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Chapter 3 - The allograft theory

3.5 A comparison between CTVT and DFTD allograft programs and undone science undone science

3.5.2 A chronology of recent CTVT studies

In 2000 Utpal and Arup Das published a review of studies into Canine Transmissible Venereal Sarcoma (a variant name for CTVT). In relation to the CTVT studies as to its type, their review cites the following studies - the 1905 study by Bashford and colleagues who concluded that CTVT was not a sarcoma, but an infective granuloma; a study by Sticker in 1906 incorrectly calling it a ‘contagious lymphoma’; and Feldman in 1929 associated the forceful nature of sexual intercourse between dogs and genital

24 Murgia C, Pritchard JK, Kim SY, Fassati A & Weiss RA, 2006, Clonal Origin and Evolution of a Transmissible Cancer, Cell, Vol 126, pp 477-487

injury in both sexes with susceptibility to transplantation of the tumour cells.25 Karlson and Mann are cited as providing proof of the transmissibility of the cancer when in 1952 they succeeded in following the passage of the tumour through 40 generations of dogs over a period of 17 years.26

During the course of the studies, it was noted that the dog tumours were found to develop at other sites, on the skin or in and around the mouth, but this was generally associated with a genital tumour. In 1966 Higgins provided an explanation, when he

‘suggested that many of the cutaneous sites where these tumours are found represent lesions caused by biting and scratching, common in stray dogs, which predispose the skin to implantation of the tumour’. 27 According to Das and Das ‘[Higgins] observed scars in the skin above … tumours, suggestive of previous wounds. 28

In 1970 Wright et al undertook genetic studies of the dog transmissible tumour cells and found there were usually between 58-59 chromosomes, whereas the normal number is 78 chromosomes in the somatic cells of dogs.29 According to Das and Das these abnormal features of the tumour cells are consistent and unique, in that they have been observed in tumours of dogs across different continents.30 Adams and Slaughter

25 Feldman WH, 1929, So-called infectious sarcoma of the dog in an unusual anatomic situation, American Journal of Pathology, Vol 5, pp 183-194 cited in U Das & AK Das, 2000, Review of Canine Transmissible Venereal Sarcoma, Veterinary Research Communications, Vol 24, pp 545-556

26 Karlson AG & Mann FC, 1952, The Transmissible venereal tumor of dogs: Observations on forty generations of experimental transfers, Annals of the New York Academy of Sciences, Vol 54, pp 1197-1213

27 Higgins DA, 1966, Observations on the canine transmissible venereal tumour as seen the the Bahamas, Veterinary Record, Vol 79, pp 67-71 cited in U Das & AK Das, 2000, Review of Canine Transmissible Venereal Sarcoma, Veterinary Research Communications, Vol 24, pp 545-556, p 548

28 Das U & Das, AK, 2000, Review of Canine Transmissible Venereal Sarcoma, Veterinary Research Communications, Vol 24, pp 545-556, p 548

29 Wright DH, Peel S, Cooper EH & Huges DT, 1970, Transmissible venereal sarcoma of dogs: A histochemical and chromosomal analysis of tumours in Uganda, European Journal of Clinical Biological Research Vol 15, p 155 in ibid.

30 Das U & Das, AK, 2000, Review of Canine Transmissible Venereal Sarcoma, Veterinary Research Communications, Vol 24, pp 545-556, p 549

confirmed the similarities between the features of the primary tumour and the secondary tumours, thus strengthening the evidence for the consistency of the abnormalities in the cells of CTVT.31 The same chromosomal patterns are also maintained in cell culture.32

In 2006 Murgia et al undertook molecular fingerprinting to identify the defective gene responsible for the cancer and matched DNA sequencing in dogs from around the world confirming the tumours to be genetically identical.33 They also identified three lines of observation they claimed confirmed CTVT as a transmissible cancer:

1. CTVT can only be experimentally induced by transplanting living tumour cells, and not by killed cells or cell filtrates;

2. Tumour karyotype is aneuploid but has characteristic marker chromosomes in tumours collected in different geographic regions

