ON THE PROLIFERATIVE CHANGES TAKING PLACE IN THE EPITHELIUM OF VAGINA AND CERVIX OF MICE WITH ADVANCING AGE AND UNDER THE INFLUENCE OF EXPERIMENTALLY ADMINISTERED ESTROGENIC HORMONES 1
V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
(From the Laboratory of Research Pathology, Washington University School 01 Medicine, St. Louis)
In the course of our studies of the effects of estrogen injections on the epithelium of the mammary gland in various strains of mice we observed the development of epithelial processes in vagina and cervix reaching downward into the underlying connective tissue and becoming carcinoma-like under the influence of this substance. Comparing these experimental animals with nor mal, non-injected mice, we found that long processes, and even processes re sembling early stages of cancer, may also develop spontaneously, without in jections of estrogen, though apparently less frequently. There exists, then, a noteworthy analogy between the behavior of the non-stimulated and that of the estrogen-stimulated mammary gland on the one hand, and the non-stim ulated and estrogen-stimulated epithelium of vagina and cervix on the other (1,2,3). In the study of these processes in vagina and cervix it is not primarily our aim to describe the precancerous or early cancerous lesions of the epi thelium, but to consider the gradual changes which take place in this epi thelium from early to advanced age, with and without the stimulation of estrogen and other hormones, in mice belonging to various strains, and the eventual transition from normal growth processes to precancerous and early cancerous proliferations. Before proceeding to the description of these changes it will be necessary to discuss briefly some facts concerning the struc ture of the vagina and especially of the cervix in the mouse. In our investigations into the alterations which take place during the sexual cycle in the vagina, cervix, and uterus of the guinea-pig (4), we found that the cervix may be divided into three parts, namely, the vaginal, inter mediate, and uterine cervix. The structure and mode of reaction of the vaginal cervix are similar to those of the vagina, while the uterine cervix re sembles the uterus in these respects, although both these parts differ to some extent from the adjoining areas in vagina and uterus. There exists thus a graded transition from vagina to uterus as far as structure and response to estrogen and lutein hormones are concerned. In the mouse the differentiation of the various parts of the cervix is less marked than in the guinea-pig, but here also there are indications that it exists.
1 These investigations were carried out with the aid of a grant from the International Cancer Research Foundation. We are indebted to the Schering Corporation for part of the estrogenic substance used in the experiments. 256 PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 257
Thus there is a gradual decrease from vagina to uterus in the height of the squamous epithelium which develops under the influence of estrogen. This may be taken as a suggestion of graded changes in the constitution of the epithelium of the cervix in the direction of the uterus; but the distinction between vaginal and medium cervix is less sharply defined than in the guinea pig. Likewise, an area of transition seems to exist between the uterus and cervix. Where the mucosa begins to be covered by a single layer of epi thelium, this cell-layer still possesses to a certain extent a tendency to become changed into a low squamous epithelium; while such a change may take place in the uterus proper, this tendency is greater in the lower uterine tissues than in those higher up. In order to simplify the description of alterations which take place in the vagina, cervix and the beginning of the uterus, we have divided this tract into four parts: The vagina up to the level at which the cervix begins is designated
FIG.!. SCHEMATIC REPRESENTATION Ol!' VAGINA AND CERVIX Ol!' MOUSE
as area 1; that part of the vagina which consists of folds running parallel to the cervix as area 2, including the portio, which connects the vaginal folds and the beginning of the uterus; the cervix proper as area 3, and the beginning of the uterus, corresponding to the cervix in the guinea-pig, as area 4. These subdivisions are shown in Fig. 1. The changes which are analyzed in this paper affect not only the vagina proper, but also the portio and the cervix, and they may extend to the beginning of the uterus. The whole tract extending from the beginning of the vagina to the beginning of the uterus is designated as VC in the tables, and occasionally also in our discussion. In order to describe the downgrowth of the' epithelium into the underlying connective tissue and the formation of epithelial processes in vagina, cervix, and the beginning of the uterus, in an approximately quantitative manner in different individuals and strains, we have adopted the following terminology: 258 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
Grade 0 signifies either the absence of downgrowth or the presence of only very short epithelial processes. Grade 1 represents epithelial processes ex tending for a moderate distance into the underlying tissue. Grade 2 signifies epithelial processes which reach to a considerable depth and which may, in addition, have begun to branch out. Grade 3 indicates a precancerous down growth, extending deeply and invading the connective tissue irregularly, with branching processes, frequently showing hyaline pearls. Grade 4 designates a marked cancer-like invasion of the underlying connective tissue and adjoin ing parts. One of the points which we wished to determine was whether a relation exists between the degree of proliferation in the vagina and cervix (VC) in different individuals and strains and that in the mammary gland. For this purpose we have recognized the following grades of proliferation in the mam mary gland. Grade 0 signifies that mammary gland ducts are seen very rarely. Grade 1 designates a mammary gland tissue consisting of frequent branching ducts, which mayor may not be surrounded by a very small number of acini. Grade 2 indicates a precancerous proliferation of the mammary gland, and Grade 3 complete carcinomatous transformation. Altogether we studied the condition of vagina and cervix in 375 mice; 128 of these served as controls, while 235 were injected with estrogenic hor mones, 52 of the latter group receiving, in addition to estrogen, preparations of anterior pituitary or lutein hormone, or of both. These 52 mice we have designated as combination-treated mice. In addition 12 mice were injected with lutein or anterior pituitary preparations without estrogen. Included in the total series of 375 mice is an injected" New Buffalo" mouse to which no VC grade could be assigned. It was found that the depth to which the epithelial processes penetrated into the underlying connective tissue varied greatly in different mice. In some almost no processes were present, while in others they were so strongly developed in places that a precancerous state or one of early cancer presented itself. Intermediate conditions of all kinds existed. Even in the same mouse the character of the epithelial processes often differed in different areas of the vaginal-cervical tract; these differences were recorded by assigning dif ferent grades to different areas. In this way it was possible to compare the average degree of the development of the vaginal-cervical epithelial processes in different individuals. Notwithstanding the presence of variations within the same animal, it was possible to correlate the degree of development of the processes with (1) the age of the mice and (2) the nature and quantity of the hormones injected. Vagina, cervix, uterus and mammary gland, and often also other organs, after the death of the animals, were studied in sections and the ovaries were cut into serial sections. The large number of mice used in our experiments made the analysis of our findings by statistical methods possible. Two difficulties, however. had to be overcome. In the first place the assigning of grades to VC in different mice depends upon the judgment of the individual observer and is therefore not entirely objective. In order to eliminate this personal factor as much as possible two persons graded the microscopic specimens independently of each other, after a third investigator had described the histologic changes. PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 259
