Biomarkers: The Clues to Genetic Susceptibility.

There are approximately 500,000 cancer-related deaths annually in the United States. Scientists believe as that many as 80% of those deaths could be prevented due to the fact that most malignancies are a result of external factors rather than inherent biological conditions. With recent advances in molecular biology, a new field that combines highly sensitive and specific techniques for detecting early damage associated with cancer has emerged. By combining knowledge about external factors related to lifestyle and environmental or occupational exposure to chemicals with knowledge of how genetic differences cause variations in human responses to environmental pollutants, scientists are developing a better understanding of questions such as why some smokers get cancer but others do not, why certain groups of people have a higher incidence of cancer after exposure to a toxicant and others do not, and why certain women are more prone to develop breast cancer than others. Scientists using biomarkers of susceptibility will be able to identify risks and prevent adverse health effects through prevention and intervention strategies.

human large bowel. However, only 1-20/o of polyps give rise to malignancy, and despite claims for dysplasia the only reliable indicator of malignancy is invasion of the muscularis mucosa. This presents problems to histopathologists, as polyps are often removed piecemeal and traumatized, and manv show pseudoinvasion.
A similar problem is the pathogenetic relationship between intestinal metaplasia and stomach cancer (Morson, 1955). Metaplasia is almost always present in stomachs bearing cancer (Correa et al., 1970) and widespread metaplasia precedes cancers in high-risk patients with previous gastric surgery (Stalsberg & Taksdal, 1971) or who suffer from pernicious anaemia (Shearman et al., 1966). Most intestinal metaplasia, however, does not lead to cancer, and attempts to define a cancer-prone group in terms of increasing dysplasia (Morson et al., 1980) have the disadvantage of being subjective.
Using immunohistochemical methods, it is now possible to study specific markers in malignant cells. These markers reflect an abnormal phenotype and are presumably acquired during the evolution of the malignant process.
They include many which occur during early embryonic differentiation. Placental markers, for example, have been reported in both germ-cell and non-germ-cell carcinoma, including gut adenocarcinomas (Azer et al., 1980;Engvall & Yonemoto, 1979). We have studied one such marker, pregnancy-specific glycoprotein (SP1), in the cells of cancer and putative premalignant conditions in human stomach and colon.
In 18 cases of carcinoma of the stomach, tissues were obtained from 9 resection specimens and 9 endoscopies. In each case, 2 pieces of malignant tissue and 1 each of mucosa from body, cardia and pyloric region were examined. Seventeen biopsy specimens of atrophic gastritis were also available for study.
Twenty cases of carcinoma of colon, 22 tubulovillous adenomas (TVA) and 8 metaplastic polyps were also studied. In each case 2-6 blocks were available.
Blocks were fixed in 4% phosphatebuffered formaldehyde solution at pH 7 0 and processed into paraffin wax.
Sections were cut at 5 jim, dewaxed and washed through graded alcohols and then 0*2M phosphate-buffered saline (PBS, pH 7.4). One section from each was stained with haematoxylin and eosin.
The rabbit anti-SPI was obtained commercially (Dako) as a single batch and an IgG concentrate prepared using Sephacryl S200 column chromatography.
The antibody was absorbed against cells of the "buffy" coat of human blood and a splenic extract, to remove nonspecific cross-reacting antibodies.
This antibody was then used, diluted 1/20, in the PAP immunoperoxidase method of Bturns (1975) with methyl green counterstain. This gave a good positive result on syncytiotrophoblast. The staining was abolished by prior incubation of the antibody with neat SP1I (antigen from Dr R. Bolhn) but not hCGA (Hoechst) or human placental lactogen.
As pure SPI was in short supply, this method for controlling the specificity of the reaction was only possible in the first sample of anti-SPI reagent. Thereafter the anti-SP] specificity was checked by prior incubation with a saline extract of fresh placenta, which abolished positive staining on control slides.
Negative controls were histologically normal gastric and colonic mucosae.
H202-alcohol blocking of endogenous peroxidase was checked by using Diaminobenzidine (DAB) alone, with negative results.
Nonspecific binding of protein and the presence of heterophile antibodies was assessed by using PAP reagent with and without non-immune rabbit Igo followed by DAB.
The results in both stomach and colon staining of positive cells was most noticeable on the membranes, but also seen in a granular distribution within the cytoplasm. The staining intensity in the placental control sections was more intense, falling to about the same level on 4-fold dilution of antibody. Whilst an accurate biochemical measure of SP1 content was not possible, tumour cells generally appeared to produce less SPI than syncytiotrophoblasts. Furthermore, not all tumour cells produced SP1: the proportion varied 5% to 750G. In general it was distributed within clusters of cells. Occasionally some stromal fibroblasts were faintly positive.   Table I shows the number of cases positive and negative for SPI in the stomach. Using a 2-way contingency test, x2=9X724, well outside the 95%o confidence limits (for 2 d.f. this gives P = 0.0077). Table II shows the results for the colon. Using the same contingency test the value for x2 = 5X568, which is just within the 950/ confidence limits (P=0 059).
Thus in the stomach, SPI shows a weak association (50%o) with cancer and the intestinal metaplasia in cancer cases. For colon cancer a similar association is not proven. SPI has been demonstrated in the serum of patients with a variety of germ-cell tumours (Javadpour, 1980) and in nontrophoblastic carcinomas (Wurz, 1979). It has been shown infrequently in the serum of patients with breast, lung and colon cancer (Searle et al., 1978;Grudzinskas et al., 1980). However, little attention has been given to its presence in tumour cells, even though immunohistochemical methods are of the same order of sensitivity as most RIA tests. The only exception has been in studies of trophoblastic and other germ-cell tumours (Horne et al., 1976;Javadpour, 1980).
In this study SPI has been shown as a marker of stomach cancer, and of the cells of intestinal metaplasia present in the stomach of cancer patients. It is not present in similar intestinal metaplasia in non-cancer cases. The positive staining does not correspond to areas of so-called dyplasia (Morson et al., 1980) or the colonic type of metaplasia described by some authors as particularly associated with cancer (Jas & Filipe, 1980). The metaplasia is small-intestinal in type, complete with goblet cells, absorptive cells and Paneth cells.
The frequency of positivity is, however, low (50%o for cancer, 23% for intestinal metaplasia associated with cancer). In cases where a diagnosis of cancer was in doubt, a positive result would be of considerable assistance. It would be better than a demonstration of CEA (Goldenberg, 1976) or changes in mucosubstances (Jass & Filipe, 1980) which are not necessarily indicative of neoplasia. A positive result in a metaplastic epithelium would identify the need for a sequential appraisal of stomach pathology.
In the colon, SPI occurs almost as frequently in so-called pre-malignant TVA as in malignant conditions, and is of no assistance in deciding whether a polypoid neoplastic mass is malignant or not. Invasion remains the only reliable marker. Nor is it a totally reliable marker of neoplasia, as the hyperplastic metaplastic polyp is also positive on occasions. However, the two positive cases, reported in detail elsewhere (Skinner & Whitehead, 1981) showed some areas more typical of tubulovillous adenomas and were large. This is an indication that metaplastic polyps require a more careful examination than has hitherto been usual. Estrada & Spjut (1980) reported adenomatous changes in 2200 of 171 hyperplastic polyps, another indication that the nature of the entity should be re-evaluated.
The finding of SPI in fibroblasts is of interest in the light of the report of SPI in the supernatants of dividing fibroblasts in culture . They are not seen in normal situations, only in tumour stroma, and it is speculated that in this situation they are dividing and proliferating as part of a tumour "desmoplastic reaction".