3. A long interspersed nuclear element (LINE-1) insertion near c-myc has been found in all tumours examined.

They also claim,

[a]lthough the tumor is highly aneuploid, the karyotype is remarkably constant in tumors from the United States, Kenya and Japan. 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.34

In terms of the instability of chromosomes they confirmed that CTVT does not appear to exhibit a mutator phenotype in terms of microsatellite instability, and neither does it exhibit progressive chromosome instability. They also stated that ‘[i]t is not evident

31 Adams EW & Slaughter LJ, 1970, A canine venereal tumour with metastasis to the brain, Pathologia Veterinaria, Vol 7, pp 498-502 cited in ibid.

32 Adams EW, Carter LP & Sapp WJ, 1968, Growth and maintenance of the canine venereal tumour in continuous culture, Cancer Research, Vol 28, pp 753-757 cited in U Das & AK Das, 2000, Review of Canine Transmissible Venereal Sarcoma, Veterinary Research Communications, Vol 24, pp 545-556

33 Murgia, C, Pritchard JK, Kim SY, Fassati A & Weiss RA, 2006, Clonal Origin and Evolution of a Transmissible Cancer, Cell, 126, pp 477-487

34 ibid, p 484

from our data whether the “infective dosage” is a single cell or a bolus of tumour tissue’

but they suspect the latter.35

In concluding, Murgia et al note that a definitive analysis based on DNA markers for DFTD, such as used for CTVT, was awaited.36 It remains to be determined if epigenetic factors affect the progressive and regressive phases of tumour growth for CTVT. The stable genome for CTVT has aided the host’s survival and onward tumour transmission ‘whereas the evolutionary dynamics of a “selfish”, dead-end tumour typically progresses toward greater autonomy and malignancy’.37 Hence DFTD contrasts with CTVT in that it is highly virulent - killing all of the affected animals.

They propose that a similarity between DFTD and CTVT may be the initial facilitation of CTVT within a partially inbred population. But today it exists within mixed-breed dogs, particularly strays. Further, in 2006 David Dingli and Martin Nowak published an article in Nature on both CTVT and DFTD concurring with Murgia et al.38 In 2009 Purohit in a review stated that CTVT is the only proven example of a naturally occurring tumour that is transmitted as an allograft by cell transplantation.39

CTVT is commonly found in dogs aged between two and five years that are sexually active. The dog cancer is benign, not fatal. CTVT appears to overcome the histocompatibility barriers to escape from the host’s immune surveillance, however a

35 ibid.

36 Murgia, C, Pritchard JK, Kim SY, Fassati A & Weiss RA, 2006, Clonal Origin and Evolution of a Transmissible Cancer, Cell, 126, pp 477-487

37 ibid, p 485

38 Dingli D & Nowak MA, 2006, Infectious tumour cells, Nature, Vol 443, pp 35-36

39 Purohit, GN, Canine Transmissible Venereal Tumour: A Review, 2009, The Internet Journal of Veterinary Medicine, Vol 6(1). Available at: http://archive.ispub.com/journal/the-internet-journal-of-

veterinary-medicine/volume-6-number-1/canine-transmissible-venereal-tumor-a-review.html#sthash.mqPhdT27.dpbs last accessed 30 September 2013

response is eventually mounted and the cancer goes into remission.40 This regression leads to tumour immunity that prevents successive occurrences.41 However in immunocompromised animals and puppies there is metastasis (secondary tumours).42

In relation to a viral hypothesis Mukaratirwa and Gruys found CTVT has the ability to be transplanted to other members of the canine family such as foxes, coyotes and wolves, which suggests a viral cause.43 But this hypothesis has been discounted elsewhere because it can only be experimentally induced by transplanting living tumour cells and not by dead cells or cell filtrates, so some scientists remain skeptical of the viral hypothesis. Meanwhile, Das and Das in two studies found oncogenic viral particles, that had not been seen through an electron microscope in the tumour cells, suggestive of the agent possibly being a type C retrovirus.44 It would appear that the viral hypothesis is still to be resolved.