'' A " and " B '' (C57 black) strains of inbred mice ( ). Preliminary observations

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1 POSSBLE METHOD OF TRANSMSSON OF SUSCEPTBLTY TO BREAST CANCER N MCE ' JOHN J. BTTNER (Research Fellow, National Cancer nstitute, U. S. Public Health Service, and Research Assouale, Jackson Memorial Laboratory, Bw Harbor, Maine) A genetic approach to the cancer problem was made possible following the development of inbred strains of mice having a high and a low tumor incidence. On making reciprocal crosses between strains which differed greatly in their incidence of mammary gland tumors, it was observed that when the maternal parents were representatives of the hightumor stock the first generation females had a high tumor incidence; when the cross was made in the opposite direction the incidence in the hybrid classes approached that of the lowtumor stock (114). When the young cast by hightumor stock females were removed from their own mothers and given to foster mothers of the lowtumor stock, the proportion of the fostered mice developing mammary tumors was reduced to a very low percentage (1522). This was comparable to the incidence in virgin females of some strains. f, however, there was little difference between the ratios in the breeding and virgin females of a hightumor strain, the incidence for fostered females used as breeders was reduced considerably below either figure (Bittner and Little, 2 1 ; Andervont and McEleney, 22 ). n the fosternursing experiments the hightumor young were permitted to nurse their own hightumor mothers from the time of birth until they were recorded the following morning. The maximum period was twentyfour hours. n no instance were the nipples sealed to prevent nursing during this interim. Two groups of lowtumor stock mice (C57 black) have been fostered to hightumor females. n one experiment a few tumors were recorded (18) ; in the other the results were negative (Andervont and McEleney, 22). One spontaneous breast tumor was observed in the control line for the experiment in which tumors were produced in the fostered class (23). By eliminating entirely any effects of nursing, by transplanting fertilized ova, Little (21) has been able to reduce the tumor incidence to a point below that observed in fostered animals of any hightumor strain. n previous reports data have been given on the breast tumor percentages recorded in virgin and breeding F, and F, females derived by crossing the '' A " and " B '' (C57 black) strains of inbred mice ( 182 1). Preliminary observations have been presented on the results of foster nursing F, hybrid animals (19). The second generation mice of the latter classes are now under observation but are too young to give any reliable data at this time. n this report we shall present additional information on the results of foster nursing the firstgeneration animals and make comparisons with the control classes. 1 Supported by a grantinaid from the National Cancer nstitute. This paper was read before the American Association for Cancer Research, Richmond, Va.,

2 ~ ~ TRANSMSSON OF SUSCEPTBLTY TO BREAST CANCER 15 TABLE : Numher of Rreedznq and Virfin Fernales ill thl "A'' and " R ' Stocks and Their Reciprocal Hyhrid Generations with the Brrast 7umor Ratios and thr Avrrage Ages Avrragv Age in Months No. Cancer Cancer NonCancer BREEDNG FEMALES A AbyBO ABFi ABFi ABFi by B 13 RbyAO RAFi RAF BAFi by A * 568t (83.6';4) 42 ( 6.4'&) 112 (94.9%) 7 (71.47) 1 (.7%) 3 (.5%) 11 (11.3%) 2 ( 1.9m (.'%1) 123 (89.87,,) VRGN FEMALES A ABFi ABFi H RAF 1 RAFl t ( 4.9'Z) 5 ( 4.5%) 2 ( 1.7'%) (./;,) (.) (."h) * Seventynine living, over eighteen nionths of agc (23) t From Little, Murray and Cloiidman (23). When hightumor " A " stock females are mated to lowtumor " B " stock males the resulting first generation mice are called ABF, hybrids. Crossing in the opposite direction gives BAF, hybrids. Hybrids derived by mating " B " females to " A " males and fostering the young to " A " stock females are designated BAF, by A. Fostered '' A " stock females (A by B ) which were tested as noncancerous mothers were mated to " B " stock males to produce the socalled ABF, by B? class. This method eliminated the nursing of " A " stock hightumor females by the first generation young for a period of twentyfour hours or less. Hybrids have been produced in this manner but are not included in this report. Breeding Females: Tables and 1 show the number of animals in each group, the breast tumor percentages, and the average breast cancer and nonbreast cancer ages. For the use of the " B " or C57 black stock control data am indebted to Dr. Little (23). The breast tumor ratios in the parental " A " and " B " stocks were 83.6 per cent and.5 per cent respectively. The proportion of ABF, mice to develop breast cancer was 94.9 per cent. Mice of this generation descended from " A " stock fostered mothers (ABF, by B ) have given.7 per cent cancer with 52 per cent of the mice still living, the youngest being older than the age attained by any ABF, control individual. The respective ratios for the BAF, and BAF, by A? classes were 1.9 per cent and 89.8 per cent. Tumors were observed in 71.4 per cent of the ABF, mice and in none of the BAF, animals. The A by B data represent the fostered mice and the descendants of the noncancerous fostered mothers (mice of only one generation were fostered).

