We hypothesized that spontaneous canine HS exhibit recurrent CNAs of genes involved in histiocytic cancerization, and that identification of these CNAs may advance our understanding of the molecular characteristics of these cancers in both canine and human patients. The pathophysiologies of several dog and human cancers share many similarities and our previous studies have demonstrated that CNAs in a variety of human cancers are evolutionarily conserved in the corresponding canine cancer [
40‐
42]. These findings support the idea of a fundamental and evolutionarily conserved association between cytogenetic abnormalities and tumor phenotype, indicating similar biological consequences in both species [
41]. In testing our hypothesis we recruited client owned BMD and FCR patients, each with a confirmed diagnosis of HS, and performed genome-integrated aCGH analysis of 104 cases to identify recurrent CNAs at 1 Mb resolution. We also performed statistical analysis of clinical and demographic data from our HS cohort in order to expand knowledge of the epidemiological basis of this disease in these breeds.
Disseminated canine HS was first described as malignant histiocytosis in the BMD [
19,
20], and while this condition has been documented in other breeds [
17], it appears that this clinical form continues to be reported more frequently in the BMD than other breeds. Conversely, Fidel et al. (2006) [
25] described that the majority of HS in the FCR were restricted to a joint and/or muscle/skin, corresponding to a localized form of HS. Epidemiological data from our cohort are consistent with previous reports. Our data showed also that the anatomical location of the histiocytic tumors differed significantly between the two breeds investigated: BMDs present more frequently with tumors of internal organs and also with a high frequency of dissemination, while FCRs more often develop a localized tumor of the skin or leg. To our knowledge this study represents the first to provide statistical significance for these parameters. The contrasting patterns of anatomical location of HS in the BMD and FCR suggest that the genetic backgrounds of these two breeds may play a key role in determining risk, location and progression of this neoplasm, which could be assessed using genome wide association analyses. Prior human and mouse studies have identified specialized DC subtypes with heterogeneous functions [
43] and so it is possible that the HS diagnosed in FCR and BMD represent malignancies of different DC subtypes, which might explain the different behavior of these cancers in the two breeds.
Identification of breed-associated genomic copy number aberrations
No statistical differences were found between CNAs detected in HS of BMDs from two distinct geographic areas (France/USA). These data suggest that it is reasonable to sample BMDs from different geographic areas to increase the number of cases available for subsequent statistical analyses. This is not surprising considering the BMD has its roots in Switzerland in the late 19
th century, was admitted to the AKC registry in 1937, and experienced numerous international 'line-exchanges' over the ensuing 74 years. These data suggest a relatively homogeneous international population, supporting the conclusion of Quignon et al. [
44] who proposed the use of international BMD cohorts for genetic studies. From a population genetics perspective, since the main populations of BMD are located in the USA and Europe, and there are no apparent differences in CNAs evident at 1 Mb resolution between the two BMD populations, we surmise that the genomic changes associated with HS are common to all BMDs regardless of geographic origin. This in turn may indicate that any risk factors for the development and progression of HS are linked tightly to the genetic makeup of the breed and independent of geography. Advances in understanding of the biological mechanism of HS in BMDs in the USA should therefore apply also to BMDs in other countries, extending the value of such studies.
Most of the CNAs identified in this study were common to both the BMD and the FCR, and so it is likely that such aberrations may also be evident in other breeds presenting with these malignancies. We identified 13 regions of the genome on seven chromosomes that showed a significant association between DNA copy number and breed of the patient (Table
1). Further, PCA indicated a significant association between specific CNAs and either breed (BMD/FCR) or anatomical location(s) of the tumor(s). Since breed and tumor location are so closely correlated, it is not possible from this study to determine whether the association between CNAs and breed was driven by breed itself or by the anatomical location of the tumor in that breed. Aberrations may be associated with location of affected organ/tissue, and/or dissemination/metastatic nature of HS. These aberrations could confer a proliferative advantage to the tumor in one particular organ, or elevate the risk of metastasis. An alternative hypothesis is that some of these aberrations are linked specifically with the genetic background of each breed. Since it has been shown previously that individual genetic backgrounds, as defined by breed in dogs, influence tumor karyotypes [
40,
45], we could hypothesize that some pathways are inactivated by germline mutations in one breed, creating a genetic risk for HS, but are inactivated by somatic modifications (genomic loss, mutation) in the second breed. Evaluation of HS in additional breeds that also present with a disseminated form of the disease, such as the Rottweiler, will aid in determining whether the apparent separation between BMD and FCR is driven by breed or by anatomical features of the cancer [
9].