TABLE I: Epithelial Processes and Age at Death in Controls of Various Strains (Total Controls: 128)
VC-o VC-l VC-2 VC-3 and VC-4
Strain Age at Age at Age at Age at Number death Number deathNumber death Number death of mice (months) of mice (months) of mice (months) of mice (months}
A 21 10.0 26 9.5 3 15.5 C57 12 8.5 12 8.3 3 17.7 D 8 12.3 1 22.0 3 16.0 1 20 C3H 5 8.4 3 16.7 CBA 4 9.6 6 10.2 New Buffalo 1 18.0 3 20.0 4 18.0 Old Buffalo 8 17.1 4 21.8
Number and av- 59 (46% . 10.9 55 (42.9% 11.4 13 (10.2% 16.9 1 (0.8% 20 erage age at of total) of total) of total) of total) death
While there were some differences between the grades given by these observers, these were, on the whole, slight. A second difficulty consisted in the fact that the age at which the mice died or were killed with chloroform depended upon certain accidental factors and differed in different groups of mice. It was necessary, therefore, in all cases to take into consideration these variable conditions in evaluating the significance of the age factor. There seem to be present, in addition, variable factors of an unknown kind which may influence the development of epithelial processes in the vaginal-cervical tract in indi vidual instances. We may therefore find irregularities in the results if we consider very small numbers of mice, but these usually disappear when the number of mice subjected to analysis becomes greater. Seven different strains were used in our investigations; they were the same which had served in our previous experiments. Three of these were high tumor strains, namely Strains A, D and C3H; in these strains spontaneous mammary gland tumors were frequent in female mice which had been allowed to breed; in the non-breeders the tumor rate was much lower. One strain, "New Buffalo," had an intermediate tumor rate, and three were low-tumor strains, namely" Old Buffalo," CBA, and C57. Table I shows the average age at death in the various strains. The mice are arranged in accordance with the grades of proliferative change in VC. The first figure in each case represents the number of individuals belonging to a group and the second.figure the mean age at time of death in this group. Thus in strain A 21 mice showed grade 0 in VC, and the mean age at death in these 21 mice was 10 months; 26 mice showed grade 1, with a mean age at death of 9J months, and 3 mice grade 2, with a mean age at death of 15.5 months. The control mice of strains C57, D, C3H, CBA, New Buffalo and Old Buffalo are analyzed in the same manner, and the means of the various grades of all the strains are given. There is not a great difference in the mean ages at which grades 0 and 1 were found, although grade 1 appeared a little later than grade O. This is probably due to the fact that the differentiation between grades 0 and 1 is not always sharp. Accidental differences, there- 260 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
TABLE II: Epithelial Processes and Age at Death in Ovariectomized Controls
VC-o VC-1 Strain Number Age at death Number Age at death of mice (months) of mice (months)
C3H 3 7.0 CBA 1 10.5 1 10.5 D 2 B.O
Number and average 6 7.8 1 io.s age at death fore, may play a certain role and complicate the results in assigning these grades. If, however, we add the 2- and 3-grade mice to the grade 1 group the difference between 0 and the other mice becomes quite definite. The average age at time of death of the group with grade 0 is 10.9 months, and of those with the higher grades 12.6. So far as grades 2 and 3 are concerned, their averages are markedly higher than those of group 0 and group 1 in the total counts. This difference is noted also in the individual strains, with a few exceptions due to the small number of mice involved. Forty-six per cent of all the control mice showed grade 0, 42.9 per cent grade 1, 10.2 per cent grade 2, and 0.8 per cent grade 3. The large majority of mice died before they reached grade 2, and only one of the mice reached grade 3. Grades VC-2 and VC-3 occurred in strains A and D, two high tumor strains, and also in strains CS7 and New Buffalo. They were found, therefore, at least in as great a percentage in low- and intermediary-mammary tumor strains as in those with a high mammary tumor rate. There is thus no indication that the frequency of mammary gland tumors is correlated with proliferative activity in vagina and cervix as far as the distribution of these changes in various strains is concerned. Among the control mice a few were ovariectomized. Among these animals, as shown in Table II, the average age at time of death was somewhat lower than among the non-ovariectomized animals. In all except one VC showed grade O. It seems that in spayed female mice the surface epithelium in VC is very inactive; otherwise no definite conclusion can be drawn from these figures. Table III shows the average age at death of mice injected with estrogen, with or without addition of other hormones; as in, Table I, these mice are arranged in accordance with the grade given to VC. Throughout this series it was found that the older the mice the greater the depth to which the epi thelial processes penetrate downward into the connective tissue. 1.'his applies to the total averages of all the mice as well as to the separate strains, except in cases in which the number included in a group is very small. In particular, the mean ages at which grades VC-2 and VC-3 appear are considerably greater than the mean ages for the lower grades. The average for grade 3 in the total number of mice is higher than that for grade 2. As in the controls, so also in the injected animals, the number of mice attaining the higher grades PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 261
TABLE III: Epithelial Processes and Age tU DeiUh in Estrogen-injected Mice, Including Ovariectomiud and CombiniUion·treated Mice (Total Injected Mice: 234)
VC-O VC-l VC-2 VC-3 and VC-4
Strain Age at Age at Age at Age at Number deathNumber death Number deathNumber death of mice (months) of mice (months) of mice (months) of mice (months)
A 30 8.7 25 11.3 7 13 13 15.4 C57 6 7.4 16 11.0 12 17.7 J 14.7 D 13 9.8 14 12.7 11 14.8 4 20.4 CJH 17 7.9 22 8.6 3 13.7 3 11.7 CBA 3 6.2 7 8.3 1 10.5 New Buffalo 3 9.8 4 13.7 3 20.3 1 5.5 Old Buffalo 5 lOA 6 14.1 1 14.0 1(VC·4) 25.0
