VIDEO

Author Katherine Belov discusses her article, including the origins of the Devil Facial Tumor Disease (DFTD), a transmissible cancer that has already caused the disappearance of 85 percent of the species and could lead to its extinction in the wild within 25 years. She explains what is known of the tumor based on its genomics, why it is transmitted between animals without causing immune recognition in the devils, and what are the conservation efforts to save the species from extinction.

INTRODUCTION

The Tasmanian devil (Sarcophilus harrisii) is a carnivorous marsupial found only on the Australian island state of Tasmania. It has had a turbulent history over the past few thousand years, becoming extinct on mainland Australia (6) and surviving two speculated population crashes in Tasmania last century (5), though perhaps its toughest battle for survival is being fought right now. A fatal transmissible cancer, devil facial tumor disease (DFTD), is ravaging the devil population and threatening this species with extinction in the wild in the next 20–30 years (28). A better understanding of this cancer is urgently required to help save Australia's largest carnivorous marsupial. As in human cancer research, studies are focusing on a genomic characterization of the tumor to determine the genetic changes that have occurred to give rise to this disease.

Initial cytogenetic characterization of devil facial tumors (DFTs) showed that tumors from different individuals possessed the same rearranged karyotype, leading to the conclusion that the infectious agent responsible for the spread of this disease was actually the tumor itself. Tumor cells are transmitted as an allograft (37) by biting during feeding and mating (27). These cells evade immune detection, resulting in tumors arising in the new host. Several karyotypic strains of the tumor have been identified (39), suggesting that new subclones of DFTs have diverged since the disease arose in the original founder devil.

Unfortunately, no genome sequencing or even molecular cytogenetic work had been carried out on devils prior to the emergence of DFTD. Hence, the first step toward characterizing the disease was to gather basic information on the normal genome to which DFT genomes could be compared; only then would it be feasible to identify and characterize the mutations that have occurred in DFTD. Fortunately, the mainstream use of next-generation sequencing technologies has made this possible, and sequencing data for both reference and tumor genomes have rapidly been obtained. These data, when combined with molecular cytogenetic analysis, have enabled the first steps to be taken toward answering many of the questions that arose after DFTD's initial cytogenetic characterization. Here we review the advancements that have been made in the genomic characterization of DFTs, speculate on what these data indicate regarding the evolution of the disease and how the tumor is able to evade immune detection, and address the implications of this work for species conservation and management.

DEVIL FACIAL TUMOR DISEASE

DFTD was first observed in 1996 near Mount William in northeastern Tasmania (Figure 1) by a wildlife photographer. Since then, the disease has spread through 85% of the devil population, and it is predicted to lead to extinction in the wild within 20–30 years (26). Populations in eastern Tasmania have already been reduced by 95% (16).

The disease is spread by biting, which occurs regularly during mating and feeding over communal carcasses. Tumor cells pass between animals and grow unimpeded by the immune system, with only 7% of tumors containing infiltrating lymphocytes (22). In 65% of cases, primary tumors metastasize (22). The incubation period of the disease is not known, but once lesions appear devils usually die within six months owing to organ failure, secondary infection, or metabolic starvation (39).

Interestingly, the disease does not affect juvenile animals, and animals are affected only after one year of age, around the time they become sexually mature. The disease has had a major impact on the age structure of devil populations. Before the disease, devils lived in the wild for five years, breeding at two, three, four, and sometimes five years of age. Today, devils rarely breed more than once, with most succumbing to DFTD at approximately two or three years of age (19).

