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

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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.

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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

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Acknowledgments

This study was funded in part by a clinical trial award from Antech Diagnostics (awarded to MB) and with funds from the NCSU Cancer Genomics Fund (MB). KP was supported in part by funds from a Comparative Biomedical Sciences Graduate Studentship, and in part by a Department of Education GAANN Fellowship. We thank Rachael Thomas for assistance in humanizing the canine CGH data and Christina Williams for sample coordination. SR was supported by T32GM081057 from the National Institute of General Medical Sciences and the National Institute of Health.

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Authors and Affiliations

  1. Department of Molecular Biomedical Science, College of Veterinary Medicine, North Carolina State University, 1060 William Moore Drive, Raleigh, NC, 27607, USA

    Kelsey Poorman & Matthew Breen

  2. Department of Pathobiology and Population Health, College of Veterinary Medicine, North Carolina State University, Raleigh, NC, USA

    Luke Borst

  3. Antech Diagnostics, 1111 Marcus Avenue, Lake Success, NY, USA

    Scott Moroff & Philippe Labelle

  4. Department of Statistics, College of Physics and Applied Mathematics, North Carolina State University, Raleigh, NC, USA

    Siddharth Roy & Alison Motsinger-Reif

  5. Center for Comparative Medicine and Translational Research, North Carolina State University, Raleigh, NC, USA

    Alison Motsinger-Reif & Matthew Breen

  6. Cancer Genetics Program, UNC Lineberger Comprehensive Cancer Center, Chapel Hill, NC, USA

    Matthew Breen

Authors
  1. Kelsey Poorman
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  2. Luke Borst
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  3. Scott Moroff
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  4. Siddharth Roy
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  5. Philippe Labelle
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  6. Alison Motsinger-Reif
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  7. Matthew Breen
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Corresponding author

Correspondence to Matthew Breen.

Additional information

Responsible Editor: Conly Rieder

Electronic supplementary material

Below is the link to the electronic supplementary material.

SOM Figure 1

Comparison 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)

SOM Table1

Targeted regions for FISH analysis with the corresponding BAC clones and locations chosen from the CHORI-82 (CH-82) canine BAC library. (XLS 32 kb)

SOM Table 2

Significant 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)

SOM Table 3

Differential chromosome regions with CN aberrations between primary canine oral melanoma (OM), primary canine cutaneous melanoma (CM), and canine benign melanocytoma (B). (XLS 70 kb)

SOM Table 4

Aberrations 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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Poorman, K., Borst, L., Moroff, S. et al. Comparative cytogenetic characterization of primary canine melanocytic lesions using array CGH and fluorescence in situ hybridization. Chromosome Res 23, 171–186 (2015). https://doi.org/10.1007/s10577-014-9444-6

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  • DOI: https://doi.org/10.1007/s10577-014-9444-6

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