Number and av- 77 (32.9% 8.7 94 (40.2% 10.9 38 (16.2% 14.5 25 (10.7% 15.7 erage age at of total) of total) of total) of total) death is relatively smaller than that with the lower grades, and the proportion of these mice becomes smaller the higher the grade. But the percentage of injected mice which reach the higher grades is much greater than that of the controls; it appears that under the influence of the injected hormones, in par ticular of estrogen, an increase in the depth of penetration of the epithelial processes into the underlying connective tissue is ultimately reached. Ac cordingly, we find that only the youngest mice have grade VC-o, and that grades VC-l, VC-2 and VC-3 appear at earlier ages than in the non-injected controls. We may thus conclude not only that the absolute number of mice in which VC reaches grades 2 and 3 is greater than in the controls, but also that the proportion of mice which reach the higher grades is greater. A comparison of Tables I and III shows it to be probable, therefore, that under the influence of estrogenic hormone the epithelial processes in VC penetrate into the underlying connective tissue with greater intensity with advancing age than in control mice. This conclusion is further confirmed by the fact that in a number of mice injected with estrogenic hormone grades 2 and 3 ap pear at an age when in the control mice only grades 1 and 2 were found. Yet there is an accidental factor involved here which is at least partly responsible for the earlier age at which the injected mice were examined. As the result of the injections of estrogenic hormones mammary gland carcinoma appears on the average at an earlier age and in a larger proportion of mice, and death occurs, therefore, at an earlier age. It may be, also, that other factors of an accidental nature caused death at an earlier age of a greater proportion of the estrogen-injected mice than of those not injected. As in the control mice, so also in the mice injected with estrogen there is no significant difference in the relative distribution of grades of VC in low and high-mammary-gland-tumor strains (Table IV). There is, furthermore, no marked difference in the mean ages at which the higher grades in VC ap pear in high- and low-tumor strains. No definite correlation exists, therefore, between the tendency to tumor formation in the mammary gland and the pro- 262 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
TABLE IY: Distribution of VC Grades in High-tumor (A, D, C3H) and Medium- and Low-tumor (C57, CBA, New Buffalo, Old Buffalo) Strains
YC-O YC-l YC-2 YC-3 YC-2+YC-3
High-tumor strains ...... 60(37%) 61(38%) 21(13%) 20(12%) 25% Medium- and low-tumor strains .... 17(23.6%) 33(46%) 17(23.6%) 5(7%) 3G.6% - liferation of the epithelium in vagina and cervix into the connective tissue in the various strains. The development of epithelial processes in vagina and cervix in ovariec tomized mice injected with hormones is shown in Table V. Apparently
TABLE Y: Epithelial Processes in Estrogen- and Combination-treated Ovariectomized Mice • (Total Number of Mice: 62)
VC-O YC-l YC-2 VC-3 and YC-4
Strain Age at Age at Age at Age at Number death Number deathNumber death Number death of mice (months) of mice (months) of mice (months) of mice (months) ---- A 9 7.7 7 8.1 1 12.0 (3 mice: 12 months; others younger) C57 I 1 7.0 I 8 5.9 2 11.25 1 16.5 (2 mice: 16 months; all others younger than 12 months) D I 2 7.0 2 9.5 (None reached age of 12 months) C3H I 10 7.15 11 7.1 1 6.5 (l mouse reached age of 12 months) CBA I 3 6.2 4 6.8 (None reached age of 12 months)
Number and av- 25 (40.3% 7.2 32 (51.6% 7.1 3 (4.8% 11.5 2 (3.2% 11.5 erage age at of total) of total) of total) of total) death
• Among the total injected ovariectomized mice, 4 reached the age of 12 mos.: 2 reached the age of 16 mos.: 6 mice reached 12 mos. or more. grades 2 and 3 are reached here in a lower percentage than in non-ovariec tomized mice, namely, VC-2 in 4.8 per cent and VC-3 in 3.2 per cent. In appraising the significance of these figures, however, we must consider the fact that the ovariectomized mice on the average were examined at an earlier age than the non-ovariectomized mice; among 62 ovariectomized injected mice only 6 reached the age of twelve months or more; 4 reached the age of twelve months, and 2 sixteen months. At present .we cannot conclude, there fore, that injection of estrogen is less effective in stimulating downgrowth of epithelium in VC of ovariectomized than of non-ovariectomized mice. A number of mice, included in Table III, were injected with anterior pituitary hormones or lutein, alone or in combination with estrogen. Table VI shows the ages at death according to grades in VC in mice treated with a combination of 1/3-10 rat units of estrogen and 0.03 c.c. of acid extract of anterior pituitary, injected alternately every five days. Again it is seen that the depth of the processes increases with increasing age of the animals. PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 263
TABLE VI: Epithelial Processes in Mice Receiving Estrogen and Anterior Pituitary Extract (Total Number of Mice: 25)
VC-o VC-l VC-2 VC-3 and VC-4
Strain Age at Age at Age at Age at Number death Number death Number death Number death of mice of mice (months) of mice (months) of mice (months) (months)
A 7 6.6 1 19.5 1 23 C57 1 5.0 2 19.5 4 16.2 D 4 7.2 3 10.5 1 24.5 1 24
Number and av- 12 (48% 6.7 6 (24% 15 5 (20% 17.9 2 (8% 24 erage age at of total) of total) of total) of total) death
In Table VII are shown the ages at death of the mice which were injected with a combination of estrogen and lutein hormone; in these, as well as in some other experiments, we used either a preparation of lutein made in our laboratory or Schering's proluton. In most cases 100 rat units of estrogen and 0.15-0.25 rabbit units of proluton, both dissolved in oil, were injected weekly in strains A and C57. In strain D smaller quantities of estrogen and lutein hormone were used. A few mice belonging to the Old Buffalo strain
TABLE VII: EPithelial Processes in Mice Receiving Estrogen and Lutein (Total Number of Mice: 20)
VC;::-O VC-l VC-2 VC-3 and VC-4
Strain Age at Age at Age at Age at Number Number death Number death Number death death of mice (months) of mice (months) of mice (months) of mice (months)
A 1 6.0 4 10.7 1 12 1 14 C57 1 7.0 4 11.6 1 16 2 16.2 D 4 10.4 1 16.0
Number and av- 6 (30% 9 9 (45% 11.7 2 (10% 14 3 (15% 15.5 erage age at of total) of total) of total) of total) death were injected with small quantities of all three hormones. These mice are included in Table VIII, in which all the mice injected with estrogen and, in addition, with one or two of the other hormones, are shown. It is seen that in all of these mice the depth of the processes increased with increasing age. On the whole, there is no marked difference between the results obtained in these groups and those in which the mice were injected with estrogen alone. If, however, we consider separately the mice of strains A and C57, which were injected with 100 rat units of estrogen and 0.15-0.25 rabbit units of proluton weekly (see Table VII) we find the following figures: VC-o, 2 mice, 6.5 months; VC-1, 8 mice, 11.2 months; VC-2, 2 mice, 14 months; VC-3, 3 mice, 15.1 months. These figures suggest that such a combination may per- 264 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
TABLE VIII: Epithelial Processes in Mice Receiving Estrogen and A nterior Pituitary Extract, and Estrogen and Lutein (Total Number of Mice: 52)
VC-o VC-1 VC-2 VC-3 and VC-4
Strain Age at Age at Age at Age at Number Number death Number death Number death death of mice (months) of mice (months) of mice (months) of mice (months)
A 8 6.5 5 12.5 1 12.0 2 18.5 C57 2 6.0 6 14.3 5 16.2 2 16.2 D 8 8.8 4 11.9 1 24.5 1 24.0 Old Buffalo • 6 12.5 1 13.0
Number and av- 24 8.7 16 13 7 16.8 5 18.7 erage age at death Number and av- 18 (40% 7.4 15 (33.3% 13 7 (15.6% 16.8 5 (11.1% 18.7 erage age at of total) of total) of total) of total) death exclud- ing Old Buffalo strain