EVIDENCE TO SUPPORT ALLOGRAFT TRANSMISSION THEORY

DFTD is a transmissible cancer—an allograft. Proof of this came initially through cytogenetic analyses of tumors from 11 individuals. Pearse & Swift (37) showed that all tumors had identical chromosomal rearrangements, resulting in a karyotype consisting of 13 chromosomes. Chromosomes 1 and 6 and the sex chromosomes were missing from tumor karyotypes. However, four additional marker chromosomes (M1–M4) were present. Moreover, they showed that one devil host was heterozygous for a chromosome 5 inversion. The tumor did not contain this inversion, indicating that the tumor arose in an individual other than the host. This work was further supported by sequencing of major histocompatibility complex (MHC) alleles by Siddle et al. (44) and genotyping of microsatellite markers (35, 44). Both studies showed that the tumors had identical genotypes that differed from those of the hosts. These studies confirmed that DFTD could not have arisen in each devil independently; rather, the cancer arose once and was then passed from animal to animal as an allograft. More recent work including mitochondrial sequencing and single-nucleotide polymorphisms (SNPs) has added further support to these findings (31).

DFTD is only the second known transmissible cancer. The other is canine transmissible venereal tumor (CTVT), a sexually transmitted disease in dogs. CTVT is the oldest known malignant cell line, having been around for at least 6,000 years (40). CTVT is believed to have arisen in wolves or an east-Asian dog breed with low genetic diversity (36). Over time, CTVT evolved strategies to become a highly efficient parasite. It evades the immune response through downregulation of cell-surface MHC molecules and generation of immunosuppressive cytokines during the progressive phase of the disease (17, 18). However, CTVT does not usually kill its hosts; instead, MHC is upregulated during the regressive phase (53), and the host's immune system mounts both a humoral and a cell-mediated immune response, leaving the host with immunity for life and providing passive immunity to immunocompromised pups (8, 54). Survival of the parasitic cell line is ensured as dogs are more sexually receptive during the progressive phase of the disease (12).

DFTD is a young cell line in comparison with CTVT, raising the question of whether DFTD will eventually evolve to be less virulent to its host (3). A number of similarities and differences between DFTD and CTVT have been identified (2, 33), and comparisons of the genomic features of these two transmissible cancers will make it possible to determine how truly similar they are and whether DFTD is likely to follow a similar trajectory as CTVT to ensure its survival as a parasitic cell line.

Unlike human cancers, where finding a common target between individuals for vaccine development is challenging, DFTD's allograft mode of transmission means that it may be possible to identify a target for vaccine development (52) but at the same time increases the risk of triggering autoimmunity (3).

ESTABLISHING A REFERENCE DEVIL GENOME FOR DEVIL FACIAL TUMOR STUDIES

The obvious first step in DFT genomics research is to determine the changes that the devil genome has undergone in DFTs. Many known cancer-associated genes are tumorigenic as a result of genomic rearrangements that cause increased transcription, produce fusion genes, or affect the regulation of genes to alter their level of transcription (15). Copy number variation (CNV), where a genomic region is deleted or copied multiple times at one locus, also plays a major role. The driver mutations of cancer must affect key pathways to cause tumorigenesis. Hence, obtaining genomic rearrangement information is critical if the biological pathways perturbed in DFTD are to be uncovered.

Genome Sequencing

The advance of sequencing technology in recent years has made it possible to sequence a mammalian genome relatively cheaply and quickly, which is enabling devil research to rapidly forge ahead. The most challenging step for devil sequencing projects lies not in obtaining the sequence but in assembling it. Recently, the genomes of two devils from different locations in Tasmania were sequenced using a combination of two sequencing platforms (31). Spirit, a male from the Forestier Peninsula in southeastern Tasmania (Figure 1), had succumbed to DFTD that had metastasized. Cedric, born in captivity to parents from northwestern Tasmania (Figure 1), was originally thought to be resistant to DFTD, having shown signs of an immune response to vaccination with DFTD cells. Cedric did eventually succumb to the disease, but it was thought that sequencing individuals with such different responses to DFTD may reveal some important sequence variants that conferred resistance or delayed onset of the disease.

