Occupational cancer in europe

This monograph summarizes the current research on the epidemiology and prevention of occupational cancer in Europe. Eleven peer-reviewed articles offer a composite view of the current status and future perspectives of this discipline at a time of major economic and political changes in Europe. The monograph includes a brief history of occupational cancer research in Europe and the current burden of cancer from occupational exposure. A large portion of the monograph is devoted to reviews of occupational cancer in various European countries or groups of countries. The first two reviews describe those regions with the strongest tradition of research in the field: the Nordic countries and the United Kingdom. These countries are characterized by high-quality cancer registries and routinely collected information on occupation, e.g., on the occasion of censuses, allowing record linkage studies. Four articles address the situation in the remaining large Western European countries--France, Germany, Italy, and Spain. Although these countries have a strong tradition in occupational health research, with the possible exception of Spain, their epidemiologic research dates back only to the 1970s and 1980s; in recent years, however, research on occupational cancer has expanded considerably. The last two articles of the geographic overview describe the occupational cancer research in the Central European countries and the European territories of the Commonwealth of Independent States. The authors of these two articles critically review the available data on occupational cancer from former Socialist countries and provide reliable information on the extent and amount of occupational exposure to carcinogens. The two final articles of the monograph address specific thematic issues: the importance of asbestos as an occupational carcinogen in Europe and the use of routinely collected data to investigate occupational cancer in women, based on the experience in England and Wales. Despite the heterogeneity of these articles, which reflects to a large extent the differences in quality and completeness of the available data on occupational cancer, they represent an original attempt to provide a systematic overview of occupational cancer research in Europe. This publication is originally a partial result of two projects on occupational cancer in Europe funded by the European Commission, Directorate General XII (grants BMH1-CT92-1110 and BMH1-CT95-1100) and has been expanded to include non-European Union countries.

It is clearly important to establish that tumours growing as xenografts retain fundamental characteristics expressed in the primary donor or, in the case of experimental tumours, on syngeneic transplantation. In this context, human tumour xenografts have been shown to continue the production of several tumour-associated materials. For example, xenografts of Burkitt's lymphoma produce IgM (Povlsen et al., 1973) colon carcinomas produce CEA (Mach et al., 1974;Houghton & Taylor, 1978b), and breast carcinoma produces calcitonin (Coombes et al., 1975). In the light of these observations with human xenografts, the present work was carried out to examine xenografts of experimental rat tumours for the continued production of their characteristic tumour-associated antigens, and to examine sera of tumourbearing mice for free circulating tumourspecific antigen, as an experimental basis for the examination of human-xenograft bearers for tumour-specific products. The rat tumours used, a carcinogen-induced sarcoma and hepatoma, have unique cellsurface neo-antigens demonstrable in vitro by indirect membrane-immunofluorescence reactions with tumour-immune rat sera. The expression of these antigens on cells of xenograft growths has been assessed, and sera of tumour-bearing mice examined for free antigen by their ability to neutralize the reactivity of tumourimmune rat serum. The development of anti-rat antibody in xenograft bearers and in mice rejecting tumour cells injected in admixture with BCG has also been examined.

MATERIALS AND METHODS
Athymic mice.-Athymic nude (CBA nu/nu or ONU nu/nu) mice and heterozygous (CBA nu/+) mice were purchased from the Medical Research Council Laboratory Animal Centre, Carshalton, Surrey. They were fed standard laboratory diet (Oxoid) and tap water ad libitumn while housed in plastic cages with sawdust bedding held in Filter Rack Ventilation Cabinets (Anglia Laboratory Animals, Alconbury, Huntingdon, Cambridge).
Tuinours. The rat tumours used were induced in adult rats of a Wistar-derived. Nottingham Inbred Strain (WAB/Not) by carcinogen administration. Sarcoma Mc7 wras induced by s.c. injection of 3-methylcholanthrene (Baldwin & Pimm, 1971) and hepatoma D23 by oral administration of 4dimethylaminoazobenzene (Baldwin & Barker, 1967a). These tumours have been maintained by routine s.c. trocar grafts in syngeneic recipients. Both possess individually distinct neoantigens, demonstrable by in vivo transplant-rejection reactions and in vitro by indirect membrane-immunofluorescence reactions with syngeneic tumour-immune rat sera (Baldwin & Pimm, 1971;Baldwin & Barker 1967a, b).
For the present studies, in vitro culture lines were established in Eagle's medium supplemented with 10% calf serum and routinely harvested in 0-2500 trypsin, washed and suspended in Hanks' Solution.