3 16 JOHN J. BTTNER TABLE 11: Number of Breeding Fembks Dying With () and Without () Breast Cancer during Each TrimontMy Age Period Total la ABF, ABFi by B , %dY9 71.4%ca?.7% ca. Age Total % ca % ca ca % ca. Age * Ninetyseven living. TABLE 1 : Number of Virgin Femak Mue Dying With () and Wirhout (N. T.) Breast Cancer during Each TrimontMy Age Period A ABFi ABFt B* BAFi BAF Total %~~ 4.574ca. l.f%ca.. ca The ratio was 6.4 per cent for the 652 mice observed. (Seven fostered A stock females were tested as transmitting breast cancer. f they and their descendants are excluded, the ratio for the others (619) is 3.7 per cent.) The number of fostered B by A stock mice and their progeny totalled 97, with a breast tumor incidenceof 11.3 per cent. Table 1 gives the number dying during each age period. The data are presented graphically in Figs. 1 to 4.

4 FG. 1. BREEDNG FEMALES: PERCENTAGE LVNG TO THE BEGNNNG OF EACH AGE PEROD OR LOXGER 71 c W u e W 5 25 MONTH8 FG. 2. BREEDNG FEMALES: PERCENTAGE OF TOTAL N EACH CLASS,DYNG DURNG EACH AGE PEROD 4c 3 P Y z," 2 w n C O FO. 3. BREEDNG FEMALES: PERCENTAGE OF TOTAL N EACH CLASS DYNO WTH BREAST CANCER DURNG EACH AGE PEROD 17

5 18 JOHN J. BTTNER Little difference was noted in the average ages of the A, ABF,, and BAF, by A classes (Table ). The groups descended from " B " mothers or nursed by " B " stock females lived longer and the average breast tumor ages were FG. 4. 'ns BREEDNG FEMALES: PERCENTAGE LVNG TO THE BEGNNNO OF EACH ACE PEROD OR LONGER TO DE WTH BREAST CANCER higher. t is of interest that the noncancerous animals of the ABF, generation survived to an average age of 18.1 months, while similar ABF, mice averaged only 12.8 months at death. The noncancer age for the ABF, by B? class will be considerably increased following the death of all the animals in this group. Virgin Females (Tables and 111): The incidence of breast cancer observed in the " A '' stock virgin females, 4.9 per cent, was not exceeded by any hybrid group. Tumors developed in 4.5 per cent of the ABF, and 1.7 per cent of the ABF, mice. Growths were not observed in the B, BAF, and BAF, classes. The number of each group dying during each age period is given in Table 111 and in Figs. 5 to 7. The average breast tumor ages in the virgin females are much higher than those recorded for the breeding females. Similar observations were made when comparing the ages of the noncancerous animals. DSCUSSON The castration experiments of Loeb (2425), Cori (26), and Murray (2 7 29) demonstrated the r61e played by the ovarian hormones in the etiology of breast tumors in mice. A reduction in the amount of secretion resulted in a reduction in the breast tumor incidence, and the earlier the animals were spayed the more significant was the decrease. Lacassagne (3) and others ' have observed that by the injection of estrogenic hormones in mice breast tumors can be produced in males of strains in which the females normally produce this type of tumor, but strains in which the normal breeding females are refractory to breast tumor development were found not to respond to the injection of the hormones with breast tumor production (Lacassagne, 32; Suntzeff, Burns, Moskop, and Loeb, 33). 2See Gardner (31) for a comprehensive review of the work on the relation of estrogenic hormones and cancer.