Identification of highly recurrent CNAs shared by the FCR and the BMD - candidate regions for human HS
Despite the high level of genome reorganization evident in canine HS and the varying anatomical location of the tumors between breeds, we identified numerous CNAs within our sample population shared between the both breeds. Several of these were classified as recurrent (≥30% frequency) or highly recurrent (≥50% frequency). Among the most highly recurrent CNAs detected were loss of regions of CFA 2, CFA 11, CFA 16, CFA 22 and CFA 31, all of which were highly frequent (50-86%) in both breeds. The presence of highly recurrent abnormalities common to both breeds, along with their presence in both the localized and disseminated forms of HS, is suggestive of an association more with the cancer phenotype than with breed. These regions likely contain genes that may play a key role in malignant transformation of histiocytes, independent of anatomical location. This is especially so for the most frequent CNA, deletion of CFA 16, an aberration that was detected in 86% of HS cases (80.4% of BMD, 96.7% of FCR).
At first glance many of the recurrent aberrations identified in this study involved large contiguous tracts of the canine genome (Figures
4 and
5), and so identification of candidate genes is challenging. Closer consideration of subchromosomal differences in aberration frequency may however be used to determine minimal regions of interest. For example, Figures
4 and
5 indicate that while the full length of CFA 16 is deleted in at least 50% of all HS cases (both FCR and BMD), there are regional differences in the frequency of deletion along the length of the chromosome. The highest frequency of recurrent deletion (86%) along CFA 16 involved a 6 Mb region (47-53 Mb) towards the telomeric end of the chromosome. There are several annotated candidate genes within this region of the canine genome
http://genome.ucsc.edu/cgi-bin/hgGateway?db=canFam2 that are known either to be involved in regulation of apoptosis, or which are suspected to be tumor suppressor genes; including
CDKN2A interacting protein (CDKN2AIP),
FAT tumor suppressor homolog1 (
FAT1),
Tumor suppressor candidate 3 (
TUSC3),
Mitochondrial Tumor Suppressor gene 1 (
MTUS1) and
pericentriolar material-1 (
PCM1). Of comparative significance specific to HS,
CDKN2AIP is known to interact with
CDKN2A/p14ARF, TP53/p53 and
MDM2, all of which have been shown to be involved in human histiocytic disorders [
4,
8,
9,
11‐
14,
46] and so this merits further investigation in future studies.
Similarly, a neighboring 2.4 Mb region of CFA 16 (41.8 Mb-44.2 Mb) was deleted in 84.9% of HS cases. Of possible comparative significance, this region of CFA 16 is in part orthologous to human chromosome (HSA) 8p22-p21.3, a region that is frequently deleted in numerous tumors, including multiple myeloma, prostate cancer, hepatocellular carcinoma and neck squamous cell carcinoma [
47‐
50]. These data indicate that in both human and canine cancers, this region exhibits a strikingly high and comparable level of CNA. Further evaluation of both human and canine patients will be required to determine whether this shared deletion contains genes and regulatory elements associated with HS, or if its presence is merely a generalized passenger aberration.
aCGH profiling of canine HS suggests that disruption of the p53 and Rb pathways is a common event
Segments of CFA 11 (q22), 22 (q11) and 26 (q25) all showed a high incidence of copy number loss in canine HS. Each of these three regions contain key cancer associated genes involved in the p53 and Rb pathways:
CDKN2A/B (CFA 11q22),
RB1 (CFA 22q11) and
PTEN (CFA 26q25).
