Cytogenetic analyses of four solid tumours in dogs

Research in Veterinary Science 1994, 57, 88-95

Cytogenetic analyses of four solid tumours in dogs B. MAYR, Institute for Animal Breeding and Genetics, M. REIFINGER, H. WEISSENBC)CK, Institute for Pathology and Forensic Veterinary Medicine, W. SCHLEGER, Ludwig Boltzmann Institute for Immuno- and Cytogenetic Research, E. EISENMENGER, Institute for Surgery, Veterinary University, Linke Bahngasse 11, A-1030 Vienna, Austria

Four solid tumours (one haemangiopericytoma, one haemangioendothelioma, one spindle-cell sarcoma and one mammary carcinoma) in dogs were analysed eytogenetically. In the haemangiopericytoma, an additional small chromosomal segment was present. Very complex changes including centric fusions and symmetric meta-centrics 1, 6, 10 and 12 were conspicuous in the highly unbalanced karyotype of the haemangioendothelioma. Complex changes, particularly many centric fusions and a tandem translocation 4/14, were features of the spindle-cell sarcoma. One centric fusion and a symmetric metacentric 13 were present in the mammary carcinoma.

FIG 2: Malignant haemangioendothelioma in dog 2. Bar = 20 #m

VERY little is known about the cytogenetics of solid tumours in domestic animals. The few reports have included transmissible venereal tumours in which the normal 78 chromosomes are reduced to 58 or 59, with many metacentrics

(Makino 1963, Murray et al 1969). There is a particular lack of information about the characterisation of solid tumours, including their chromosome banding, and in the case of canine tumours there are difficulties with the karyotype of this species. However, there have been descriptions of single cases of haemangiopericytoma (Mayr et al 1990d, 1992a), haemangioendothelioma (Mayr et al 1991a), mammary adenocarcinomas (Mayr et al 1990a, c, 1991b, 1993) and mammary sarcomas (Mayr et al 1992c) in dogs. This paper describes several cytogenetic anomalies in new cases of haemangiopericytoma, haemangioendothelioma, spindle-cell sarcoma and mammary adenocarcinoma in dogs. Materials and methods A seven-year-old Yorkshire bitch (dog 1) was admitted in October 1991 with a tumour 5 cm in diameter in the dorsal region of its toes. The tumour was removed surgically, the dog made a good recovery and the tumour has not recurred.

FIG 1: Haemangiopericytoma in dog 1. Characteristic are the concentric layers of spindle-shaped cells around blood vessels. These cells are presumably derived from pericytes or from fibroblasts following the route of the vessels. Bar = 220 gm

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Cytogenetic analyses of tumours in dogs

FIG 3: Spindle-cell sarcoma in dog 3. Bar 20 gm

A histological examination showed the tumour to be a haemangiopericytoma (Fig 1). A seven-year-old German short-hair bitch (dog 2) developed a tumour 10 cm in diameter in the stifle joint. It was removed surgically and the dog recovered well. The tumour was a haemangioendothelioma (Fig 2). A 10-year-old German shepherd mixed-breed bitch (dog 3) developed a tumour 6 cm in diameter in the inguinal region. After its surgical removal this spindle-cell sarcoma (Fig 3) regrew and the animal had to be euthanased after a few weeks. A nine-year-old German shepherd bitch (dog 4) developed a papillary adenocarcinoma with widespread progression to anaplastic carcinoma of the mammary gland (Fig 4). The mmour was removed but regrew and the dog died after a few weeks. The diagnosis was based on the international histological classification of tumours and dysplasias of the mammary gland (Hampe and Misdorp 1974). In all four cases, explant cell cultures from the tumour were set up by mincing the solid tissue into small fragments (less than 1 mm). The fragments were transferred into sterile flasks containing 8 ml RPMI 1640 medium with L-glutamine, antibiotics (50 iu penicillin and 50 gg streptomycin ml-I) and 10 per cent fetal calf serum (all from Gibco). The cultures were maintained in 5 per cent carbon dioxide/air at 37°C for three to 14 days. Colcemid was added to a final concentration of 0-1 gg ml -I five hours before harvesting. The metaphases were analysed by conventional Giemsa staining and by G-banding as described by Wang and Fedoroff (1972). The chromosomal nomenclature followed an earlier description

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FIG 4: Papillary adenocarcinoma with widespread progression to anaplastic carcinoma in dog 4. The tumour shows small areas of well differentiated papillary adenocarcinoma (inset) and widespread areas of anaplastic carcinoma: numerous solitary tumour cells and clusters of tumour cells in fibrous stroma, moderate infiltration with lymphocytes. Bar = 30 gm, inset bar= 180 g.m

(Selden et al 1975). However, major discrepancies appear in the numbering of chromosomes used in a Giemsa banding study of Stone et al (1991). Results