In this case, de novo assembly of the devil genome sequence data was considered a better option than using an existing marsupial genome assembly as a reference, as only two other marsupial genomes have been sequenced—those of the South American gray short-tailed opossum (Monodelphis domestica) (30) and the tammar wallaby (Macropus eugenii) (41), both of which are only distantly related to the devil. The de novo assembly was achieved by combining sequence data from both sequenced devils, resulting in an assembly spanning 3.3 Gb and 148,891 sequence supercontigs. However, Miller et al. (31) acknowledged that the de novo assembly of this type of data is too new a field for the generation of a definitive reference genome assembly. Nonetheless, the sequence data represent a valuable resource for identifying polymorphic sites within the genome to assess genetic variation across the devil population (31).

A second devil genome sequencing project was recently completed on a female devil using the Illumina platform for sequencing short and large insert libraries (34). Although the total number of bases covered for this project was similar to that achieved for the male genomes described above, the sequence has been assembled into larger contigs and supercontigs in the female assembly (Table 1). As part of a clever approach to assign sequences to chromosomes and improve the overall assembly, flow-sorted chromosomes were sequenced, permitting the assignment of 99% of supercontigs to chromosomes. Conservation with the opossum genome was used to order the supercontigs on devil chromosomes. One aspect of concern, though, is that this sequence was obtained from a fibroblast cell line rather than tissue. This cell line was trisomic for chromosome 6, which needs to be taken into consideration when using it as a reference genome. An additional male genome was also sequenced as part of this project, although not to the same depth as the female, and it was not included in the reference genome assembly (34).

Table 1

Genome assembly statistics for the male (31) and female (34) devil sequencing projects

Annotating genes within the reference genomes is essential for downstream comparisons with sequence from DFTs, and particularly for the identification of candidate genes involved in tumorigenesis. Miller et al. (31) used the opossum gene annotations to identify genes in the male genome assembly. Genes in the female reference assembly were identified using the Ensembl genome annotation pipeline and data from the sequencing of 12 devil transcriptomes, resulting in the annotation of 18,775 protein-coding genes and 363 microRNAs (34). Armed with this valuable information, researchers can now begin characterizing the genomic changes present in DFTD.

Devil Chromosomes and Anchoring Genome Sequence

The devil diploid genome is packaged into six pairs of autosomes and a pair of sex chromosomes, with XX females and XY males. Prior to the emergence of DFTD, only a very basic level of chromosome characterization had been performed to describe the size and morphology of devil chromosomes (25). However, the devil belongs to a family of marsupials known for their extremely well-conserved karyotypes (25, 43, 55), permitting molecular cytogenetic work on one species from this family to be easily translated to the devil. For instance, cross-species chromosome painting has been used to identify homologous chromosome segments in two other members of the Dasyuridae family, Sminthopsis macroura (13) and Sminthopsis crassicaudata (42). Homologous chromosome segments identified between dasyurid species and the chromosomes of the opossum and tammar wallaby enable predictions to be made about the gene composition of each devil chromosome.

Although predicting which genes are present on each devil chromosome is useful, it is essential that gene order on devil chromosomes be established for the reference genome. As mentioned above, sequencing of flow-sorted chromosomes has made it possible to assign sequences to chromosomes (34) but does not provide information on gene order. This has been achieved by constructing a cytogenetic map of the devil genome. Genes have been isolated from a bacterial artificial chromosome (BAC) library constructed using DNA extracted from one of the sequenced individuals (Spirit) and then mapped to normal devil chromosomes through fluorescence in situ hybridization (FISH). Genes were chosen from the ends of wallaby-opossum conserved gene blocks with the intention of generating a virtual map of the genome. A comparison of the order of 105 genes assigned to devil chromosomes revealed a surprising level of rearrangement among the devil, opossum, and wallaby genomes, making it difficult to construct a virtual map based solely on mapping the ends of wallaby-opossum conserved blocks (10). This level of rearrangement further confirmed the necessity of de novo assembly of devil genome sequence and the need for more detailed gene mapping for an anchored genome assembly, rather than relying on conservation with other marsupial genomes.