• Injected with all three substances. haps be more effective in causing a marked epithelial proliferation in VC than estrogen alone. Before such a conclusion can be reached, however, further experiments, with a larger number of animals, will have to be made. So far we have considered the relation between the penetration of epi thelial processes into the connective tissue of VC and the age of the mice examined. We have found that with increasing age the processes penetrate more deeply into the connective tissue, until in the end they produce precan cerous or early cancerous changes in a number of animals. We have seen, furthermore, that injections of estrogen alone or in combination with certain other hormones in all probability intensify and accelerate this process. But in these calculations we have disregarded the amount of estrogenic substance injected. We shall now inquire whether a relation exists between the amount of this substance injected into the various mice and the depth of the processes which developed and their change into precancerous proliferations. For this purpose we have arranged the mice injected with estrogen, or with combina tions of estrogen and the other two hormones, into three classes. Class I com prises those animals that were injected with 100 rat units of estrogen dissolved in oil once every week, with the addition, in some instances, of 50 rat units of estrogen dissolved in water, making altogether 150 rat units weekly. Class II comprises all those mice which received daily (except Sundays), or at least several times a week, a dose of estrogen less than 100 rat units dissolved in water. The maximum dose in this class was 50 rat units six times a week; but the large majority of mice belonging to this class received a smaller amount. It is to be noted that many mice belonging to Class II received an amount of estrogen at least as large as those in Class I, but dissolved in water instead of in oil. Since estrogen is apparently absorbed and eliminated much more rapidly when dissolved in water than when dissolved in oil, the TABLE IX: Epithelial Processesin Class I Estrogen-injecwd Mice (Total Number of Mice: 111)
VC-o VC-l VC-2 VC-3 and VC-4
Strain Age at Age at Age at Age at Number death Number deathNumber death Number death of mice (months) of mice (months) of mice (months) of mice (months)
A 13 7.5 11 8.0 2 10.8 3 10.8 C57 3 6.8 11 i.s 3 9.3 3 14.7 D 2 7.0 3 8.8 3 13.0 1 21.5 C3H 14 7.3 16 8.5 1 11.0 3 11.7 CBA 3 6.2 7 8.0 1 10.5 New Buffalo 2 8.8 1 7.0 1 17.0 1 5.5 Old Buffalo 2 5.0 1 12.0
Number and av- 39 (35.1% 7.2 50 (45% 8.1 11 (9.9% 11.5 11 (9.9% 12.6 erage age at of total) of total) of total) of total) death
TABLE X: Epithelial Processes in Class II Estrogen-injecwd Mice (Including Class III) (Total Number of Mice: 130)
VC-O VC-l VC-2 VC-3 and VC·4
Strain Age at Age at Age at Age at Number death Number death Number death Number death of mice (months) of mice (months) of mice (months) of mice (months)
A 17 9.6 14 13.9 5 13.9 10 \6.8 C57 3 R.O 5 18.4 9 20.6 D 11 10.3 11 13.8 8 15.4 3 20.0 C3H 3 11.0 6 10.3 2 15.0 New Buffalo 1 12.0 3 16.0 2 22.0 Old Buffalo 9 13.0 6 14.3 1 14.0 1 25.0
Number and av- 44 (33.8% 10.5 45 (31.2% 14 27 (21% 17.3 14 (11% 18.1 erage age at of total) of total) of total) of total) death
TABLE XI: Epithelial Processes in Class III Estrogen-injected Mile (Total Number of Mice: 26)
VC-O VC-l VC·2 VC-3 and VC·4
Strain Age at Age at Age at Age at Number death Number death Number death Number death of mice (months) of mice (months) of mice (months) of mice (months)
A 1 12 2 6.5 2 8.8 3 13.8 D 1 15.0 3 14.7 C3H 3 11 6 10.3 2 15.0 New Buffalo 2 14.0 1 20.0
Number and av- 4 (15.4% 11.2 11 (42.3% 10.7 8 (31% 13.9 3(11.5% 13.8 erage age at of total) of total) of total) of total) death
265 266 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
TABLE XII: Estrogen-treated Mice: Average Age at Death of All Mice and Mice with Mammary Tumors
Average age No. with Average age Strain No. of at death mammary at death mice (months) gland tumors (months)
CLASS I A 29 8.9 2 9.25 (9-9.5) C57 20 8.8 0 D 9 11.2 5 8.8 (7.5-10) C3H 34 8.1 16 10 (6-15.5) CBA 11 7.9 0 New Buffalo 6- 9.1 1 7.5 Old Buffalo 3 7.3
Total 112 8.7 24 9.6 Total with exclusion of strains in which mammary tumors did not appear 78 8.6 24 9.6
CLASS II A 46 12.9 17 15.6 (t 1-20) C57 17 17.7 0 D 33 13.6 19 16 (9-24.5) C3H 11 11.3 9 12.2 (8-15) CBA NeW Buffalo 6 17.3 2 22 (20-24) Old Buffalo 17 14.2 0
Total 130 14.0 47 15.4 Total with exclusion of strains in which mammary tumors did not appear 96 13.0 47 15.4
- In strain It New Buffalo" one mouse is here added in which no grade could be assigned to VC.
amount which could become effective and influence the genital tract was therefore presumably much greater in Class I. In Class III we consider sep arately those mice that received 50 rat units dissolved in water six times weekly, the largest dose of estrogen administered. If we consider Class I, which received the large doses of estrogen dissolved in oil, and Class II, which received smaller but as a 'rule more frequently re peated doses of estrogen dissolved in water, we find that the mice in Class II throughout all the grades reach, on the average, a greater age; but whereas with VC-o the difference between Class I and Class II is only three months, with the higher grades it is about six months; in other words, if estrogen is administered in a more effective manner the higher grades appear at an earlier age. In Class III (Table XI), representing mice injected six times weekly with 50 rat units of estrogen dissolved in water, the number of mice is relatively PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 267 small; but the age of the different grade groups is intermediate between those of Class I and Class II. The proportion of mice reaching the higher grades is greater in Classes II and III than in Class I, but this is probably due to the greater number of mice reaching a more advanced age in these classes as com pared with Class I. Table XII shows that the average age at death of all the mice belonging to Class II is much higher than those belonging to Class I, a fact which is true also of the separate strains. It is shown, furthermore, that this difference is not due to mammary gland tumors which develop in mice injected with estrin. Owing to the greater age reached by mice of Class II, the tumors are more numerous in that class than in Class I. In Class I the average age at which mammary gland tumors appear varies greatly in the different strains, but in the total of all the Class I mice the age at death of mice bearing mammary gland tumors is slightly higher than for the total number of Class I mice. In Class II the average age of the mice affected by mammary gland tumors ex ceeds definitely the average age at death of the total of Class II mice. This is true of the individual strains as well as of all the Class II mice combined. The fact that the average age at death of mice belonging to Class I is lower than the age at death of mice belonging to Class II is probably to be explained, at least partly, by the relatively large amounts of oil which in our earlier experiments had to be injected weekly in order to administer 100 rat units of estrogen. These large amounts of oil were not well tolerated. In later experiments a more concentrated preparation of estrogen in oil was used. However, the increasing differences between Class I and Class II which we find in the higher grades of VC as compared with the differences existing in the lower grades, make it probable that large doses of estrogen in oil given weekly are more effective than smaller doses in water given more frequently. This conclusion is confirmed by data which we shall present in subsequent tables. As stated above, the great differences which exist between different strains as regards their readiness to give origin to mammary gland tumors, do not exist as far as their tendency to develop precancerous or early cancerous le sions in vagina and cervix is concerned. In addition, it can be shown that within the same strain no definite correlation exists between the incidence of tumors in the mammary gland and of precancerous or early cancerous changes in Vc. We have compared for this purpose the degree of prolifera tion in the epithelial tissue of the mammary gland and of the downgrowth of the surface epithelium in VC in a number of mice in which marked prolifera tions occurred either in one site or the other. Mg signifies grades in mam mary gland tissue, VC signifies grades in vagina and cervix. The age at time of examination is stated in one or the other.
