Our results, based on several independent genetic markers in tumor-bearing dogs living on five continents, show that CTVT arose from a common ancestral neoplastic cell. Early in its evolution, the clone diverged into two subclades, each of which now has a broad geographic distribution. Many breeds of dog tend to be homozygous for DLA class II genes (Kennedy et al., 2002a
), and CTVT is also homozygous for these genes when they are diploid as in subclade 1. Our microsatellite and DLA typing indicate that CTVT first arose in a wolf or in a dog related to the “old” East Asian breeds.
The precise date when CTVT first occurred is difficult to determine. From its indistinguishable histopathology and its ability to grow as an allograft, it is likely that Novinski (1876)
studied the same clone, and CTVT could have become established centuries before this date. Our analysis of divergence of microsatellites indicates that the tumor arose between 200 and 2500 years ago. Whether this time period represents the time the tumor first arose or whether it represents a later bottleneck in the tumor’s dispersion as a parasite cannot be resolved. While this estimated date indicates a relatively recent evolutionary origin, CTVT represents the oldest known mammalian somatic cell in continuous propagation, having undergone countless mitoses and host-to-host transfers.
Although the tumor is highly aneuploid, the karyotype is remarkably constant in tumors from the United States, Kenya, and Japan (Murray et al., 1969
; Oshimura et al., 1973
; Weber et al., 1965
). Therefore, its genome diversity at the chromosomal level appears to have stabilized early in its emergence as a transmissible parasite, and our studies revealed only moderate diversification of microsatellite DNA sequences. CTVT has active telomerase (Chu et al., 2001
), and we surmise that if telomerase activation occurred after the generation of aneuploidy, the subsequent maintenance of the remaining telomeres may have stabilized the abnormal karyotype. Long-established human tumor cell lines, such as HeLa cells, may be similar in this regard. Other than expression of c-myc
(Katzir et al., 1987
), activation of oncogenes and deletion of tumor-suppressor genes have not yet been studied in CTVT.
Based on our analysis of 73 widely dispersed microsatellites, there is no evidence of significant genome loss or progressive genome instability in this longest lived of all known tumor clones. CTVT does not appear to exhibit a mutator phenotype (Raptis and Bapat, 2006
) in terms of microsatellite instability, and neither does it exhibit progressive chromosome instability (Brumer et al., 2006
) following the gross rearrangements early in its emergence.
Both naturally and experimentally transplanted CTVT exhibit an initial stage of rapid and progressive growth, which is typically followed by spontaneous regression 3 to 9 months later, unless the dog is elderly, is in poor condition, or is immunosuppressed (Cohen, 1985
). After tumor regression, the host is immune to rechallenge, and passive transfer of serum from a recovered dog also confers immunity. Experimentally, CTVT can be transplanted into immunocompetent animals of other canine species, such as foxes, coyotes, and jackals (Cohen, 1985
), as well as into immunodeficient mice (Harmelin et al., 2001
; Holmes, 1981
). CTVT is a histiocytic tumor (Marchal et al., 1997
), and histiocytic tumors with markers of the myeloid dendritic cell lineage that express DLA class II antigens are relatively frequent in dogs of several breeds (Affolter and Moore, 2002
). What has led a single clone to become sexually transmissible as an allograft remains obscure. A recent study (Hsiao et al., 2004
) shows that, during progressive growth, secretion of TGF-β1 by CTVT acts as a potent local inhibitor of host immune responses, as does the downmodulation of DLA class I and II expression observed by us and others (Cohen et al., 1984
). Thus, the evasion of host immune responses has enabled the tumor to survive and grow until it can be further transmitted.