6 TRANSMSSON OF SUSCEPTBLTY TO BREAST CANCER 19 That the carcinogenic action of the estrogenic hormones is not specific for the development of mammary cancer has been shown by Cori (26), Gardner, Smith, Strong, and Allen (34), Loeb, Burns, Suntzeff, and Moskop (3536) c W 2 u W n FG \ \ \.. VRGN " ' t MONTHS VRGN F~MALES: PERVENTACE OF TOTAL N EACH CLASS LVNG TO THE BEGNNNG EAmi AGE PEROD OR LONGER OF 5 2 W 2s L W n P t27 MONTHS FG. 6. VRGN FEMALES: PERCENTAGE OF TOTAL N EACH CLASS DYNG DURNG EACH AGE PEROD r c 2 W u n W FG MONTHS VRGN FEMALES: PERCENTAGE OF TOTAL N EACH CLASS LVNG TO TfE BEGNNNG OF EACH AGE PEROD OR LONGER TO DE WTH BREAST CANCER and Lacassagne (3738), who found that some animals, usually from lowbreasttumor lines, may develop any of several types of tumor not characteristic of the strain.

7 11 JOHN J. BTTNER TABU V: Comparison oj Observation witb in Brccdittg FiCnOlcs "A" Q Q.. X _.. "B" $3 Milk susceptibility * Hormonal Expectation 1 bserva t ion 84% ABFi 1% BAFr By AQ Milk Susceptibility * Hormonal Expectation 1 Observation 95% ABF, Milk Susceptibility 3:1 Hormonal Expectation 75% Observation 71% * Based on one factor. By BQ 'r ABF, 3:1? BY BQ 'i". * BAF, 3:1 BY A :1 BAFr 75%? nherited breast cancer constitution and ovarian secretion have been considered as necessary for the development of breast tumorsat least in mice. Let us consider the above results, assuming that one additional " influence " is necessary (13). This factor may be: (1) A " breasttancerproducing influence " transmitted in the milk of potentially cancer stock females and obtained by the young while nursing. Only a smal amount is needed, as even young which were allowed to nurse their hightumor stock mothers for two days before being transferred to lowcancer foster mothers gave a high tumor ratio. The " influence " may be transferred by the inoculation of normal tissue from fourweeksold animals of a hightumor strain (18). The nature of the " influence " has not been determined; that it acts as a catalyst and occurs in many if not all of the organs seems probable. t may also be that it can multiply in the tissues of the host. (2) A breasttancersusceptibility factor which is transmitted as a dominant complex. The evidence supports the theory that only one factor is involved, but other explanations are possible (Wright, 44). (3) A hormonal stimulation which may or may not result from the production of young. A sufficient amount to stimulate the growth of the mammary gland tissue to a point necessary for the cancerous change to take place, in the presence of the other essential influences, is obviously present in mice of strains which have a high incidence in virgin females. Hightumor strains of mice inherit the breast cancer constitution, while the breastcancerproducing influence is transmitted through the milk. Whether or not a sufficient amount of the hormonal secretion is present in virgin females