I. STRAIN A: Controls. (1) Mg:3, VC:o; 13.5 mos. (2) Mg:3, VC:O; 17 mos. (3) Mg:2, VC:l; 23 mos. (4) Mg:l, VC:2; 24.5 mos. Injected mice. (1) Mg:3, vc.i. 12.5 mos. (2) Mg:3, VC:l; 17 mos. (3) Mg:3, VC:2; 16 mos. (4) Mg:3, VC:3; 11 mos. (5) Mg:3, VC:1; 12 mos. (6) Mg:l, VC:3; 18 mos. (7) Mg:3, VC:1; 12 mos. (8) Mg:3, VC:1 +; 18 mos. (9) Mg:3, VC:1; 17 mos. (10) Mg:3, VC:2; 18 mos. (11) Mg:3, VC:1-; 11 mos. (12) Mg:3, VC:3; 18 mos. (13) Mg:l, VC:3; 18 mos. (14) Mg:3, VC:3; 19 mos. (15) Mg:l +, VC:3; 16 mos. (16) Mg:l, VC:3; 11 mos. 268 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
TABLE XIII: Ages of VC-3 Mite at Death at:Cording to Classes
Strain Class I Class II
A 8.5, 9.5, 14 mos. 11, 18, 18, 18, 19, 20, 16.5, 11, 14, 23 mos. C57 11.5, 16.5, 16 mos. D 21.5 mos. 20, 16, 24 mos. C3H 6.5, 13, 15.5 mos. New Buffalo 5.5~mos. Old Buffalo 25 mos.
II. STRAIN C57: Injected mice. (1) Mg:l, VC:3; 11.5 mos. (2) Mg:l +, VC:3: 16.5 mos. (3) Mg:l, VC:3; 16 mos. III. STRAIN D: Controls. (1) Mg:3, VC:3 +; 20 mos. (2) Mg:3, VC:2; 12 mos. (3) Mg:3, VC:2; 22 mos. Injected mice. (1) Mg:3, VC:O; 13 mos. (2) Mg:3, VC:l; 17 mos. (3) M~:3, VC:l; 17 mos. (4) Mg:3, VC:O; 14 mos. (5 and 6) Mg:3, VC:2; 11 and 18 mos. (7) Mg:3, VC:3; 20 mos. IV. STRAIN "NEW BUFFALO": Injected mice. (I) Mg:3, VC:2; 24 mos. (2) Mg:3, VC:2; 20 mos. (3) Mg:3, VC:l +; 17 mos. (4) Mg:l, VC:3; 5,5 mos. V. STRAIN" OLD BUFFALO '': Injected mice. (1) Mg:l, VC:4; 25 mos. These examples are sufficient to prove that in individual mice the tenden cies to the development of mammary gland cancer and of precancerous or early cancerous changes in VC are not identical. In some animals, however, marked growth in both of these organs may be found, as is to be expected especially since old age and large quantities of estrogen promote tissue proliferation in both places. Other factors differ, due presumably to differences in the hered itary tendency to the development of mammary gland carcinoma and of pre cancerous lesions in VC. This conclusion is also supported by the fact, pre viously mentioned, that the sharp differences which exist between some of the strains studied by us, as regards the hereditary tendency to the develop ment of mammary gland carcinoma, do not exist in the case of the proliferative activity of VC. But there still remains the possibility that some differences in hereditary proliferative tendencies may also obtain in the case of VC. We shall now analyze still further the incidence of VC-2 and VC-3 changes. It is the VC-3 proliferations which particularly interest us. In Table XIII we have divided the mice belonging to VC-3 into Class I and Class II. In Class I 8 mice were below the age of 16 months, one as young as 5.5 months, and 3 mice were 16 months or older. In Class II 11 mice were 16 months or older and only 3 were younger than 16 months, the youngest being 11 months old. These figures make it very probable that VC-3 actually occurred more frequently at an early age in mice injected with the large dose of estrogen dissolved 'in oil once a week than in those injected with the smaller quantities of estrogen dissolved in water. In Tables I and III we compared the percentages of grade 3 and grade 2 mice in reference to the total number of control and estrogen-injected animals without regard to age. Inasmuch, however, as grade 3 and also grade 2 occur for the most part in older mice, it might be more correct to determine the incidence of these two grades with reference to mice 12 months of age or older. PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 269
TABLE XIV: Controls: Number and Percentage of Mice More than Twelve Months Old with Grades 2, 3, and 4 in VC [ ) = Grade 3 and grade 2 mice below age of sixteen months
Percentage Percentage Age at death of grades Percentage of grades 2, Grades Grade 3 and 4 of grade 2 3 and 4 Strain 3and4 2 mice 12 mice 12 mice 12 mos. or 12-13 14-15 16 mos. mos. or mos. or mos. mos. or older older older older
A 12 3 5 0 3 [2] 15% [10%] C57 3 2 3 0 3 [1) 37% [12.5%1 D 1 3 6 1 [0] 3 [2] 10% 30% [20%] 40% C3H 0 2 1 0 0 CBA 0 2 0 0 0 New Buffalo 2 0 6 4 [21 50% [25%] 50% Old Buffalo 4 4 4 0 0
Total 22 16 25 1 13 (7) 1.6% 20.6% [11%] 22.2%
TABLE XV: Estrogen-injected Mice: Number and Percentage More than Twelve Months Old with Grades 2, 3, and 4 in VC [) = Grade 3 and grade 2 inice below age of sixteen months
Percentage Percentage Age at death of grades Percentage of grades 2, of grade 2' Strain Grades ' Grade 3 and 4 3 and 4 3and4 2 mice 12 mice 12 mice 12 12-13 14-15 16 mos. mos. or mos. or mos. or mos. mos. or older olderolder older '- - --- A 4 5 18 11 [5) 6[3] 41% [28.5%] 22% [11%1 63% C57 1 0 7 1 7 (2) 13% 87% [25%1 100% D 2 3 12 3 (0) 10(6) 18% 59% [35%) 77% C3H 4 6 0 3 3 30% 30% 60% eBA 0 0 0 0 1 New Buffalo 3 1 4 3 [0] 12.5% 37.5% 50% Old 1 • Buffalo 2 8 1 1 1 9% 9% 18%
Total 16 23 42 20 (5) 31 (11) 24.7% [6.2%1 38.3% [13.5%] 63%
Tables XIV, XV, and XVI show the number and percentage of mice with grades 2, 3 and 4 in VC in the control, estrogen-injected, and combination treated series, the percentage being figured with reference to the number of mice 12 months or older. The figures in brackets represent the absolute and percentage numbers of grade J and grade 2 mice which were below the age of 16 months, the percentage again being determined with reference to the number of mice 12 months or older. In the estrogen-injected series the per centage of grade 2 and grade 3 mice is considerably greater than iI;l the non injected control series. In the combination-treated series the results are similar to those in the estrogen-injected series; again, we see that the sharp 270 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
TABLE XVI: Combination-treated Mice I mInding Those Receiving Estrogen-Lutein and Anterior P~'tuitary Preparations: Number and Percentage MOTe than Twelve Months Old with Grades 2, 3, and 4 in VC () = (omitting "Old Buffalo" combination-treated mice (3 combinations). [) = Grade 3 and grade 2 mice below age of sixteen months
Percentage Age at death Percentage of Percentage of of grades 2, Grades Grade grades 3 and 4 grade 2 mice 3 and 4 Strain 3 and4 2 mice 12 mos. 12 mos. or mice 12 12-13 14-15 16 mos. or older older mos. or mos. mos. or older older
A 3 1 3 2 [1) 1 28% [14%1 14% 43% C57 1 8 2 (0) 5 [1) 22% 55.5%[11%1 77.5% D 4 1 [0) 1 (0) 25% 25% 50% (Old Buffalo 6 0 0)
Total 9 (3) 2 15 5 [1) 7 [1) 19% (25%) 27% (35%) 46% (60%) [5%1 [5%) Total es- trogen and cornbina- tion-treated mice 25 (19) 25 57 25 38 23.3% (24.7%) 35.5% (37.6%) 58.8% (61%) division which exists between mice belonging to high- and to low-mammary gland-tumor strains is not found as far as proliferative activity in VC is con cerned. As is to be expected, the percentage figures for grade 3 and grade 2 mice are considerably higher in Tables XIV, XV, and XVI than in Tables I, III, and VI. Table XVII shows that there is no marked difference between the fre quency with which grades 3 and 2 appear in mice treated with combinations of estrogen and anterior pituitary on the one hand, and combinations of estrogen and lutein on the other. Table XVIII shows that if we determine the percentage of mice reaching grades 2 and 3 below the age of 16 months on the basis of all mice, the difference between controls and estrogen-injected mice is maintained in the same way as when the percentages of mice below the age of 16 months reaching grades 2 and 3 is determined in reference only to mice below that age.