CHARACTERIZATION OF DEVIL FACIAL TUMORS

Since the chromosomes of DFTs were first described (37), there has been intense interest in the molecular characterization of DFTs, with the aim of identifying the mutations responsible for driving tumorigenesis. Sensitive molecular cytogenetic techniques have been used to identify the origins of DFT marker chromosomes. Chromosome painting was used to identify gross homologies between normal and DFT chromosomes, and gene mapping provided a higher level of resolution (10). Sequencing of DFTs has permitted an even more comprehensive analysis of DFT mutations. Three tumors have been sequenced: two from individuals captured in the Forestier Peninsula region in southeastern Tasmania (Spirit and 87T) (31, 34) and one from a lung metastasis sampled from a north-coast devil (53T) (34) (Figure 1). Both the cytogenetic and sequencing approaches have provided important insight into the characteristics of DFTs.

Devil Facial Tumor Chromosomes

Chromosome painting, a technique where flow-sorted chromosomes from a normal female devil were hybridized to DFT chromosomes, determined that the marker chromosomes of the first tumor strain described by Pearse & Swift (37) consisted largely of genetic material from chromosomes 1 and 5 and the X chromosome (10) (Figure 2). Gene mapping of the same 105 BACs used in the construction of the physical map of normal devil chromosomes supported this finding and provided a finer level of resolution of the chromosomal rearrangements in DFTD. The giant marker chromosome M1 is basically a rearranged chromosome 1 plus the addition of a small amount of X chromosome material to the short arm. The other copy of chromosome 1 appears split between M2 and M3; M2 contains genes mainly from the long arm of chromosome 1 and appears to have fused with one copy of chromosome 5 and some chromosome 4 and X genes, whereas M3 consists of one copy of the X chromosome fused to chromosome 1 short-arm genes. Gene mapping showed that M4, the smallest marker chromosome, was made up of genes from chromosomes 1 and 4, but chromosome painting detected homology to chromosome 5. This discrepancy between the two techniques may be due in part to the scarcity of chromosome 5 genes mapped overall (10). Further gene mapping is required to verify the gene content of this chromosome.

Conflicting with the original DFT karyotype reported by Pearse & Swift (37), both chromosome painting and mapping data identified two copies of chromosome 6 present in DFTD along with one intact copy of chromosome 5, with the other copy distributed across marker chromosomes (10). Reshuffling of gene order on both copies of chromosome 6 and the addition of a small amount of X chromosome material to one homolog would have made it difficult to accurately determine the identity of these chromosomes based solely on G-banding. Similarly, the size difference between the homologs of chromosome 2 was initially explained as a deletion from the long arm of one homolog, whereas chromosome painting and gene mapping were able to show that the size difference is due to the addition of chromosome 1 and X chromosome genes to the short arm of one homolog. The remaining chromosomes appear to have undergone very little change in the tumor based on assessment by cytogenetic techniques.

One of the interesting findings is that, unlike many human cancers, the DFTD genome appears to be largely diploid. Each chromosome is present in two copies, despite some of them being rearranged into marker chromosomes. Based on the resolution of FISH mapping, the exceptions to this are 12 genes that are present in one copy and three genes mapping to an additional location. It is interesting to note that most of these deleted genes are from chromosome 1 or the X chromosome, the two chromosomes most rearranged in the DFTs (10). Tumor sequencing supports this finding, with copy number analysis across the genome predominantly showing two copies, although selected regions have been subject to a deletion to one copy or an increase to three copies (34).