CDKN2 encodes three distinct tumor suppressor genes (
ARF, p15
INK4b
p16
INK4a
) that code for proteins regulating cell cycle progression via the Rb and p53 pathways. While p15
INK4b and p16
INK4a regulate the Rb-pathway, ARF inactivates MDM2 protein and so regulates p53 [
51,
52]. The human region orthologous to CFA 11q22 is HSA 9p21, which is among the most frequent sites of DNA copy number loss in human cancers [
53]. Genes within this region, especially
p16
INK4a
, have also been shown to be inactivated in several dog cancers including lymphoma, melanoma, hemangiosarcoma and osteosarcoma [
42,
54‐
59]. Since direct inactivation of
p16
INK4a
by point mutation, deletion or promoter methylation is evident in approximately one third of human hematopoietic tumors [
53,
60], it is not surprising to find this locus is involved in human histiocytic disorders. Significantly, monosomy of HSA 9, including the
CDKN2 locus, has been observed in different human dendritic proliferations including plasmacytoid DC sarcoma [
8] and follicular DC sarcoma [
9]. Moreover deletion of HSA 9p has been reported as the second most frequently observed aberration in Langerhans cell histiocytosis (LCH) of the lung [
10]. In mice, loss of
INK4a allows macrophages to bypass senescence [
61] and Pten and Ink4a/Arf have a cooperative role in restricting macrophage growth. The same is true in human HS where inactivation of
PTEN and
INK4a/ARF tumor suppressors are critical steps in the pathogenesis of this cancer [
4,
11]. It is therefore of interest that in this study >50% of HS cases presented with a deletion of the
CDKN2 locus. Other mechanisms, such as DNA sequence mutations or methylation, may also inactivate these tumor suppressor genes. In future studies it will be important to investigate whether HS cases presenting with no apparent deletion of
CDKN2 have an increased rate of DNA sequence mutation of this locus, resulting in aberrant expression for reasons other than gene dosage.
Also belonging to these key pathways is the gene
Retinoblastoma 1 (
RB1), which is disrupted in a variety of human solid tumors including pituitary adenomas, esophageal carcinoma, gliomas and ovarian cancer [
62]. Deletion of HSA 13q14, containing
RB1, is also a common event in a wide variety of acute/chronic myeloid disorders as well as in human dendritic sarcomas (plasmacytoid DC and follicular DC sarcomas) [
8,
63]. In our study, loss of the
RB1 locus on CFA 22 was highly recurrent across the cohort (55.8% of HS cases) although it was detected twice as frequently in HS tumors of FCRs than of BMDs (83.3% of FCR cases vs 41.1% of BMD cases).
Deletion of HSA 17p, containing
TP53, has been described in human LCH [
10,
12], while other studies reported an elevated expression of p53 in this disease [
13,
14]. In the present study, 36% of canine HS cases demonstrated CNA of
TP53, representing copy number loss in 9.3% cases and gain in 26.7% of cases. Further studies are needed to assess if gene dosage of
TP53, as well as other mechanisms, result in altered expression of p53 that may be correlated with elevated expression of this protein in Langerhans cell proliferation.
Deletion of CFA26 was present in approximately 20% of canine HS cases, but the telomeric end of this chromosome, a region encoding the
PTEN locus, was deleted in ~41% of cases, regardless of breed.
PTEN plays a significant role in inducing cycle arrest and programming apoptosis. It is an antagonist of the
PI3K/AKT pathway, and in turn regulates the
Rb pathway [
64]. It also controls p53 protein levels and transcriptional activity through both phosphatase-dependent and independent mechanisms [
65].
PTEN has been shown to be deleted or mutated in a wide range of human tumors [
64] and also in several canine tumors [
58,
66]. While
PTEN influences p53 transcriptional activity and p53 stability [
65], no association was found in our data between gain/loss of
TP53 and
PTEN loss (Fisher's Exact test, data not shown).
Copy number aberrations common to both breeds, combined with their likely consequential impact on the same pathways in human and canine HS, support the relevance of the dog as a model of HS. In addition to the genes discussed above we suspect that other tumor suppressors on CFA 2, CFA 16 and CFA 31 (deleted in 50%, 86% and 61.6% of HS tumors, respectively) also may play an important role in histiocytic cancerization. Their identity likely will become apparent with increased resolution and functional analysis of genes within these regions.