Growth of xenografts.-Tumour growths wvere initiated by subcutaneous injection in the right flank of cells harvested from tissue culture. In some cases mice received one or two injections of cells in direct admixture with 500 tg moist weight of BCG organisms (Glaxo Percutaneous Vaccine, B.P., Glaxo Laboratories, Greenford, Middlesex); these inocula wvere consistently rejected (Pimm & Baldwin, 1975). Mice were bled by cardiac puncture under ether anaesthesia, blood clotted at room temperature and stored at +4°C for 18 h. Cellular contamination was removed from serum by centrifugation at 500 g for 15 min, and serum clarified by centrifugation at 1500 g for 30 min and stored at -20°C.
Allogeneic, anti-WAB/Not sera was raised in KX/Not rats by repeated implantation of viable D23 tumour tissue, these grafts being consistently rejected.
Mouse anti-rat serum was raised in heterozygous CBA nu/+ mice by i.p. injections of viable rat sarcoma Mc7 cells, these being consistently rejected.
Assay for antibody.-Rat tumour cells were harvested from tissue culture or prepared by trypsin digestion of solid xenograft growths from athymic mice (Baldwin & Barker, 1967b). 2 x 106-5 x 106 cells were incubated for 20 min at room temperature w%Aith 0-1 ml serum, washed x 4 in Hanks' solution and resuspended in 01 ml of fluorescein-labelled antiglobulin, incubated for a further 20 min, washed x 4 in Hanks' solution and finally suspended in 1:1 (v/v) glycerol:phosphatebuffered saline (pH 7.2). For tests wAith rat antisera, normal rat serum was used as a negative control, and the fluorescent antiserum was rabbit anti-rat IgG, prepared in this laboratory; tests with mouse sera had normal mouse serum (nu/nu or nu/+l) as negative control, and fluorescein-labelled goat anti-mouse globulin (Nordic Diagnostics, London, diluted 1/30). Cells were examined with a Reichert Zetopan fluorescence microscope (x 600) and cells showing complete or partial membrane fluorescence scored as positively stained. A fluorescence index (FI) wAas calulated for each serum as: Fl-00 cells unstained by control sera-00 cells unstained by test serum 00 cells unstained by control serum Assay for serum antigen.-Rat tumourspecific antiserum wias incubated with serum from mice bearing rat tumour xenografts for 30 min at room temperature, at a ratio of 0-1 ml antisera: 0'02-0-2 ml tumour-bearer serum. The incubated mixture was assayed for free antibody as outlined above by immunofluoreseence reaction against appropriate target cells. Untreated serum and serum mixed with normal mouse serum A-ere included as controls in the tests.

Tumour-specific antigen expression on xenograft growths
To confirm that serologically defined tumour-specific antigens, known to be expressed on cells of sarcoma Mc7 and hepatoma D23 in syngeneic transplants, were similarly expressed in xenografts in athymic mice, indirect membrane immunofluorescence (MIF) reactions were carried out with rat tumour-immune sera and In some cases, however, reactivity against cells prepared from xenografts was not as complete as that with culture-derived cells, as shown by FIs less than 1-00. This may reflect contamination of the xenograft preparation with mouse stromal or bloodvessel cells, although no attempt was made to actively demonstrate these host cells, and indeed the technique of cell preparation and handling (Baldwin & Barker, 1967b) may not be suitable for their recovery.
Anti-rat antibody in xenograft bearers and in mice rejecting cells after BCG treatment Sera from groups of mice bearing xenografts of D23 and Mc7 were tested for antirat antibody by MIF reactions against D23 and Mc7 target cells (Table II). In no case was a significant reaction detected, using sera from mice with 24-46-day-old growths  (Pimm & Baldwin, 1975) and the mice were subsequently tested for anti-rat antibody by the MIF reaction. Some mice received 2 injections of cells and BCG, at 30day inter-vals. The sera from none of these groups of mice had antibody detectable by the MIF reaction (Table III) but again sera from heterozygous mice, rejecting tumour cells alone, reacted strongly. Detection of tumour-specific antigen in the serum of xenograft-bearing mice The sera of athymic mice bearing xenografts were assayed for antigen by their ability to neutralize the reaction of rat tumour-immuine antisera against appropriate target cells in the MIF test. The results of these assays (Table IV)    that 4/5 pools of sera from mice with Mc7 xenografts contained antigen, and both of 2 serum pools from D23 bearers were also positive. For example, in the first test, sera from mice with 46-day-old xenografts of Mc7 reduced the FI of Mc7 antiserum from 0-55-0-59 to 0-14-0-25, when 0-1 ml tumour-bearer serum was mixed with 0-1 ml antiserum, while normal mouse serum (nit/nu) had no neutralizing effect (FI of mixture, 0.59). The 2 D23 tumour-bearer sera reduced the Fl of D23-immune serum from 0-44-0-67 to zero. A cross test carried out with the second of these D23 tumourbearer sera showed that this neutralization of antiserum was specific, the D23-bearer seruim failing to neutralize the reactivity of Mc7 antiserum against Mc7 target cells in 2 separate tests (FIs of untreated serum: 0-55, 0-72; FJ after mixture with D23 bearer serum: 0-56, 0.63).