NATURE OF THE PRECANCEROUS OR EARLY CANCEROUS CHANGES (GRADES 3 AND 4) The localization of the precancerous and early cancerous changes in vagina and cervix is shown in Table XIX, which includes proliferations ranging in intensity from 3 - to 4. All types of transition are found, from a normal state, as represented by grade 2 to 2 +, through beginning precancerous changes, grade 3, to proliferations of the epithelium reaching far down and extending in a lateral direction in the underlying connective and muscle tissue, grades 3 + and 4. In addition to the varying degrees of invasion of the un derlying tissue by the proliferating surface epithelium, a variety of other PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 271
TABLE XVII: Combination-treated Mice: Number and Percentage More than Twelve Months Old with Grades J and 2 in VC
Age at death Percentage Grade Grade Percent- Percent- Strain 12-13 14-15 16 mos. 3 2 age of age of of grades mos. mos. or older grade 3 grade 2 2 and 3
Estrogen-anterior pituitary A 3 1 33% C57 5 4° 80% D 2 °1 1 50% 50% 100%
Total 10 2 5 20% 50% 70%
Estrogen-lutein . A 3 1 1 1 25% 25% 50% C57 1 3 2 1 50% 25% 75% D 2 ° ° Total 3 2 5 3 2 30% 20% 50%
TABLE XVIII: Total Number and Percentage of Grades 3 and 2 Mice Below Age of Sixteen Months - Age at death Percentage Grade Grade Percent- Percent- Series of grades 12-13 14-15 16 mos. 3 2 age of age of 2 and 3 mos. mos. or older grade 3 grade 2
Percentages determined in reference to all mice Controls 22 16 25 0 7 0% 11% 11%
Total 63 Estrogen- and com- bination-treated 19 25 57 11 17 11% 17% 28%
Total 101 Percentages determined in reference to mice below age of sixteen months Controls 22 16 0 7 0% 18% 18% Estrogen- and com- t bination-treated 19 25 12 18 27.3% 41% 68.3%
changes occur which are characteristic of carcinomatous growth, such as ir regular size of cells and nuclei and formation of giant cells from the epithelial tissue. The latter changes, however, are found in only a few instances. Mi toses occur, but they vary in number in different animals and in the same ani mal in different places. In some mice the downgrowth is so extensive and irregular, that if a similar picture were seen in a human being the diagnosis of carcinoma would be made. Of great interest is the distribution of these proliferative changes. In only 4 of the 26 animals studied did we find them limited to a single precan cerous or early cancerous proliferation, as follows. ( 1) Mouse 95-89, Strain A, which had been injected with 100 rat units of estrogen in oil weekly for 272 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
TABLE XIX: Proliferative Changts: Grade 3 and Grade 4
Strain and mouse Sil1i1e Multiple Generalized Notes 1. A. 59, 79 3 foci, grade 3. In portio; arade 2 + In location 1, 3,4 2. A. 58,79 Grade3- in Grade 2 + in location 2 location 1 (Including folds) 3. A. 57, 79 ~r Grade 2 in ro:rtio Grade 1 In location I b. Grows into muscle b) Grade 3 in ocatlon I .tissue 4. A. 183,79 Grade 31n rade 2 In folds Grade I In location 1 (up portio to arade 2-) S. A. 121,89 Grade 3 multlr,le In portio; Grade 2-3 in vagina arade 3 In fa d; arade 3 growing Into folds 6. A. 95, 89 Grade 3-4 In portio 7. A. 32, 89 Grade 2-3 in folds (up to ~e 3), also in cervix. rade 2 In vagina (up to arade 3) 8. A. 123,89 Grade 3 ID portio; grade 2 In vagina and folds 9. A. 121,91 (a) Grade 3-4 in portio. Proliferation with arades grade 2 In portio 3-4 Is almost generalized (b) Grade 3-4 multiple In In portio and cervix. In both sides of cervix. sUbmrltonea1 tlssue a Grade 3 In locations 3 pap llama Is found in and 4 area corresponding to the cervix 10. A. 54, 82 Grade 3 - and arade 3-4 in Grade 1-2 in vagina va8,na and lolds 11. A. 66, 82 ~a~ rade 2-3 In fold Grade 2 in vagina b Generalized arade 2 passes Into grade 3 In sev- eral places In vagina, lead- Ing almost to generalba. tlon of arade 3. Grade 1-2 in cervix 12. A. 57, 82 Grade 2 in vagina passes into multiple changes. Grade 2 in cervix. Grade 3 almost generalized (grade 3 or grade 3 -) In portio; in one place grade 3+ 13. A. 180, 82 Grade 3 at junction of por- Generalized grade 2-3 Elsewhere little prolifera- tlo and Inner layer of fold tion 14. C3U. 73, 99 Grade 3-4 In outer layer of Grade 3-4 in Inner layer of Elsewhere not many proc- fold fold extending to portio essea, In places only one layer of surface epithelium IS. C3H. 138,99 Grade 3 in- ner layer of fold 16. C3H. 75, 99 (a) In vagina grade 3-4 (elsewhere grade 1-0) very near to urethra (b) At junction of cervix and ~rtlo (c) A normal gland out- growth In location 4 17. 0.96.82 Multiple grade 3 In fold (a) Grade 2 in vagina prog- rell8C!8 to arade 2-3 in places (b) In fold generalized grade 2 ces to grade 3 In one p ace nine months and was 9% months old at the time of death; (2) Mouse 138-99, Strain C3H, which received 150 rat units of estrogen for 5lh months and was 6% months old at the time of death; Mouse 148-99, Strain New Buffalo, which received one injection of 100 rat units of estrogen in oil weekly over a period of 5 months and was 50 months old at the time of death; Mouse 183-79, Strain A, which was injected daily with 10 to 30 rat units of estrogen during a period of 21 months and was 23 months old at the time of death. Three of these animals were thus relatively young at the time of examination and had been injected for a relatively short period of time. The fourth mouse was older and had been injected for a long time. In every other instance the proliferative changes attaining grade 3 or 4 were multiple, either near PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 273
TABLE XIX: Proliferatifle Changes: Grade J and Grade 4-Continued
Strain and mouse Slnllle Multiple Generalized Notes 18. D. 169, 82 (a) Grade 2 + in one place (a) Around portio Ileneral In valdna Ized lI1ade 3 (b) In lnner and outer lay (b) In squamous part of ers of fold Ilrade 3-4 cervix Ilenerallzed Ilrnde (c) In outer layer of other 3-4 fold lI1ade 3 (c) In cylindrical epithe lium of cervix much llandular outll1owth. Glands with partly cy lindrical and partly squamous epithelium 19. D. 157, 76 (a) MUltiple Ilrnde 3 in va gina. Grade 2 In some places in vagina (b) In cervix grade 2 + (c) In fold Ilrnde 2 + (d) In top of fold Ilrnde 3-4 20. D. 187,79 (a) In inner layer of fold (a) Generalized Ilrnde 2 + Ilrnde 3-4 In vagina in direction to (b) In other wall of va&lna outer layer of fold IJ1'!de 4 (b) All portio generalized (C) Grade 3 at be&lnnlng of grade 3-4 squamous epithelium in (c) Generalized Ilrnde 2, cervix &lant cells, abnormal nuclei, In outer layers of fold (d) In cervix generalized lI1ade 2-3 21. D. Control (a) Grade 3-4 in cervix TI88ue with main Ilrade 188,78 (squamous epithelium) 3-4 penetrates into mus (b) In folds of vagina grade cle tissue 2 in places (c) In cervix hlilher up grade 2-3 22. New Buffalo Grade3-ln 148,99 uterine cer vix 23. Old Buffalo Multiple to generalized out Generalized grade 4 ell 131,78 growth tending from valdna to beldnnlng of uterus with marked 1I10wthinto ad joining tlsaues and with alternations between squamous and cylin drical epithelium Ilrnded In accordance with cbanges In location. In 1I10wthof epithelial cells and Illands into vessels 24. C57.43,91 Grade 3 - (a) In va&lna, (b) near border between IQuamous and cylindrical epithelium of cervix 25. C57. 130,91 Grade 3 at border of inner In vagina Ilenerallzed Between vaKina and fat layer of fold and portio Ilrnde 2; In folds Ilrnde 2; tiseue containing mam In lower part of cervix mary Illand structures a grade 2 sebaceous Illand ade noma was found 26. C57. 