Abstract
Canine histiocytic malignancies (HM) are rare across the general dog population, but overrepresented in certain breeds, such as Bernese mountain dog and flat-coated retriever. Accurate diagnosis relies on immunohistochemical staining to rule out histologically similar cancers with different prognoses and treatment strategies (e.g., lymphoma and hemangiosarcoma). HM are generally treatment refractory with overall survival of less than 6 months. A lack of understanding regarding the mechanisms of disease development and progression hinders development of novel therapeutics. While the study of human tumors can benefit veterinary medicine, the rarity of the suggested orthologous disease (dendritic cell sarcoma) precludes this. This study aims to improve the understanding of underlying disease mechanisms using genome-wide DNA copy number and gene expression analysis of spontaneous HM across several dog breeds. Extensive DNA copy number disruption was evident, with losses of segments of chromosomes 16 and 31 detected in 93% and 72% of tumors, respectively. Droplet digital PCR (ddPCR) evaluation of these regions in numerous cancer specimens effectively discriminated HM from other common round cell tumors, including lymphoma and hemangiosarcoma, resulting in a novel, rapid diagnostic aid for veterinary medicine. Transcriptional analysis demonstrated disruption of the spindle assembly complex, which is linked to genomic instability and reduced therapeutic impact in humans. A key signature detected was up-regulation of Matrix Metalloproteinase 9 (MMP9), supported by an immunohistochemistry-based assessment of MMP9 protein levels. Since MMP9 has been linked with rapid metastasis and tumor aggression in humans, the data in this study offer a possible mechanism of aggression in HM.
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Abbreviations
- BAC:
-
bacterial artificial chromosome
- BMD:
-
Bernese mountain dog
- CFA:
-
Canis familiaris
- oaCGH:
-
oligo-array comparative genomic hybridization
- CNA:
-
copy number aberration
- CYT:
-
histiocytoma
- ddPCR:
-
droplet digital polymerase chain reaction
- DNA:
-
deoxyribonucleic acid
- FCR:
-
flat-coated retriever
- FFPE:
-
formalin fixed paraffin embedded
- FISH:
-
fluorescence in situ hybridization
- HEM:
-
hemangiosarcoma
- HM:
-
histiocytic malignancy
- IHC:
-
immunohistochemistry
- LSA:
-
lymphoma
- MCT:
-
mast cell tumor
- MEL:
-
melanoma
- OSA:
-
osteosarcoma
- PL:
-
plasmacytoma
- STS:
-
soft-tissue sarcoma
- TMA:
-
tissue microarray
- TVT:
-
transmissible venereal tumor
- UC:
-
urothelial carcinoma
- WCPP:
-
whole chromosome paint probe
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Acknowledgements
The authors wish to thank the North Carolina State University College of Veterinary Medicine Histopathology department for their expertise in performing immunohistochemistry evaluation of canine tumors. The authors also wish to thank the American Kennel Club Canine Health Foundation (Grant 01557) and the North Carolina State University Cancer Genomics fund for their generous financial support. We thank the Bernese Mountain Dog Club of America, Berner-L, and the Flat-Coated Retriever Foundation for continued support of HM research at NC State University.
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M.B. conceived of the study, participated in its design and coordination, and helped to draft the manuscript. K.K. carried out molecular genetic studies and drafted the manuscript. R.T. assisted with data interpretation and manuscript preparation. J.D. reviewed tumor histopathology and immunohistochemistry. A.M.-R. supervised and performed statistical analyses with T.J. All authors read and approved the final manuscript.
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K.K. is an employee of Sentinel Biomedical, which currently holds the commercial license to the ddPCR assay described in this paper. M.B. and R.T. are founders of Sentinel Biomedical.
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Supplemental Table 1
ddPCR assay sequences and locations. Sequence and location information for each set of primers and probes for the regions of interest. (PDF 30 kb)
Supplemental Table 2
ddPCR CFA 31 assay sensitivity and specificity by disease. Results of a 100-fold cross-validation assessment of the sensitivity and specificity for the ddPCR chromosome 31 assay when comparing HM to each of 10 other tumor types individually, and when comparing HM to those tumors that would be in the top differential (hemangiosarcoma, lymphoma, mast cell tumor, extramedullary plasmacytoma, benign histiocytoma, and amelanotic melanoma). (PDF 17 kb)
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Kennedy, K., Thomas, R., Durrant, J. et al. Genome-wide DNA copy number analysis and targeted transcriptional analysis of canine histiocytic malignancies identifies diagnostic signatures and highlights disruption of spindle assembly complex. Chromosome Res 27, 179–202 (2019). https://doi.org/10.1007/s10577-019-09606-0
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DOI: https://doi.org/10.1007/s10577-019-09606-0