Genome Restructuring and Devil Facial Tumor Disease

Traditionally, tumors were thought to originate from the gradual accumulation of mutations, leading to changes in genes involved in cell growth, the apoptotic pathway, genome stability, and/or proto-oncogenes (50). It is becoming more apparent that a single cataclysmic event responsible for the generation of multiple concurrent mutations may also lead to tumorigenesis (29). One such recently described mechanism, termed chromothripsis, may be responsible for the origin of DFTD. Chromothripsis is a phenomenon whereby a chromosome segment, an entire chromosome, or several chromosomes are shattered and rejoined by the nonhomologous end-joining DNA repair mechanism, resulting in extensive rearrangements of limited regions of the genome (49). In DFTD, extensive chromosomal rearrangement has been limited to just three chromosomes (1, 4, and X). Furthermore, other signatures of chromothripsis are evident, such as very little change in copy number and the use of microhomology-mediated end joining in DFTs (10, 34).

Gene mapping data suggest that if DFTD did arise via a chromothripsis event, one copy of chromosome 1 was shattered into at least 16 pieces that then joined in a completely different order, with several chromosome fragments being lost (10) (Figure 3a). The fact that M1 is essentially a reordered chromosome 1 would suggest that this event may have taken place on its own and was perhaps the original event driving tumorigenesis. It is interesting to note that a large region corresponding to the proximal region on the long arm of chromosome 1 is preserved in gene order on M1 (10). Because this represents the only region protected from rearrangement, it is tempting to speculate that a shortening of telomere length may have played a role. Stephens et al. (49) suggested that breakage-fusion-bridge cycles associated with telomere loss could result in the extensive genomic restructuring characteristic of chromothripsis.

A similar event appears to have occurred for one copy of the X chromosome, shattering it into at least seven fragments, which have dispersed and joined five different chromosomes (Figure 3b). Chromosomes M2 and M3 are essentially derived from a break occurring just below the centromere on chromosome 1 and the joining of one half to one copy of the X chromosome and the other half to chromosome 5 (Figure 3c). Perhaps at least one copy of chromosome 5 and the X chromosome in the DFTD progenitor had lost their telomeres and fused to the broken chromosome 1 to rectify this problem.

Evolution of Devil Facial Tumors

Since the first report of the tumors from different individuals possessing the same karyotype, a number of different karyotypic strains have been discovered (28). These strains resemble the original DFT karyotype reported by Pearse & Swift (37) (designated strain 1) but are characterized by additional cytogenetic rearrangements consistent with ongoing tumor evolution as the disease continues to spread through the population (10). Two additional strains derived from primary DFTs sampled from individuals trapped in various locations throughout Tasmania (Figure 1) have been characterized in detail by chromosome painting and gene mapping to determine the distinguishing features of each. Strain 2 is characterized by an additional small marker chromosome (M5). Strain 3 karyotypes were a little more complicated than strains 1 and 2, with three different variants of strain 3 detected among four samples. Variations include the presence of the additional marker chromosome (M5) as found in strain 2, deletions of part of the short arm of one or both homologs of chromosome 3, and rearrangements of chromosome 4 and M2.

It is intriguing that differences between strains were minimal and limited to particular regions of the genome—namely, regions from chromosomes 4 and 5 and the X chromosome—whereas other regions appear stable, at least at the karyotypic level. Gene mapping would suggest that chromosome 1 is the most rearranged in DFTs, but it would appear that following the initial rearrangement, the chromosomal arrangement of chromosome 1 material has remained unchanged (10). The karyotypic strains described above are all actually subclones derived from the original DFT. It appears from both cytogenetic and sequencing analysis that DFTs are continuing to accumulate karyotypic, copy number, and sequence variants, but compared with most human cancers, DFTs are remarkably stable (10, 34). Perhaps selection is working to maintain the tumorigenic properties of the DFT genome, in particular the arrangement of M1, while permitting genomic instability and sequence substitutions in regions not critical for the survival of the DFT cell (10).