115,91 (a) In vagina near -skin, In upper urethra near Ilrnde 2 vq!na cbanlle&-very (b) In upper vagina grade 3 marked pearl formation (c) In portio and fold and irrellUlar strands of Ilrnde2+ epithelial tissue (d) In cervix Ilrnde 2; at junction of portio with cervix bellinmnll lI1ade 3 one another in the same area, or occurring in different parts of the genital tract. The number of proliferative areas of grade 3 or 4 in an animal varied; some mice had only two, while in others there were four or more. In a number of cases proliferations of grade 3 or 4 developed from generalized changes 2 or 2 +, and transitions to grades 3 -,3, or 4 sometimes occurred at various points in such generalized areas. These proliferations may be so close to gether that in the end the precancerous or early cancerous changes give the impression of being generalized. Generalization under these conditions means a very high degree of multi plicity of the proliferative processes. An extreme instance of such a general- 274 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB ization was attained in "Old Buffalo" mouse 131-79, in which there was a generalized cancer-like proliferation beginning in the vagina and extending to the point where the cervix joins the uterus. Reference has been made to this case in an earlier paper (2). In three instances we observed a somewhat increased proliferative activity in organs adjoining the genital tract. ( 1) In mouse 121-91, Strain A, there was an almost generalized carcinoma-like change affecting portio and cervix. In a direction parallel to the cervix a marked papilloma had developed in the peritoneal tissue. (2) In mouse 130-91, Strain C5 7, an adenoma-like devel opment of a sebaceous gland was observed in the fat tissue at the side of the vagina, not far from the skin adjoining the entrance into the vagina. (3 ) In mouse 115-91, Strain C57, there was found an irregular gland-like down growth into the connective tissue with pearl formation, starting from a place near the junction of urethra and bladder and originating either from the surface epithelium of these structures or glands located in this region. In some other cases.ialso, certain abnormalities had occurred in this area, as well as in the mammary gland, which extended in some instances near to the wall of the lower vagina. Thus we observed in one instance in this location a cyst which presumably had its origin in a mammary gland duct. In another case, not included among the 26 mice with grade 3 or 4 proliferations of vagina and cervix, a sarcoma developed in the tissue surrounding the wall of the vagina. We shall describe this tumor in a subsequent paper. It is prob able that the proliferative processes which take place in certain tissues may stimulate adjoining tissues to proliferate; this may perhaps be true in the A mouse in which the peritoneal papilloma was observed. In other cases the simultaneous occurrence of abnormal proliferations in different tissues in the same vicinity may be accidental. The data given in Table XIX prove that while precancerous or carcinoma like proliferations may occur anywhere in the vaginal-cervical tract, they are found much more frequently in some locations than in others. These are, in the first place, the portio and the adjacent points where the portio joins the inner layer of the vaginal folds or the cervix. Other sites are the upper parts of the vagina, especially those areas adjoining the outer layer of the folds and parts of the vaginal wall which protrude into the lumen of the vagina, where they may rub against the opposite wall of vagina or portio; the lower portion of the cervix and the region near its junction with the cylindrical cell cervix. Only rarely are cancerous and precancerous proliferations seen near the junction of the vagina with the skin. These situations suggest that places where epithelial surfaces tend to rub against each other are most liable to give rise to an abnormal cancer-like downgrowth of the epithelium. This applies particularly in the case of the portio, which is especially exposed to friction. The probability that mechanical factors, such as friction, are somehow involved in the origin particularly of the more extreme proliferative changes, is further suggested by the fact that the surface epithelium covering a pre cancerous or cancer-like proliferation is, as a rule, devoid of keratin and is even thinned out, evidently as the result of friction. The reduction in thick ness of the epithelium may go so far that only one or two layers are left of the squamous epithelium, a condition found especially in the portio. Two PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 275 interpretations of this phenomenon are, however, possible. It may represent ( 1) a primary occurrence preceding or accompanying the downgrowth of the epithelium, or (2) a secondary occurrence, following rather than preceding the proliferative changes, due to the protuberance in the surface epithelium which formed as the result of the precancerous or early carcinomatous growth
FIG. 2. VAGINA IN MOUSE 54-82, STRAIN A. AGED ELEVEN MONTHS A. Generalized proliferation 2. B. Generalized proliferation 2 +. C. Localized proliferation 3. processes. The second of these two possibilities cannot be entirely excluded; it may playa certain part. But it is probable that the first alternative is the essential factor. This follows from the fact that a partial or total loss of keratin is often found in the portio, cervix, and also in certain parts of the vagina at a time when an abnormal downgrowth of epithelium has not yet taken place; on the other hand, in the lower part of the vagina, where ab- 276 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB normal proliferations are rare, the keratin is often well preserved, as an indi cation that here friction is lacking. It is therefore probable that long continued friction of the surface epithelium is a factor which contributes to the development of a precancerous or carcinoma-like downgrowth of the epi thelium. Friction can be only a secondary factor, however; the most im portant factor is the rhythmic growth-stimulation of the epithelium by estro gen, which functions as a growth hormone. That this proliferative effect
FIG. 3. MULTIPLE PROI.IFERATlVF: CHA;I1GES IN VAGINA A!'1D CERVIX OF MOUSE 59-79. STRAIN A A. Portio. B. and C. Precancerous proliferations 3. D. Cervix. E. Proliferation 2 + in cervix. alone is sufficient to produce abnormal downgrowth of the epithelium is indi cated by the fact that the latter process may sometimes, although rarely, take place, even in cases in which the squamous epithelium is still fairly well pre served and covered by keratin. Where the epithelium is thinned out in the vagina and portio and not protected by a layer of keratin, we usually find polymorphonuclear leukocytes and sometimes also lymphocytes infiltrating the connective tissue and migrating through the surface epithelium, and this may hold good also in the case of abnormal precancerous or carcinoma-like downgrowths of epithelium. Some of the changes which take place in vagina and cervix are illustrated in the accompanying photomicrographs." Fig. 2 represents a part of the wall