In dog 1 (haemangiopericytoma, Fig 1), all 12 metaphases analysed had the normal canine number of chromosomes (2n=78) plus one additional small chromosomal segment probably representing a partial trisomy (Fig 5). However, it was not possible to attribute it to a specific chromosome. In dog 2 (haemangioendothelioma, Fig 2), only three of the 15 metaphases analysed had the normal canine karyotype, and the other 12 had 72 chromosomes (Fig 6). In all these cells symmetric metacentric chromosomes 4, 8, 10 and 12, centric fusions 1/11, 4/36, 9/13, 13/27, 14/20, 21/34, 24/25 and 30/35 and trisomy 28 were observed. In dog 3 (spindle-cell sarcoma, Fig 3), only one of the 10 metaphases analysed had the normal canine karyotype and the other nine had 65 chromosomes (Fig 7). In all these cells there were centric fusions 2/18, 4/11, 6/22, 6/29, 8/16, 10/13, 10/24, 16/32, 22/34, 23/33, 24/28, 25/27 and 31/35, a symmetric metacentfic 15, and a tandem translocation 4/14. In dog 4 (papillary mammary adenocarcinoma with widespread progression to anaplastic carcinoma, Fig 4), six of the 12 metaphases examined had the normal karyotype, but the other six cells had a centric fusion 4/8 and a symmetric metacentric

90

B. Mayr, M. Reifinger, H. Weissenb6ck, W. Schleger, E. Eisenmenger

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chromosome 13 (Fig 8); four of these six had 2n=76 (Fig 8) and two had 2n=77 (owing to the presence of an additional small unidentifiable chromosome).

Discussion

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normal chromosome count represents a novel finding in canine haemangiopericytomas. In earlier cases (Mayr et al 1990d, 1992a) trisomy 2, deleted chromosome 1 and a few centric fusions were the predominant feature. Although the pres-

ence of a partial trisomy 2 is not certain it cannot be excluded as an explanation of the findings. Molecular genetic data, including genetic mapping, will be necessary to solve the question. Unfortunately, such knowledge is lacking for the

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canine genome and pioneering attempts are just beginning. In the haemangioendothelioma, four metacentric chromosomes with a symmetric G-banding pattern (chromosomes 4, 8, 10 and 12) were a consistent finding in all the cytogenetically abnormal cells. These could have been either isochromo-

somes consisting of homologous arms that were mirror images of one another and resulting from the misdivision of the centromere (transverse breakage), or centric fusion chromosomes. Further consistent abnormalities in all these cells were the centric fusions 1/11, 4/36, 9/13, 13/27, 14/20, 21/34, 24/25 and 30/35 which, together with the

Cytogenetic analyses of tumours in dogs

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8b FIG 8: (a) Trypsin G banded metaphase of dog 4 (2n=76). (b) Karyotype of the metaphase of (a). Note symmetric metacentric 13/13 and a centrie fusion

94

B. Mayr, M. Reifinger, H. Weissenb6ck, W. Schleger, E. Eisenmenger

symmetric metacentrics, caused the reduction in the chromosome count from 78 to 72, in spite of the presence of third copies of several chromosomes (chromosome arms). The presence of these additional copies gave the haemangioendothelioma a highly unbalanced genetic make up. However, such a highly unbalanced karyotype is not surprising, because failures of accuracy of chromosome disjunction, leading to aneuploidy, are a general feature of tumour cells (Holliday 1989). A similar, although much less pronounced aneuploidy was also the predominant feature in a haemangioendothelioma investigated earlier (Mayr et al 1991a). The present study revealed complex cytogenetic changes in the spindle-cell sarcoma, a type of tumour for which no data are available. Several biarmed chromosomes (centric fusions and a symmetric metacentric) led to a reduction of the chromosome count from 78 to 65. A further consistent aberration was a third copy of chromosome 4 involved in a tandem translocation. Such types of rearrangement may possibly enhance the expression of oncogenes in the relevant genomic regions and/or lead to structurally recombined oncogenes, anti-oncogenes or other genes. In any case, it is reasonable to assume that increasing complexities in the karyotypes are correlated to some degree with the characteristics of tumour progression, for example, their speed of growth and invasiveness, and/or with the prognosis. However, at present there are insufficient data to support this idea unequivocally in the domestic dog. In the papillary adenocarcinoma with widespread progression to papillary carcinoma, the symmetric metacentric chromosome 13 may have represented an isochromosome or a centric fusion chromosome. Other symmetric metacentrics (1 and 6) were present in a mammary adenocarcinoma investigated by Mayr et al (1990a). Given that they represent isochromosomes, the idea of a net loss of genes, including the loss of heterozygosity at the tumour suppressor loci, becomes important. Again, the development of the canine gene map is extremely poor and nothing is known about the genomic localisation of tumour suppressor loci. Another type of net loss of chromosomal material has been reported in a few other cases of mammary tumours; they concerned deletions of autosomal (Mayr et al 1992b) and X-chromosomal (Mayr et al 1991b) material. An asymmetric metacentric 13 has been found in an osteochondrosar-

coma in the mammary region (Mayr et al 1990b). However, at present it would be unjustified to draw any conclusions about causal relationships between this finding and the present finding in a mammary carcinoma. Wolfe et al (1986)reported the loss of chromosomes in three of six canine mammary cancer cell lines maintained in culture. Rutteman et al (1988) considered the implication of chromosome loss in canine mammary carcinomas; in their studies hypodiploid cancers seemed to be more frequent than in the human beings (Barlogie et al 1987, Cornelisse et al 1987). The distribution of genes coding for vital cellular functions over a larger number of chromosomes (78 vs 46 chromosomes) might enable canine cells to withstand the loss of chromosomes more successfully (Rutteman et al 1988). The dog plays an important role for veterinary and comparative oncology, yet the first successful approaches to solving the band identification of its solid turnout karyotypes have only just begun. However, a thorough band identification is an indispensable prerequisite for a deeper understanding of their origin and evolution. The present cases should therefore provide a useful contribution for future canine tumour research.

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Received July 14, 1993 Accepted January 18, I994