Assessment of the genetic diversity of DFT subclones suggests a linear radiation of DFTs, with a wide distribution of different subclones and the coexistence of different subclones within the same geographical locality (34) (Figure 1). In contrast, it appears that the DFT population on the Forestier Peninsula may have been subject to a selective sweep (34). This DFT lineage was founded by a single subclone that has diverged to give rise to several subclones found only within the Forestier Peninsula. This was followed by an increase in the prevalence of one subclone in the past few years. Intriguingly, the Forestier Peninsula was the site of a disease suppression trial, where animals infected with DFTD were culled in an attempt to manage the disease in the population (21). Culling of infected individuals had no impact on disease prevalence in the area (21) but may have led to severe selection pressure on DFTs, resulting in the selective sweep. The implication of these findings is that human intervention may actually be detrimental to devils, promoting the evolution of more virulent or faster-growing DFT subclones.

ORIGINS OF DEVIL FACIAL TUMOR DISEASE

The intriguing and unusual nature of DFTD, being a transmissible cancer, has sparked interest in its origins. Fortunately, molecular analysis of DFTs is permitting some questions regarding DFTD's origin to be answered, and although much remains unknown, an impressive effort has been made to determine its origin despite a lack of samples from the individual in which the disease arose.

The first sighting of DFTD was in 1996 in northeastern Tasmania (Figure 1), and it is presumed that the disease arose in an individual from this region (6). Sequencing of mitochondrial genomes from normal and tumor samples demonstrated that mitochondrial haplotypes of tumors were most similar to a common, widespread devil haplotype but was unable to further narrow down the geographical origin of DFTD (31, 34). However, sequencing of more than 100 DFTD genomes provided further evidence for the allograft theory of transmission as all were either identical to or derived from a single devil haplotype, presumably that of the founder (34). Evidence from molecular cytogenetic analysis and genome sequencing suggests that this founder individual was a female. A lack of both Y chromosome hybridization on DFTD chromosomes and SRY sequence in the tumor and the presence of two copies of most X chromosome genes support this theory (10, 34).

A reconstruction of the genome of this founder devil and DFTD progenitor has been attempted by identifying common sequence variants between two DFTD subclones (53T and 87T), allowing the exclusion of variants that arose within a subclone. A comparison of these common variants with two normal genomes determined that at least 80% of the 700,436 SNPs and more than 90% of the 251,257 common insertions/deletions (indels) are likely to be germline variants of the founder. The remaining variants would either be germline variants specific to the founder (i.e., not present in DFTs) or mutations acquired during DFTD progression (34).

Determining the tissue origin of DFTD proved challenging with traditional microscopic techniques, which described the tumor simply as a poorly differentiated soft-tissue malignancy (22). Immunohistological staining for a range of markers suggested a neuroendocrine origin (23). Sequencing of the DFTD transcriptome was able to further refine the cellular origin: A gene expression pattern resembling that of myelinating cells and, more specifically, expression of the Schwann cell–specific gene PRX encoding periaxin protein involved in the maintenance of peripheral nerve myelin suggested a Schwann cell origin (35). Intense and specific immunohistological detection of periaxin in nearly all DFT cells supported this theory and provided a diagnostic marker for DFTD (35, 51).

CANDIDATE GENES INVOLVED IN DEVIL FACIAL TUMOR DISEASE TUMORIGENESIS

There are many different ways a driver mutation for tumor development may arise. In some instances it can be a single amino acid change in a proto-oncogene, the movement of a tumor suppressor gene to be under the control of a different regulatory element, or a translocation resulting in the fusion of two gene products. In addition to the driver mutation(s), passenger mutations may also be present in the tumor genome. These mutations have not caused tumor development but may or may not play a role in tumor progression. Tumors accumulate many somatic passenger mutations, and separating the drivers from the passengers can be challenging. For DFTD, several different approaches have been taken to identify the candidate genes that may bear driver mutations (Table 2).

