DURING the course of a scrutiny of mammae of old breeding females of several inbred strains of mice in which either very few or no spontaneous mammary tumours had arisen, foci were found in one of the strains of apparently spontaneous squamous differentiation. This differentiation took the form of squamous epitheliosis in ducts and in acini. It was frequently, but not invariably, accompanied by scattered or massed secretory acini in mammary glands that elsewhere were completely involuted. The strain affected was the R III X, a subline of the Paris R III strain, which had previously been freed from the Bittner agent of heritable mammary tumours. A close scrutiny was being made to determine whether mammae of mice free of the agent and of heritable mammary carcinomas were devoid also of adenomas. The result of this first objective is incomplete since it entails survival of a sufficient number of spayed mice to determine the part played by ovarian hormones, but will later be reported. Another experiment in progress during the period of observation included an examination of mammae of young breeding females of the same R III X agent-free subline which had been treated by skin painting with methylcholanthrene with the object of inducing mammary tumours. The experience of many authors indicated that mammary tumours of various sorts would arise (Dobrovolskaia

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THE presence and activity of the milk-borne agent of mammary tumours of mice discovered by Bittner (1936) are detectable only by the growth of a mammary tumour. No other criterion of its presence is yet generally accepted, though an opinion is widely held that certain localized nodular adenomatous mammary hyperplasias are an expression of the combined activity of the milk-borne tumour agent and a mammogenic hormone. Focal or nodular adenomatous hyperplasias in the mammae of dealer's mice were first described by Apolant (1906) and their cause investigated by Haaland (1911). Haaland inquired but failed to decide whether the nodules ( Fig. 1 and 2) were tumours from the beginning, or whether they were the basis from which tumours develop. Similar nodules occur in some inbred strains of mice. Thus Gardner, Strong and Smith (1939) found their incidence to be greatest in mice of the high cancer strains D and C3H, and intermediate in the low mammary tumour strain CBA and high mammary tumour strain A. Objections to accepting nodular hyperplasia as evidence of activity of the milk-borne tumour agent derive from two sources. In the first place these nodules-have occasionally been found in strains that do not develop mammary tumours. Gardner et al. found two nodules in one mouse out of nine of the N strain in which mammary tumours had never occurred. Also when efforts have been made to exclude the milk-borne agent from high cancer strains by cross-suckling, nodules have nevertheless been found. Thus Bittner, Huesby, Visscher, Ball and Smith (1944) examined the mammae of fostered breeding females (fostered by cancer-free maternal stock) from high cancer strains A and C3H. All the females had given birth to at least three litters and ranged from 11 to 16 months old, thereby providing suitable conditions for pathogenic activity of the milk-borne agent. Nodules were found occasionally, though never more than one to a gland, Thus nodules were still present though in smaller numbers when efforts were made to exclude them than in mice which had been suckled on their own cancer-prone mothers. This evidence indicated that nodule incidence is not eliminated by cross suckling with the same regularity that is attainable in regard to tumour incidence. Huesby and Bittner (1946) record that they found no nodules in the mammae of 6 virgin, and X per gland in 5 out of 12 breeding Zb females; none in 6 virgin and X per gland in one out of 12 breeding Strong AX females; none in 10 hybrid virgin females and per gland in 2 breeding females of the F.1 generation derived from these two strains, both deprived, previous to crossing, of the milk-borne agent.

SINCE the discovery of the milk factor of mammary tumours in mice (Bittner, 1936), it has often been supposed that inbred strains with low incidences of mammary tumours are free of milk factor and those with high incidences are infected with it. Subsequent reports of actual tests of tumour material or by cross-suckling and breeding experiments have shown that such assumptions are not always valid. In the case of CBA strains variable incidences have been recorded (Heston, 1945) and not all the progeny of Bittner's original fostering of Strain A sucklings on a CBA mother were tumour free (Bittner, 1937(Bittner, , 1939(Bittner, , 1952, though it was from one of these cross-suckled mice that the AX low cancer subline and strain were developed. At the request of Dr. P. R. Peacock, 2 mammary tumours from 2 CBA mice bred in these laboratories were tested for the presence of milk factor by injection of tumour extracts into susceptible young females, themselves free of agent. Twenty-one mammary tumours arose in 33 test mice, 11 from one and 10 from the other tumour extract. The mammary tumour incidence of breeding females of the strain from which the test mice were derived was less than 2 per cent. The high incidence of mammary carcinomata, 63 per cent, among the injected progeny of this strain, is therefore attributed to milk factor contained in the inoculated tumour extracts.METHODS. Test mice.RIlIb (Syn. RIII X, Pullinger, 1952) females less than 1 month old were used. Comparison of results was made with pure line females of the breeding stock of the same strain.Preparation and injection of extracts.Two mammary tumours which had arisen in 2 CBA breeders were weighed and ground in mortars with sterile distilled water to give 20 per cent suspensions. The fluids were decanted for further grinding into 2 glass homogenisers. The suspensions were then spun at 14,000 g. for 15 minutes in a Servall centrifuge. Seventeen young suckling mice were used for inoculation of each tumour extract. Volumes of 0-25 ml. of the supernatant fluids were injected intraperitoneally into the test mice.The young mice were replaced with the mothers until weaned. They were then mated with RJJJb males and divided over 4 breeding boxes. The females were left in the breeding boxes throughout the experiment to ensure rapid breeding.

HYPERPLASIA, atrophy, chronic inflammation and scarring have all been described as antecedents of spontaneous cancer. More often no previous pathological change has been detected. Of the known antecedents, atrophy is the most surprising because it is a retrogression believed to be irreversible. The association of cancer combined with atrophy thus raises the question of the order of events. Did neoplastic change occur before or after observed atrophy? It ought to be possible to devise experiments to decide whether new growth can be initiated and imposed in cells already in a state of atrophy or whether this change, if it occurs, precedes the atrophy. In experimental pathology the antecedent of cancer most commonly observed is hyperplasia. Prolonged hyperplasia is sometimes succeeded by atrophy and tumour growth. A nutritional atrophy combined with fatty infiltration due to choline deficiency is associated with hepatoma in rats but in neither circumstance is the order of events known relative to actual initiation of new growth. The term "initiation " is used in the sense introduced by Friedewald and Rous (1944). Experiments designed to decide the order of events would be done in two stages: first an endeavour to bring about true atrophy using for the purpose some non-carcinogenic agent or method, and second the application of a specific carcinogen to the region made atrophic. Attempts by the author to carry out this sequence revealed a constant tendency of mouse skin-the most suitable tissue to use-to undergo hyperplasia in response to all varieties of injury that were tried. Hair follicles may be destroyed, for example by repeated excision of the same area of skin, giving an appearance of local atrophy, but the epidermis itself always thickens. A trial was next made with an agent known from human pathology to cause both atrophy and new growth in the expectation that the order of events might be deduced by the difference in dosage required for the two effects.The experiments here recorded made use of mammary epithelium and repeated X-irradiation. By means of prolonged whole body irradiation with radium Lorenz, Eschenbrenner, Heston and Uphoff (1951) increased the prevalence and decreased the time of appearance of mammary tumours in C3Hb mice without milk-factor but attributed these tumours in part to stimulation of the mammary gland by secretion from granulosa-celled tumours of the ovary which were due to and arose during the course of irradiation. To avoid this possible additional factor, local surface X-irradiation of 2nd and 3rd right nipple regions, which overlap, was carried out. Five palpable mammary carcinomas and four which were visible during microdissection and after bulk-staining arose in 27 virgin female