ongoing development of a genome sequence assembly for the cat.⁴ Meanwhile, molecular cytogenetic data for feline cancers are now emerging, through application of two key techniques.

Fluorescence In Situ Hybridization Analysis

Fluorescence in situ hybridization (FISH) analysis is used for studying the organization of chromosomal DNA within a genome.⁵ In its simplest form, a DNA sequence representing a chromosome region of specific interest is first tagged with a fluorescent dye, to generate a probe. The probe is applied to cells from the patient, where it will bind to complementary sequences on the corresponding chromosomes. This reveals the structural and numerical status of that specific DNA sequence in the patient's genome, which is then compared to that of a normal individual. In cancer studies, the probe typically represents a DNA sequence known to be associated with a given tumor type, for which the status in the patient is assessed by FISH analysis as a means for diagnosis and/or prognosis.

Array Comparative Genomic Hybridization Analysis

Array comparative genomic hybridization analysis (aCGH) is a fundamental tool used for discovering chromosome abnormalities in cancer.^6,7 DNA is isolated from all chromosomes (the entire genome) of the tumor specimen, and is tagged with a fluorescent dye. Independently, total genomic DNA is also isolated from a normal, healthy donor (the 'reference' sample), which is tagged with a different fluorescent dye. The two fluorescent probes are then combined and applied to a glass slide (array), on which many thousands of short segments of normal chromosomal DNA have been printed, each with a known genomic location. The tumor and reference probes then compete to bind to the corresponding chromosomal DNA sequence on the array. A laser scanner is used to measure the amount of each fluorescent dye (tumor versus reference) that has bound to each arrayed DNA sequence. If these values are not equal, this indicates an abnormal gain or loss of that particular chromosomal region in the tumor.

aCGH analysis therefore enables identification of abnormal gains and losses among many thousands of different chromosomal DNA sequences in a single experiment, for each tumor. The pattern of gains and losses is then compared between different patients, to identify common and unique features that are associated with specific tumor subtypes.

FISH and aCGH analyses have been used extensively in human cancer studies, and more recently in other species including the domestic dog. Since both rely in part on the availability of a genome sequence for the species of interest, their application to the cat has lagged behind. With funding support from the Morris Animal Foundation we used emerging data from the feline genome sequence assembly to develop a framework panel of probes for FISH analysis, distributed along the length of each cat chromosome. Using these reagents we then constructed a first-generation platform for aCGH analysis of feline cancers, and demonstrated their application to the study of feline injection-site sarcomas and abdominal lymphoma.

Application of Molecular Cytogenetics to Feline Sarcomas and Lymphomas

Since the early 1990s there have been numerous reports of tumor formation at the site of a routine vaccination or other injection event in domestic cats.^8–11 While opinions vary, these injection site-associated sarcomas (ISAS) have a tendency to be more highly invasive and prone to recurrence than spontaneous sarcomas (non-ISAS). Furthermore, ISAS typically warrant more radical surgery and intensive clinical management with regular monitoring for progression, and confer a poorer long-term prognosis. At the time of initial diagnosis, however, it can be challenging to distinguish conclusively between these different sarcoma subtypes based on clinical and histological factors alone.

We sought to establish whether there are specific chromosome abnormalities that are statistically more frequent in one sarcoma subtype compared to the other, which could be developed as additional biomarkers for tumor classification at the time of diagnosis. We performed aCGH analysis of 46 feline sarcomas, comprising 19 ISAS and 27 non-ISAS, revealing numerous chromosome abnormalities that were highly recurrent throughout the cohort. Among these were two chromosome regions for which deletions were significantly associated with the non-ISAS subtype.¹² High-resolution characterization of these two chromosome regions, and their relationship with clinical and histological parameters, is now underway.

We are applying similar principles to the study of feline lymphoma, focusing primarily on tumors within the abdominal cavity. The main goal is to identify DNA-based markers of specific tumor subtypes as a means to refine subclassification of this highly heterogenous cancer.¹³ The resulting data are being compared with information from comparable tumors of human patients, providing opportunities for reciprocal exchange of information between two species with similar cancers. Ultimately, through correlation of genomic data with clinical parameters in retrospective cases, this approach may expand our ability to predict tumor behavior and outcome. Knowledge of the effect of specific chromosome abnormalities on gene expression and function will provide insights into the underlying basis of tumor pathogenesis, and in the longer term may identify new preventive measures and novel therapeutic targets.

Summary

Molecular cytogenetics has played an extensive role in advancing diagnostic, prognostic and therapeutic strategies in human medicine, particularly through the correlation of chromosomal abnormalities with specific cancer subtypes. It is becoming increasingly evident that there are tremendous opportunities for these concepts to be extrapolated to veterinary oncology; however, efforts to date have focused heavily on cancers of the dog. Taking examples from our ongoing studies, we demonstrate how the field of feline molecular oncology is now gaining momentum.

References, Reviews and Suggested Reading

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2.  Koretzky GA. The legacy of the Philadelphia chromosome. J Clin Invest 2007;117:2030–2032.

3.  Breen M and Modiano JF. Evolutionarily conserved cytogenetic changes in hematological malignancies of dogs and humans - man and his best friend share more than companionship. Chromosome Res 2008;16:145–154.

4.  Pontius JU, Mullikin JC, Smith DR, Lindblad-Toh K, Gnerre S, Clamp M, Chang J, Stephens R, Neelam B, Volfovsky N, Schaffer AA, Agarwala R, Narfstrom K, Murphy WJ, Giger U, Roca AL, Antunes A, Menotti-Raymond M, Yuhki N, Pecon-Slattery J, Johnson WE, Bourque G, Tesler G and O'Brien SJ. Initial sequence and comparative analysis of the cat genome. Genome Res 2007;17:1675–1689.

5.  O'Connor C. Fluorescence in situ hybridization (FISH). Nat Educ 2008;1:1.

6.  Pinkel D, Albertson DG. Array comparative genomic hybridization and its applications in cancer. Nat Genet 2005;37:S11–17.

7.  Theisen A. Microarray-based comparative genomic hybridization (aCGH). Nat Educ 2008;1:1

8.  Doddy FD, Glickman LT, Glickman NW, Janovitz EB. Feline fibrosarcomas at vaccination sites and non- vaccination sites. J Comp Pathol 1996;114:165–174.

9.  Vaccine-Associated Feline Sarcoma Task Force. The current understanding and management of vaccine-associated sarcomas in cats. J Am Vet Med Assoc 2005;226:1821–1842.

10. Hendrick MJ. Musings on feline injection site sarcomas. Vet J 2011;188:130–131.

11. Martano M, Morello E, Buracco P. Feline injection-site sarcoma: past, present and future perspectives. Vet J 2011;188:136–141.

12. Thomas R, Valli VE, Saylor K, Smith E, Bell J, Cullen C, Langford CF, Breen M. Microarray-based cytogenetic profiling of feline injection-site sarcomas reveals recurrent and subtype-associated genomic aberrations. Chromosome Res 2009;17:987–1000.

13. Louwerens M, London CA, Pedersen NC, Lyons LA. Feline lymphoma in the post-feline leukemia virus era. J Vet Intern Med 2005;19:329–335.