Table 2

Candidate genes involved in devil facial tumor disease

Sequencing of tumors has flagged several genes with nonsynonymous mutations as potential driver mutations. ANTXR1, a gene known to regulate the “guardian of the genome,” P53, is one such gene (31). Murchison et al. (34) also identified an amino acid substitution in the proto-oncogene RET, a tyrosine kinase expressed in the peripheral nerve system that has been linked to tumors derived from neural crest cells (38). A mutation resulting in an amino acid change has been identified in a member of the Fanconi anemia complementation group, FANCD2 (34), which plays a role in genome stability (11). Modifying amino acid changes in at least three genes (PRKCH, GALNS, and CCNA-like) linked to metabolic pathways associated with cancer in humans have been put forward as potentially perturbed pathways in DFTD warranting further investigation (31).

Homozygous deletions of at least two genes have been discovered in DFTD: MAST3 (a serine/threonine kinase) and BTNL9 (a member of the butyrophilin gene family). Unfortunately, little is known about the functions of these two genes, but there is some indication that BTNL9 may be involved in immune modulation (34). Gene mapping has identified genes involved in human cancers occurring near regions with hemizygous deletions in DFTD. The tumor suppressor gene NF2, a gene whose loss of function is associated with tumors of the central nervous system, particularly benign tumors of Schwann cells called schwannomas (1), stands out as a candidate gene given the Schwann cell origin of DFTD (10). Two other common tumor suppressor genes (APC and MLH1) have also been found close to deleted regions in DFTD (10).

Tumor sequencing has also uncovered several rearrangements involving genes associated with cancer, such as a balanced translocation involving the gene encoding platelet-derived growth factor alpha (PDGFA) (34). Interestingly, no in-frame fusion genes resulting from genome rearrangements have been detected in DFTD (34).

All of the candidates described above warrant further investigation to determine which mutation or mutations have acted as drivers of DFTD tumorigenesis.

HOW DOES DEVIL FACIAL TUMOR DISEASE EVADE IMMUNE RESPONSE?

Devils have low genetic diversity in the MHC (46). The MHC plays a key role in self/nonself discrimination and is the key region of the genome that is matched in donors and recipients for organ transplantation. We originally believed that DFTD was able to spread because of low diversity in the MHC of devils (46), and we predicted that devils would not be able to “see” DFT cells as foreign because the devils and tumors share the same MHC antigens. However, recent studies suggest that the tumor itself is capable of immune evasion (20).

The MHC is a dynamic region of the genome that evolves through gene duplication, deletion, and conversion. Therefore, different species have different numbers of MHC genes. The devil MHC has only recently been characterized by sequencing of BAC contigs from two individuals—Spirit and Cedric (7). Sequencing revealed up to five class I loci and four class II loci. Both class I and class II molecules could play a role in DFTD immune evasion (Table 3). MHC class I molecules are expressed on the surfaces of all nucleated cells and platelets and are involved in the presentation of endogenous peptides to CD8+ cytotoxic T cells. MHC class I molecules are composed of a heavy chain encoded within the MHC and a β₂ microglobulin encoded outside the MHC. Interestingly, three CNVs were observed between the two sequenced haplotypes within the MHC class I region. One of these deletions rendered the class I locus Saha-UA a pseudogene in Cedric. The effect of the other two indels is not known, but it is possible that they may impact gene expression by interrupting promoter regions. The role of these indels and CNVs in DFTD resistance needs to be investigated.

Table 3

Candidate proteins that may be involved in immune evasion in devil facial tumor disease

MHC class II molecules are found only on a subset of specialized antigen-presenting cells, including B cells, some macrophages and monocytes, Langerhans cells, and dendritic cells. MHC class II molecules present extracellular peptides to CD4⁺ helper cells, resulting in the generation of an appropriate immune response. MHC class II molecules are composed of an alpha chain and a beta chain, both of which are encoded by genes within the MHC. As with MHC class I, MHC class II diversity in devils is low (44, 45).