To determine the amount of tumourbearer mouse serum required to neutralize a standard volume of tumour-immune serum, tests were carried out with 0-02-0-25 ml Mc7 xenograft bearer serum mixed with 0-1 ml rat tumour-immune serum (Fig.). With 3 individual tumour-bearer sera, increasing amounts of serum pro-MC7 106 fc7 106 Mc7 106 M\c7 106 D23 105 D23 1(i duced progressive neutralization of reactivity. DISCUSSION Human tumours growing as xenografts in congenitally athymic or immunosuppressed mice have been reported to continue the production of characteristic tumour-associated secretory products. Thus, a xenograft of Burkitt's lymphoma continued the production of IgM (Povlsen et al., 1973), human colon-tumour xenografts produce characteristic epithelial mucin (Houghton & Taylor, 1978b) CEA and AFP, these latter demonstrable both within the xenograft and free in the host serum (Mach et al., 1974;Houghton & Taylor, 1978b;Miwa et al., 1977). Similarly, a choriocarcinoma synthesized HCG (Kamneya et al., 1976), and human mammary carcinomas continue to produce calcitonin (Coombes et al., 1975) and tumourassociated glycolipid (absent from nonmalignant tissue) and shed this into the serum (Nordquist et al., 1978). Similar studies with animal-tumour xenografts have demonstrated the synthesis of myeloma protein by a mouse plasmacytoma, insulin by a hamster pancreatic-islet-cell tumour, and SV40 T-antigen by SV40transformed mouse, rat and rabbit cells (Freedman et al., 1976). The objective of the present studies was to examine the continued production in xenografts of the unique cell-surface tumour-associated neoantigens of 2 carcinogen-induced rat tumours, and here it has been demonstrated that these antigens are expressed during xenograft growth. Moreover, free antigen, assayed by its ability to specifically neutralize tumour-specific antibody, was demonstrable in the serum of xenograft bearers. Antigen is similarly shed during growth of these tumours in syngeneic rats, but is then frequently complexed with tumourspecific antibody (Bowen & Baldwin, 1976;reviewed by Price & Baldwin, 1977). Assays carried out in the present study were with sera of mice with large tumours (1-2 cm in diameter) and no attempt has yet been made to correlate the level of serum tumour antigen and tumour size. It is probable that the serum of tumour-bearing mice contains other rat tumour products in addition to tumourspecific antigen, including species-specific antigens, but assays for these have not been carried out.
The present tests were unable to detect antibody to tumour-specific antigens or rat species antigens in the serum of mice developing xenografts. These findings do not exclude the possibility of the presence in tumour-bearer serum of antibodyantigen complexes in antigen excess, although mice rejecting 2 inocula of tumour cells prevented from growth by the local application of BCG, also had no detectable antibody. With virally induced tumours, athymic mice bearing growths of SV40induced tumours similarly fail to develop antibody to SV40 T antigen, assayed by irnmunofluorescence reactions, or virus neutralizing antibody (Tevethia et al., 1977) although inoculation of polyoma virus evokes antibody to virus, detectable by haemagglutination inhibition (Stutman, 1975). With human tumour xenografts, anti-human antibody was demonstrable in the serum of athymic mice with a Burkitt's lymphoma xenograft (Povlsen et al., 1973) by both an indirect membraneimmunofluorescence test similar to that used here, and by complement-dependent cytotoxicity. However, antibody against membrane-associated EBV virus was not demonstrable, and the precise antigenic components of the human cells in the athymic mice were unresolved. Similar studies with mice bearing xenografts of the human bladder carcinoma line T24 failed to detect antibody by the membrane-immunofluorescence test (Pimm & Baldwin, 1978).
Essentially the present studies extend those of others who have demonstrated continued production or excretion of tumour-associated materials, showing that individually distinct tumour-specific components are also produced during xenograft growth. Taken as a whole, these studies indicate that many fundamental characteristics of malignant cells remain unchanged during xenograft development, strengthening the case that xenografts may be used as models for the assessment of therapeutic protocols and as a source of tumour tissue or antigen, and that the serum of tumour-bearing animals may be a suitable source of tumour products.