Molecular cytogenetic characterization of canine histiocytic sarcoma: A spontaneous model for human histiocytic cancer identifies deletion of tumor suppressor genes and highlights influence of genetic background on tumor behavior

Abstract

Background: Histiocytic malignancies in both humans and dogs are rare and poorly understood. While canine histiocytic sarcoma (HS) is uncommon in the general domestic dog population, there is a strikingly high incidence in a subset of breeds, suggesting heritable predisposition. Molecular cytogenetic profiling of canine HS in these breeds would serve to reveal recurrent DNA copy number aberrations (CNAs) that are breed and/or tumor associated, as well as defining those shared with human HS. This process would identify evolutionarily conserved cytogenetic changes to highlight regions of particular importance to HS biology.

Methods: Using genome wide array comparative genomic hybridization we assessed CNAs in 104 spontaneously occurring HS from two breeds of dog exhibiting a particularly elevated incidence of this tumor, the Bernese Mountain Dog and Flat-Coated Retriever. Recurrent CNAs were evaluated further by multicolor fluorescence in situ hybridization and loss of heterozygosity analyses. Statistical analyses were performed to identify CNAs associated with tumor location and breed.

Results: Almost all recurrent CNAs identified in this study were shared between the two breeds, suggesting that they are associated more with the cancer phenotype than with breed. A subset of recurrent genomic imbalances suggested involvement of known cancer associated genes in HS pathogenesis, including deletions of the tumor suppressor genes CDKN2A/B, RB1 and PTEN. A small number of aberrations were unique to each breed, implying that they may contribute to the major differences in tumor location evident in these two breeds. The most highly recurrent canine CNAs revealed in this study are evolutionarily conserved with those reported in human histiocytic proliferations, suggesting that human and dog HS share a conserved pathogenesis.

Conclusions: The breed associated clinical features and DNA copy number aberrations exhibited by canine HS offer a valuable model for the human counterpart, providing additional evidence towards elucidation of the pathophysiological and genetic mechanisms associated with histiocytic malignancies. Extrapolation of data derived from canine histiocytic disorders to human histiocytic proliferation may help to further our understanding of the propagation and cancerization of histiocytic cells, contributing to development of new and effective therapeutic modalities for both species.

Figure 1

Distribution of the age of diagnosis of HS in US-resident BMD (68 cases) and FCR (45). The mean age of HS diagnosis is significantly different between BMD (7.7 yrs) and FCR (8.6 yrs) (pval = 0.01, Mann-Whitney U test).

Figure 2

Anatomical distribution of histiocytic tumors in FCR and BMD. Anatomical location of HS is significantly different between the two breeds (p value < 0.001, Fisher Exact test).

Figure 3

Molecular cytogenetic evaluation of a canine histiocytic malignancy using aCGH and FISH. A. Example of whole genome aCGH profile of a HS in a five year old female FCR. Log₂ ratios representing thresholds of genomic gain and loss are indicated by horizontal bars above (green line) and below (red line) the midline (orange line), which represents normal copy number. The chromosome copy number status line for the tumor appears as an orange overlay of the center-line when there is a normal copy number, and as either green (gain) or red (loss) in the regions where genomic imbalances were apparent, as determined by the aCGH Smooth algorithm [29]. The aCGH profile is annotated with the clone address of nine BAC clones from the 1 Mb array that were used in subsequent FISH analysis of this case. Three of these nine clones have been shown previously to contain the full coding sequence of a key cancer-associated gene (CDKN2A, RB1, PTEN) [26]. The color of the text denotes the fluorochrome with which the BAC clone was labeled. B, C Targeted FISH analysis of tumor metaphase chromosome spreads from the same case using nine differentially labeled BAC clones (highlighted in A) combined in two separate groups. The modal copy number for each clone is indicated. D. Summary of copy number data of all nine loci evaluated by FISH analysis of at least 30 tumor interphase nuclei or metaphase spreads. The aCGH copy number status of these regions (gain, loss, balance) are indicated, demonstrating concordance between FISH data and aCGH data.

Figure 4

Whole genome CNA penetrance plot showing the percentage of all HS cases in the cohort (BMD+FCR; n = 86 cases) that presented with detectable DNA copy number gain (green) and loss (red) along the length of each dog chromosome (CFA 1 to CFA 38), surveyed at 1 Mb intervals. The asterisks indicate those seven chromosomes (CFA 10, 15, 22, 25, 30, 35 and 36) that have regions of CNAs differing significantly between BMD and FCR.

Figure 5

Distribution of the size of the CNAs identified by aCGH analysis of HS of BMD and FCR.

Figure 6

Whole genome CNA penetrance plots separated by breed A) FCR and B) BMD, where each indicates percentage of HS cases identified with DNA copy number gain (green) and deletion (red) along the length of each dog chromosome (CFA 1 to CFA 38) in 1 Mb intervals. The dashed horizontal red and green lines indicate thresholds of recurrence (≥33% incidence) and high recurrence (≥50% incidence).

Figure 7

Principal Component Analysis (PCA) to identify potential population substructure between French and American BMD cases of HS. A). Scree plots of the eigen values first 10 components. B). Three dimensional plot of the first three components. American BMD patients are indicated in red while French BMD patients are indicated in blue.

Figure 8

Principal Component Analysis (PCA) to assess global variation in aberration frequency between BMD and FCR. A) Scree plots of the eigenvalues first 10 components. B) Three dimensional plot of the first three components. BMD patients are indicated in red and FCR patients are indicated in blue.