In dogs cancer is the most common cause of non-traumatic death [1]. Several types of canine malignant neoplasms—based upon pathobiology—can serve as useful models for rare cancers in humans, including osteosarcomas (OS) and soft tissue sarcomas (STS) [2,3,4], both types being relatively common in the dog [5,6].

As in humans, the prognosis of canine tumor patients is related to tumor location and, for malignant tumors, clinical stage [7]. A key factor in this assessment is the histological phenotype including malignancy grade [4,8,9], but there is a significant morphological overlap between malignant, benign, and reactive mesenchymal lesions in the human [10,11]. For both species, classification and management of mesenchymal tumors is complicated by the fact that these form a very heterogeneous group [9,12]. In order to improve the prognostic value of histological examination of Malignant Tumor of Bone (MTB) and of STS, several classification and grading systems have been established in humans [13], and later adopted for use in the dog [8,9], but the prognostic value of these systems for many individual tumors is limited [4,9,14]. Therefore, objective prognostic criteria are urgently needed.

Determination of the DNA-ploidy status may help to discriminate non-neoplastic or benign neoplastic lesions from (pre)malignant neoplastic conditions [15], including lesions of mesenchymal origin [16,17,18,19]. In humans, DNA-aneuploidy is more common in high-grade sarcomas than in those of low- or intermediate-grade [16,18,20,21]. These potential discriminatory values still need to be examined in dogs. In addition, DNA-aneuploidy has been reported to have prognostic value in several cancer types in humans [22,23,24]. However, in some sarcoma subtypes, this is not always the case [16,21,25,26,27,28,29,30,31,32].

Besides the diagnostic value that the DNA-ploidy status can have, the DNA index (DI) distribution may help to comprehend the nature of genomic changes during tumorigenesis [33,34,35]. It reflects gross chromosomal changes [36] that, as already hypothesized one century ago [37], appear to play a mechanistic role in tumorigenesis, the extent of which is subject of debate [33,34,35,36,38,39,40,41,42,43,44,45,46,47,48,49]. In approximately two-thirds of human malignant solid cancers, the evolution of karyotypic alterations over many cell divisions until cancer clinically manifests, is thought to be reflected by the increase of total nuclear DNA content, which peaks at 1.6-fold of the normal amount [33,34,35,50]. In the other one-third of human cancers, the deviation of the DI is much less prominent, or cannot be discriminated from normal due to only minute or balanced chromosomal changes [15]. In general, a significant decrease in the DNA-content or DNA-hypoploidy is rare in most human solid cancers, with a few exceptions such as chondrosarcomas [51].

Loss of function of the P53 pathway has been hypothesized to be one important factor in the development of aneuploid cancers [52]. As in humans, most cancers in dogs, such as thyroid and mammary carcinomas, are DNA-aneuploid, but the extent of the aberration of the DI in DNA-aneuploid cancers is less and DNA-hypoploidy more common in such carcinomas in the dog than in humans [53,54,55]. In fact, these observations led us to hypothesize that there is an interspecies evolutionary variation in the manifestation of DNA-aneuploidy in tumors, when comparing humans and dogs [44].

In continuation with our earlier research [53,54,55], we now examined the DNA-ploidy distribution pattern as determined by flow cytometry (FCM) in a series of fresh frozen samples of canine benign and malignant mesenchymal lesions. Preliminary results of this study have been presented as poster at the ESF conference (ESF Conference, Dresden, March 2010). For sarcomas, the data were compared with information on clinical stage, histological subtype and grade and the mutational status of the P53 gene, and with published data in human sarcomas. Comparison of observations in the current and earlier studies on canine neoplasms to those published on human cancers, points to interspecies variation in the driving force of aneuploidy in the development of malignant tumors.

Our study indicates that the existence of aneuploidy in canine mesenchymal proliferative lesions is suggestive but no proof of a malignancy, in accordance with observations in humans [16,58,59,60], although it must be recognized that a diploid status does not rule out malignancy. This finding may lead to additional cytological/histological testing of canine mesenchymal lesions in which the results of routine diagnosis remain ambiguous.

For sarcomas in humans, a relationship between histological grade of malignancy and ploidy pattern has often been reported [16,20,59,61], but no such relationship was evident in our study. In part, this may be related to the relatively high frequency of hypoploid cancers, and the related presence of smaller nuclei, which might be judged lower grade by pathologists.

As in earlier studies in mammary malignant tumors in dogs [22,53,55] and in humans [22,23,30], we were also able to demonstrate a relation between ploidy status and risk of metastases in STS. Many studies in humans [28,30,31,62] reported a similar correlation, although this is not universal [58]. Striking was the absolute lack of a relation between ploidy status and risk of metastasis in OS, which is in contrast to OS in humans [32,63]. In diploid cases within particular types of cancer more detailed cytogenetic analyses are required to discern those changes that predict metastatic behavior [62,64].

Consistent with early research [65,66,67], clear similarities were found with respect to aneuploidy occurrence amongst STS (53%) and MTB (57%) when compared to such sarcomas in humans [50,58,60]. For the subtypes of MPNST and leiomyosarcoma a diploid status is common in both dogs (as seen in our study) and humans [68]. However, in most human sarcomas hypodiploidy is rare [10,50], with the highest reported rate being 11% [58], while chondrosarcomas are an exception since they are frequently hypoploid [69]. We observed a remarkable overall hypoploidy incidence of 26% (48% of aneuploid cases). One other study in canine OS reported a somewhat lower figure [67]. In addition, both studies indicate that for many subtypes of canine sarcomas the net increase in DNA content in hyperploid cases is modest when compared to their human counterparts [16,58,70] albeit that some sarcoma types, such as the three liposarcomas in our study, form an exception, since these were all (hyper)tetraploid.