However, low MHC diversity alone does not fully explain the rapid spread of DFTD under the radar of the immune system. Recent skin graft experiments were performed between four devil pairs with highly similar MHC repertoires—three that were identical at their MHC peptide binding sites (typically highly polymorphic regions of MHC molecules) and one with only two amino acid mismatches within the peptide binding sites. This experiment showed that even MHC-similar devils are capable of allorecognition. Lymphocytes from the MHC-matched pairs were not able to trigger in vitro responses following mixed lymphocyte reaction assays, yet grafts in MHC-matched devils were still rejected. Rejection involved infiltration of CD3⁺ T cells into the graft site, a direct contrast to what is seen in DFTD (20). So what triggered allorecognition in these experiments? It is possible that allogeneic responses were directed against minor histocompatibility antigens (24, 32, 47, 48) or that the skin was more allogeneic (4) than a more immune-privileged Schwann cell. But it is equally likely that DFT cells have evolved a mechanism of immune evasion.

CTVT may provide some clues as to how DFT could have achieved this. During the progressive phase of CTVT, only 2%–5% of CTVT cells express MHC class I, and expression of β₂ microglobulin is low (9, 36). At this time immunosuppressive cytokines, including TGF-β1, are produced (17). Given that the CTVT cell line is genetically stable, it has been suggested that epigenetics may drive the transition from progressive to regressive phase (14). Whether a similar process also occurs in DFTD is not known. MHC class I transcripts are expressed in DFT cells (44), but to determine whether stable MHC antigens are found on the surfaces of DFT cells, specific immunological reagents need to be developed. This work is currently in progress. Table 3 provides other immune evasion candidate genes that are also under investigation.

POPULATION GENETICS

The sequencing of the devil genomes has allowed the identification of more than 1 million polymorphic sites in the devil genome, roughly a quarter of the diversity seen between two human genomes (31). This information can now be used to help manage the genetic diversity of devils in captivity as part of the devil insurance program—Australia's largest captive breeding program, which aims to capture and maintain all existing genetic diversity in captivity until it is safe to release devils back into the wild, after either the disease has run its course or a vaccine has been developed. A preliminary study of 1,536 SNPs in 87 devils across Tasmania was able to refine devil subpopulations from two to seven, and representation of individuals from each of these subpopulations in the program will help maximize the genetic diversity in captivity. Genomic technologies will help maintain this genetic diversity and minimize genetic adaptation to captivity over time, allowing us to release the most genetically fit population of devils back into the wild.

CONCLUSIONS

Traditionally, genomic approaches to disease characterization have been limited to humans and key model species owing to the prohibitive costs associated with data collection. However, advancements in genome technology are making it possible to use such approaches to study diseases in wildlife. Research into DFTD is leading this field owing to the urgency of the work—not only does the disease threaten the entire species with extinction, but the uniqueness of the cancer offers novel insights into tumor transmission and stability as well as mechanisms of immune evasion that may improve our understanding of human cancer.

The latest technologies in genomics and molecular cytogenetics have allowed us to rapidly gain a vast amount of knowledge about DFTs, but we still have a long way to go. Devils are likely to go extinct in the wild within the next 25 years unless we can find a way to treat the disease, develop a vaccine, or isolate devils from DFTD. Genomics offers hope of identifying gene targets for therapies and helping to identify novel tumor antigens for use in a vaccine.

Beyond an understanding of DFTD, comparative genomics provides hope of identifying novel pathways and/or genes involved in human tumorigenesis (Figure 4). DFTD arose once and, rather than dying with its host, has been continuously passed through different individuals across different host genotypes in different geographic locations. Tracing evolutionary changes in DFTs over time and across different genetic backgrounds should allow us to pinpoint how the tumor managed to remain so stable, maintaining its transmissibility and viability. Do the stable chromosomes hold the clues? Finally, DFTD will also provide new insights into the evolution of more aggressive cancers, especially following immune therapies, through the discovery of new mechanisms of immune evasion.

FUTURE ISSUES

disclosure statement

The authors are not aware of any affiliations, memberships, funding, or financial holdings that might be perceived as affecting the objectivity of this review.