8 TRANSMSSON OF SUSCEPTBLTY TO BREAST CANCER 111 TABLE V: Comparison oj Observation and Expectation in Virgin Females A Milk Susceptibility * Hormonal Expectation Observation 5% Milk Susceptibility * Hormonal Expectation Observation 5% BAFl Milk Susceptibility * Hormonal Expectation Observation *Based on one factor. ABF2 3:1 2% O r BAF2 3:1 depends upon the stock. The average age at which tumors are observed may be an expression of the amount secreted, the other factors being equal. Little (21) by transplanting fertilized ova and Andervont and McEleney (22) by foster nursing found that the ratio could be reduced in breeding females of strains which showed high ratios in virgin females (22, 7), confirming the work on a strain having a low virgin incidence. A line of C57 black mice, which had one breast tumor in the control stock (18; 23), produced a few tumors following foster nursing by hightumorstock females. Andervont and Mc Eleney (22) obtained negative results in another test. Similar results were found by Bagg (3941) and Little and Pearsons (42) when two groups of B stock mice were subjected to the functional test. n Tables V and V we have compared the breast tumor data for the hightumor A and the lowtumor B strain breeding and virgin females with the theoretical expectations according to this theory. First generation mice produced by crossing these strains would inherit the breast cancer susceptibility from the (A stock parent of either sex. Assuming one dominant factor to be involved, all of the F, generation and 75 per cent of the F, hybrids should be cancerous as far as this complex is concerned. Pure stock and hybrid females used as breeders would have the hormonal influence which would be lacking in all virgin females. All the animals which were nursed by A stock females received the socalled breastcancerproducing influence through the milk. Conversely, all mice nursed by B stock females would lack this influence. ( A stock breeding females should theoretically be breast cancerous, as they have all the essential influences (milk, inherited, hormonal). The observed incidence for multiparous females of this strain was 84 per cent. The B strain animals would lack the inherited susceptibility factor and the milk influence and should give no cancer, as compared with the recorded ratio of

9 112 JOHN J. BTTNER.5 per cent (23). A stock mice fostered to B stock females and B strain animals nursed by A line females should all be negative. The percentages were 6 per cent and 11 per cent. (The ratio of 4.9 per cent observed for virgin (( A stock females might be used for the expected incidence for fostered A stock breeding females.) All first generation females would inherit the breast cancer susceptibility factor from their maternal or paternal A stock parent. Hybrids nursed by A stock females and used as breeders (ABF, and BAF, by A ) would all be expected to be cancerous (95 per cent and 9 per cent observed respectively), whereas the groups nursed by B strain females should have no cancer. The recorded results were 2 per cent for the BAF, and 1 per cent for the ABF, by B classes (as stated above the ABF, by B hybrids were produced by mating A by B? females to ( B males). Seventyfive per cent of the ABF, and BAF, hybrids should have the genetic cancer constitution, assuming one factor to be transmitted for susceptibility (3 : 1 ). Both classes were used as breeders and thus the mammary tissue was insured the hormonal stimulation. The milk influence was common to ABF, animals (from the A stock grandmothers) but was absent from BAF, mice. The observed breast cancer ratio in ABF, breeding females was 71 per cent (75 per cent expected) and in the BAF, animals. (ABF? by B and BAF, by A hybrids are under observation, as are the third generation animals of all four classes.) Virgin mice of the A and B stocks and their hybrid generations would be wanting the hormonal stimulation needed by the hightumorstock mice for tumor development. n addition the 3 strain mice and BAF, and BAF, hybrids would lack the milk influence. Thus, all the pure stock mice and their hybrids should be free of breast cancer when maintained as virgins. The observed tumor ratios for { A, ABF, and ABF, mice were 4.9 per cent, 4.5 per cent and 1.7 per cent respectively. None of the B, BAF, or BAF, virgin females developed mammary tumors. n inbred strains of mice having a high breast tumor ratio the progeny descended from nonbreast cancerous mothers do not show a low breast tumor ratio (43, 11). Such mothers are able to transmit this type of neoplasm to their descendants although they themselves have not developed mammary cancer. n lowbreastcancer strains which have been inbred the progeny of breast cancerous mothers do not show a higher incidence than is characteristic of the stock. As these neoplasms are not transmitted in lowtumor lines, further work must determine if they have a genetic basis. CONCLUSONS (1) The development of breast carcinoma in highcancer strains of mice is probably due to three influences : (a) breastcancerproducing influence transmitted in the milk of breastcancer stock mothers; (b) an inherited breast cancer susceptibility of one or more dominant factors; (c) a hormonal stimulation influence. (2) The breast tumor incidence of a hightumor stock may be reduced by fostering one generation of young to lowtumor stock females.