Since similar observations have been made in other types of cancer in the dog such as thyroid and mammary carcinomas and malignant lymphomas [53,54,56,57], it seems that the dog is particularly prone to the development of aneuploid tumors associated with either chromosome loss or low number chromosome gain, and less frequently to hypertriploid tumors that are common in humans [33,34,40,50]. This feature seems at variance with ploidy evolution patterns described in humans. In humans, this evolution can be divided in stages with early on the development of tetraploidy which is followed by chromosome loss and later on leads to a major proportion (amongst aneuploid cases) of cancers manifesting an increase in DI of approximately 1.6–1.7 [33,34,35,40,45,50]. Still, in a few sarcomas of the current study and in mammary carcinomas [53,65] tetraploidization (in the primary cancer) followed by an appearance of hypotetraploid stemlines (in its metastases) has been observed. A possible explanation for this difference in ploidy evolution could be the presence of a more powerful defense mechanism in humans against the tumorigenic effects of hypodiploidy or low level hyperploidy [33,71]. While for many human cancers a greater destabilization over multiple phases of destabilizing events seems necessary to arrive at a fully malignant state, such state may be reached with less destabilization in the dog, as hypothesized earlier [44]. How some cancers can reach at a fully malignant state while remaining DNA-diploid is uncertain. More subtle and sometimes balanced chromosomal gains and losses have been observed by cytogenetic analysis in some such human cancers [34] including sarcomas [19,68], and also in canine sarcomas [72,73] and carcinomas [74]. In other human diploid cancers however, structural chromosomal abnormalities are absent, and the transformation to malignancy seems to be driven by defects in DNA repair pathways leading to microsatellite instability, with microsatellite instability and aneuploidy being mutually exclusive phenomena [46].

It must be recognized that the karyotypic alterations that occur in dog cancer are to some extent at variance with those described for human cancers. The autosomes in normal dog cells are acrocentric/telocentric, and changes in canine cancers often concern centric fusions [74,75,76,77] albeit that alterations such as trisomies or monosomies and translocations are also frequent [72,75,78,79] as well as smaller structural abnormalities that have been detected with comparative genomic hybridization studies [73,79,80,81] in the past few years. Still, as mentioned in an extensive review by Breen and Thomas, “tumors of the same histological types in both species present with equivalent cytogenetic lesions” [82].

In cancers that harbour significant chromosomal alterations, even if not recognized as DNA-aneuploid, the cause underlying this chromosomal instability has been subject of study and aberrations in many pathways, in particular those related to sister chromatid cohesion and segregation have been suggested as possible causes [46,47,49,83,84,85]. Contrasting views exist as to whether loss of P53 function is essential for the development of aneuploidy [52,86,87,88]. In the current study, no relation between P53 mutations and DNA-ploidy status was found. Although it must be recognized that analysis for P53 mutations concerns only part of the many elements involved in the P53 pathway, our results are in clear contrast with a study in human sarcomas that demonstrated a relation between the presence of P53 mutations and DNA-aneuploidy [61].

Regarding whether and how aneuploidy may be essential for the development of cancer [38] it is essential to agree on the definition of malignancy. Most oncologists agree that proof of metastatic potential is not the only prerequisite for a tumor to be considered malignant and that tumors with extensive infiltrative destructive growth and low or late risk of metastases such as leiomyosarcomas and MPNSTs in the dog should also be regarded as malignant [7]. In our study, these sarcomas were only rarely found to be aneuploid. There is no doubt that many malignancies with full malignant potential are aneuploid, reflecting gross quantitative genomic destabilization. Several major disturbances must have taken place in order to overcome restraints, which are—in part tissue specific—against malignant transformation. Other cancers, such as the diploid OS in the current study, can reach this state of progression with much less genome destabilization. Functional changes that may allow malignant behavior in such cancers may include P53 inactivation or MDM2 amplification [89,90] as well as many other genetic alterations. Such changes may be called high hierarchy changes. This is opposite to many more subtle low hierarchy changes that can accumulate during progressive genomic destabilization and, by chance or number, can also disrupt crucial control mechanisms. To some extent and for some types of sarcomas (such as MPNST and leiomyosarcoma), a diploid status is then related to low metastatic potential. For OS in the dog a highly metastatic state can be either reached by appearance of a relatively low number of high hierarchy genetic changes, and low level of genomic destabilization in the form of chromosomal instability, while in others a high level of genomic destabilization, involving more lower hierarchy changes, may lead to this state.

The debate on gene mutation versus aneuploidization as cause of cancer cannot be resolved, since both phenomena may be linked and cooperative in tumor development [36,48].

Once a malignant tumor has developed, the same DI can often be found in both the primary and the metastatic lesion. Sometimes, haploidization or duplication of such stemlines occurs, with variation between metastases. In other tumors in the current and earlier studies [53,65,67] stemlines were found which really looked like an unrelated clone, able to propagate as independent subpopulation, as hypothesized previously [91].

In conclusion, aneuploidy is frequent in various canine malignant lesions, with prominent variation amongst the different sarcoma types. The overall frequency of aneuploidy is largely in accordance with findings in humans [50]. However, the high frequency of tumors with hypoploidy and low-level hyperploidy in various canine cancers [53,54,57] when compared to human malignancies including sarcomas [50], is striking. In human solid cancers hypoploidy is rare and the majority of cases are hypertriploid thought to arise from a tetraploid intermediate (33–35, 46, 50). Our findings therefore are suggestive of interspecies variation in aneuploidy evolution and genomic destabilization during carcinogenesis.