Allorecognition of nonself from self predates the evolution of the highly polymorphic MHC system and is seen in yeast mating types, sponges, and cellular slime molds. However, natural chimeras (Buss, 1982
) do occur in metazoans including colonial urochordates (Rinkevich, 2004
), and CTVT can be regarded a special case of somatic cell chimerism. The driving selection for the evolution of the MHC system and cell-mediated adaptive immunity in early jawed vertebrates may have been as much to protect against malignancy as to protect against infectious disease because invasive and metastatic tumors develop only in vertebrates, whereas infections are universal. Although recent discussion of cancer immunosurveillance has focused on recognition within the host (Dunn et al., 2002
), the rejection of malignant allografts may have been a factor in MHC evolution. Nonetheless, CTVT has evolved into a cellular parasite that has gained independence from and long outlived its original host. Since CTVT is an asexually reproducing cell that cannot “cleanse” its genome of accumulated deleterious mutations through recombination, it may be expected that, over evolutionary time, its genome may suffer from slow degradation through the process of Muller’s ratchet (Muller, 1964
). However, there is no evidence that Muller’s ratchet has yet exerted an effect.
In humans, occult tumors in donor organs have emerged on rare occasions in immunosuppressed transplant recipients (Barozzi et al., 2003
; Kauffman et al., 2002
; MacKie et al., 2003
), and choriocarcinoma represents a malignant version of the hemiallogeneic fetal trophoblast. We are not aware of any reports on the sexual transmission of tumor cells (for example, prostate or cervical carcinoma) between humans, but the possibility merits investigation in transplant recipients and immunodeficient individuals with AIDS. Cohen (1985)
suggested that the emergence of CTVT may have been favored because of the copulatory and postcoital tie in canid species that provides a tight contact between injured vaginal and penile mucosae for a sufficient time to allow the implantation of tumor cells. It is not evident from our data whether the “infective dosage” is a single cell or a bolus of tumor tissue; the latter seems more likely from a report (Holmes, 1981
) that only ~13% of experimentally injected tumor cells survive to develop into a tumor.
Given that MHC expression is downregulated in many tumors (Khong and Restifo, 2002
), it is not clear why parasitic tumors have not emerged more frequently. However, the natural transmissibility of CTVT does not appear to be unique. Based on karyotype, a transmissible tumor was reported in a colony of Syrian hamsters (Cooper et al., 1964
) and can even be transmitted via mosquitoes (Banfield et al., 1965
); like CTVT, this tumor is histiocytic. The recent emergence of a contagious tumor spread by biting in the Tasmanian devil (Pearse and Swift, 2006
) also appears to represent an example of cellular transmission according to karyotype, although a definitive analysis based on DNA markers such as we used for CTVT is awaited.
As a sexually transmitted cell, CTVT would not have been able to colonize dogs worldwide if it killed them too quickly; the host must survive in a fit state long enough to transmit the tumor, which in the case of females probably entails an estrous cycle. Thus, it will be interesting to model the restraints preventing the emergence of more aggressive subclones within the host and whether epigenetic factors affect the progressive and regressive phases of tumor growth. CTVT cells with their stabilized genomes may reflect kinship selection and reduced virulence, thus aiding host survival and onward tumor transmission (Frank, 1996
), whereas the evolutionary dynamics of a “selfish,” dead-end tumor typically progresses toward greater autonomy and malignancy (Greaves, 2002
; Michor et al., 2004
In contrast to CTVT, the Tasmanian devil facial tumor is highly virulent, killing most of the affected animals by obstructing their ability to feed (Pearse and Swift, 2006
). If the devil facial tumor does not eradicate its entire host population, it will be interesting to investigate whether the newly emerged tumor cell lineage eventually evolves toward a less aggressive phenotype.
In the hamster and Tasmanian devil examples, the tumors spread among animals that have little genetic diversity (Cooper et al., 1964
; Jones et al., 2004
; Owen and Pemberton, 2006
). The fact that CTVT is nearly homozygous in each of the DLA class II loci and also has closely related class I alleles may similarly have facilitated the origin and spread of CTVT within a partially inbred population, but today its chief reservoir is among mixed-breed dogs, particularly strays. Thus, CTVT is not a temporary, localized outbreak within a high-kinship group of animals; rather, it represents the evolution of a cancer cell into a successful parasite of worldwide distribution.