10 TRANSMSSON OF SUSCEPTBLTY TO BREAST CANCER 113 (3) No significant increase in the breast tumor ratio was observed from fostering lowcancer stock young to highcancer females. (4) The incidence of breast tumors in first generation hybrid mice which inherit the breast cancer susceptibility and have the hormonal stimulation may be increased or decreased as the result of nursing. (5) Breast tumors which are not transmitted may result from other causes. LTERATURE CTED 1. STAFF, JACKSON MEMORALABORATORY: Science 78: 465, STAFF, JACKSON MEMORAL LABORATORY: Am. J. Cancer 27: 551, KORTEWEG, R.: Nederl. tijdschr. v. geneesk. 78: 24, KORTEWEG, R.: Genetica 18: 35, KORTEWEG, R.: Nederl. tijdschr. v. geneesk. 8: 48, KORTEWEG, R.: Acta, nternational Union Against Cancer 2: 136, MURRAY, W. S., AND LTTLE, C. C.: Genetics 2: 466, MURRAY, W. S., AND LTTLE, C. C.: Science 82 : 228, MURRAY, W. S., AND LTTLE, C. C.: Am. J. Cancer 27: 516, BTTNER, J. J.: J. Heredity 27: 391, BTTNER, J. J.: Proc. SOC. Exper. Biol. & Med. 34: 42, BTTNER, J. J. : Quart. Rev. Biol. 13: 51, BTTNER, J. J.: Pub. Health Rep. 54: 1113, CLOUDMAN, A. M., AND LTTLE, C. C.: J. Genetics 32: 487, BTTNER, J. J.: Science 84: 162, BTTNER, J. J.: Am. J. Cancer 3: 53, BTTNER, J. J.: Am. J. Clin. Path. 7: 431, BTTNER, J. J.: J. Heredity 28: 363, BTTNER, J. J.: Am. J. Cancer 35: 9, BTTNER, J. J.: Am, J. Cancer 36: 44, BTTNER, J. J.. AND LTTLE, C. C.: J. Heredity 28: 117, ANDERVONT. H. B., AND MCELEYEY, W. J.: Pub. Health Rep. 53: 777, LTTLE, C. C., MURRAY, W. S., AND CLOUDMAN, A. M.: To appear in Genetics. 24. LOEB, L.: Science 42: 912, LOEB, L.: J. M. Research 4: 477, COR, C. F.: J. Exper. Med. 45: 983, MURRAY, W. S.: Science 66: 6, MURRAY, W. S.: J. Cancer Research 12: 18, MURRAY, W. S.: J. Exper. Med. 63: 893, LACASSAGNE, A.: Compt. rend. Acad. de sc. 195: 63, GARDNER, W. U.: Arch. Path. 27: 138, LACASSACNE, A. : Am. J. Cancer 2 7 : 2 17, SUNTZEFF, V., BURNS, E. L., MosKoP. M.. AND LOEB, L.: Am. J. Cancer 27: 229, GARDNER, W. U., SMTH. G. M., STRONG, L. C., AND ALLEN, E.: Arch. Path. 21: 54, LOEB, L., BURNS, E. L., SUNTZEFF, V., AND MOSKOP, M.: Canad. M. A. J. 35: 117, LOEB, L., BURNS, E. L., SUNTZEFF, V., AND MOSKOP, M.: Am. J. Cancer 3: 47, LACASSACNE, A.: Compt. rend. SOC. de biol. 126: 19, LACASSACNE, A.: Bull. Assoc. franc. p. '6tude du cancer 27: 96, BAGG, H. J.: Science 83: 374, BAGG, H. J.: Am. J. Cancer 27: 542, BAGG, H. J., AND JACKSEN, J.: Am. J. Cancer 3: 539, LTTLE, C. C., AND PEARSONS, J.: Am. J. Cancer 38: 224, STRONG, L. C.: Am. J. Cancer 25: 599, WRGHT, S.: Genetics 19: 537, 134.