Chromosome Research

, Volume 23, Issue 2, pp 171–186 | Cite as

Comparative cytogenetic characterization of primary canine melanocytic lesions using array CGH and fluorescence in situ hybridization

  • Kelsey Poorman
  • Luke Borst
  • Scott Moroff
  • Siddharth Roy
  • Philippe Labelle
  • Alison Motsinger-Reif
  • Matthew Breen
Article

Abstract

Melanocytic lesions originating from the oral mucosa or cutaneous epithelium are common in the general dog population, with up to 100,000 diagnoses each year in the USA. Oral melanoma is the most frequent canine neoplasm of the oral cavity, exhibiting a highly aggressive course. Cutaneous melanocytomas occur frequently, but rarely develop into a malignant form. Despite the differential prognosis, it has been assumed that subtypes of melanocytic lesions represent the same disease. To address the relative paucity of information about their genomic status, molecular cytogenetic analysis was performed on the three recognized subtypes of canine melanocytic lesions. Using array comparative genomic hybridization (aCGH) analysis, highly aberrant distinct copy number status across the tumor genome for both of the malignant melanoma subtypes was revealed. The most frequent aberrations included gain of dog chromosome (CFA) 13 and 17 and loss of CFA 22. Melanocytomas possessed fewer genome wide aberrations, yet showed a recurrent gain of CFA 20q15.3–17. A distinctive copy number profile, evident only in oral melanomas, displayed a sigmoidal pattern of copy number loss followed immediately by a gain, around CFA 30q14. Moreover, when assessed by fluorescence in situ hybridization (FISH), copy number aberrations of targeted genes, such as gain of c-MYC (80 % of cases) and loss of CDKN2A (68 % of cases), were observed. This study suggests that in concordance with what is known for human melanomas, canine melanomas of the oral mucosa and cutaneous epithelium are discrete and initiated by different molecular pathways.

Keywords

Canine Oral melanoma Cytogenetics Array Comparative genomic hybridization 

Abbreviations

BAC

Bacterial artificial chromosome

BRAF

V-raf murine sarcoma viral oncogene homolog B1

C-KIT

V-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog

C-MYC

V-myc myelocytomatosis viral oncogene homolog (avian)

CCND1

G1/S-specific cyclin-D1

CDK4

Cyclin-dependent kinase 4

CDKN1A

Cyclin-dependent kinase inhibitor 1A

CDKN2A

Cyclin-dependent kinase inhibitor 2A

CFA

Canis familiaris (also used as a prefix to chromosome numbers)

CNA

Copy number aberration

FFPE

Formalin-fixed paraffin-embedded

FISH

Fluorescence in situ hybridization

H&E

Hematoxylin and eosin

HSA

Homo sapiens (also used as a prefix to chromosome numbers)

MAP-K

Mitogen-activated protein kinases

oaCGH

Oligo-array comparative genomic hybridization

PTEN

Phosphatase and tensin homolog

RAS

Rat sarcoma gene

RB-1

Retinoblastoma 1

SLP

Single locus probe

SPRED1

Sprouty-related, EVH1 domain containing 1

TACC3

Transforming, acidic coiled-coil containing protein 3

TP53

Cellular tumor antigen p53

TRPM7

Transient receptor potential cation channel, subfamily M, member 7

Supplementary material

10577_2014_9444_MOESM1_ESM.pdf (2.2 mb)
SOM Figure 1Comparison of fresh frozen and formalin fixed paraffin embedded tissues from the same tumor biopsy by oaCGH. To demonstrate that both fresh frozen punch biopsies and macrodissected fixed biopsy specimens could be in used in the same study, DNA from several sample pairs was assessed. In this example oaCGH profiles are from DNA isolated from A) a snap frozen punch biopsy and B) 3 x 25μm sections of the corresponding FFPE specimen, after macrodissection to exclude surrounding non-neoplastic regions of tissue and enrich for tumor cells. Analysis was completed in Agilent Genomic Workbench. Chromosomes are presented along the x-axis with log2 ratio of copy number changes presented along the y-axis centered at y=0. (C) Shows an overlay of the two oaCGH profiles in A (blue) and B (red). The blue and red bars above and below the combined profiles indicate the size of called aberrations in the fresh and fixed tissue profiles, respectively. These data demonstrate that while the amplitude of called events was slightly higher in the marcodissected FFPE specimen, both fresh and fixed tissue presented with the same called aberrations. (PDF 2252 kb)
10577_2014_9444_MOESM2_ESM.xls (32 kb)
SOM Table1Targeted regions for FISH analysis with the corresponding BAC clones and locations chosen from the CHORI-82 (CH-82) canine BAC library. (XLS 32 kb)
10577_2014_9444_MOESM3_ESM.xls (56 kb)
SOM Table 2Significant genome wide DNA copy number aberrations using GISTIC for three subtypes of canine melanocytic lesions, oral melanoma (OM), benign melanocytoma (B), and cutaneous melanoma (CM). In each case regions with significant copy number gain are presented before regions with significant copy number loss. The G-score considers the amplitude of the aberration as well as the frequency of its occurrence across samples. False Discovery Rate q-values are then calculated for the aberrant regions (XLS 56 kb)
10577_2014_9444_MOESM4_ESM.xls (70 kb)
SOM Table 3Differential chromosome regions with CN aberrations between primary canine oral melanoma (OM), primary canine cutaneous melanoma (CM), and canine benign melanocytoma (B). (XLS 70 kb)
10577_2014_9444_MOESM5_ESM.xls (40 kb)
SOM Table 4Aberrations with at least 60% penetrance for three subtypes of canine melanocytic lesions, oral melanoma (OM), cutaneous melanoma (CM), and melanocytoma (B) lesions after recoding as human (HSA). (XLS 40 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Kelsey Poorman
    • 1
  • Luke Borst
    • 2
  • Scott Moroff
    • 3
  • Siddharth Roy
    • 4
  • Philippe Labelle
    • 3
  • Alison Motsinger-Reif
    • 4
    • 5
  • Matthew Breen
    • 1
    • 5
    • 6
  1. 1.Department of Molecular Biomedical Science, College of Veterinary MedicineNorth Carolina State UniversityRaleighUSA
  2. 2.Department of Pathobiology and Population Health, College of Veterinary MedicineNorth Carolina State UniversityRaleighUSA
  3. 3.Antech DiagnosticsLake SuccessUSA
  4. 4.Department of Statistics, College of Physics and Applied MathematicsNorth Carolina State UniversityRaleighUSA
  5. 5.Center for Comparative Medicine and Translational ResearchNorth Carolina State UniversityRaleighUSA
  6. 6.Cancer Genetics ProgramUNC Lineberger Comprehensive Cancer CenterChapel HillUSA

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