2 We are indebted to Dr. H. A. McCordock for these photomicrographs. PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 277 of the vagina in mouse 54-82, Strain A, aged 11 months. The nipples were ligated after one pregnancy. Ten rat units of estrogen dissolved in water were injected daily for a period of seven months. The illustration shows a generalized proliferative activity extending over a considerable area of the vaginal wall (A and B). The intensity of these proliferations varies between grades 2 - and 2 +. Along the edge of the picture are seen grade 1 proc esses. In one place (C) a marked precancerous change (grade 3) has devel-
FIG. 4. MORE ADVANCED. PROLIFERATIVE CHANGE IN MOUSE SHOWN IN FIG. 3 A. Vaginal fold lined with squamous epithelium. B. Squamous epithelium. C. Carcinoma like proliferation 3 + (early stage). oped from the generalized grade 2 condition. The surface epithelium from which this downgrowth takes its origin is covered by squamous epithelium which has been thinned out by friction. In several other places in the vagina, not shown here, there are beginning changes representing grade 3. Figs. 3 and 4 show multiple proliferative changes in vagina and cervix of mouse 59-79, Strain A, which had been injected daily with 20 rat units of estrogen dissolved in water for the greater part of a period of 16 months and 11 days, though at first smaller doses had been used. The animal was 18 278 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB
FIG. S. UPPER PORTION OF VAGINA AND PORTIO OF OLD BUFFALO MOUSE 131-79 A. Portio. B. Epithelium of vagina. C. Remnant of folds. D. Carcinoma-like proliferations. E. Part of section shown in higher magnification in Fig. 6. months and 21 days old at the: time of death. Fig. 3 shows two precancerous conditions (B and C) in the portio, one on each side of its junction with the cervix. Keratin has been rubbed off from the surface epithelium. In the beginning of the cervix a marked downgrowth of the surface epithelium (E) into the underlying connective tissue has taken place. The lumen of the cervix is filled with polymorphonuclear leukocytes which have invaded the epithe lium. Lack of keratin here was responsible for the attraction exerted on the leukocytes. Fig. 4 shows a farther advanced proliferative change in the inner layer of the cervical fold. The epithelial processes penetrate rather deeply into the underlying connective tissue and branch in various directions. Again we find desquamation of the surface epithelium covering this area. PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 279
FIG. 6. HIGHER MAGNIFICATION OF AREA E 1N FIG. 5 A. Beginning of vaginal fold. B. Lumen of fold. The fold is surrounded by carcinoma-like proliferations.
Figs. 5 and 6 show the upper portion of the vagina and the portio of mouse 131-79, "Old Buffalo" strain. This mouse at the time of death was 25 months old and had been injected with from 10 to 30 rat units of theelol and theelin dissolved in water daily over a period of 24V:! months. In the lower part of Fig. 5 an area of the upper vagina can be seen covered partly with low squamous epithelium. The upper part of the vagina is lined with a single layer of cylindrical epithelium instead of squamous epithelium. The vaginal folds are only partly open; the pressure of the carcinoma-like proliferation has obliterated portions of them. On both sides of the wall of the vagina carcinoma-like downgrowths into the connective tissue can be seen, and these changes extend also into the folds. These proliferations consist mainly of squamous epithelium, but at certain points gland-like extensions are noted, especially at the base of the carcinoma-like formations. From the portio and the vaginal folds diffuse outgrowths take place, partly resembling adenocarci noma, partly squamous-cell carcinoma, and these gland-like and squamous cell structures pass into each other at various points. Changes of a similar nature extend far upward into the cervix and the beginning of the uterus. Fig. 6 represents a portion of the right fold of the vagina and the sur rounding tissue in higher magnification. A layer of cylindrical cells lines the 280 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB fold. In the lower half of the photomicrograph the outgrowth on the vaginal side is shown. Squamous-cell growth predominates, but some gland-like structures composed of one layer of cylindrical cells also occur. Similar is the appearance in the upper half, extending into the portio; but here the gland-like structures are perhaps somewhat more prominent. These carci noma-like cell strands penetrate deeply over wide areas into the tissue sur rounding vagina and cervix and reach "the neighborhood of the ureter. Here they penetrate into vessels, also, as is shown in Fig. 7, where strands of the adenocarcinoma-like tissue are seen in the lumen of a vessel. In one other place, not reproduced here, a strand of the carcinoma-like tissue was seen in a vessel lumen, and it is probable that other strands also filled the blood or lymph vessels. In certain places in these subperitoneal areas the tumor strands are surrounded by hyaline tissue which exerts pressure on them and presumably prevents adequate nourishment from reaching them, so that they become atrophic and undergo degenerative changes. But before this occurs they show abnormal proliferation, as is indicated by nuclear enlargement and an increase in the amount of chromatin in the nuclei, as well as an increase in amitotic and mitotic nuclear proliferation. Such a combination of degen erative processes and preceding abnormal growth processes occurs also in other conditions; it is quite common in peripheral areas of experimental pla centomata. We may assume that before the stage is reached when unfavor able environmental conditions exert their destructive effect, they may modify and, in certain respects, exaggerate growth processes in cells in which either intrinsic or external proliferative stimuli are active. Very effective doses of estrogen, particularly large doses dissolved in oil, given over long periods of time, cause extensive deposits of hyaline substance in the vagina, cervix, and uterus. This substance seems, at least at first, to be rather soft in consistency and easily organized by growing cellular connec tive tissue. However, the more dense hyaline tissue which forms in the course of time, as stated, exerts pressure on the carcinoma-like epithelial strands and may cause their atrophy. The hyaline tissue which develops in vagina and cervix in old age may in a similar manner exert pressure on the epithelial processes which reach down into the connective tissue and thus cause their atrophy. This applies to processes of grade 2 as well as to those of higher grades. We have noted that from the strands of squamous epithelium growing down from the surface epithelium of the vagina to the connective tissue, gland like processes may develop at the base of the squamous-cell nests, and that, conversely, in the cervix proper and in the uterine cervix or beginning of the uterus, strands of squamous epithelium and hyalinizing pearls may develop. Even in the uterine glands situated near the uterine-cervical junction there may be found squamous epithelium which undergoes hyalinization. But while the vagina, various parts of the cervix, and the beginning of the uterus have these various potentialities, they have them in different degrees. The nearer the tissue approaches, in its normal situation, the vaginal orifice, the greater is the tendency of the epithelium to grow in the form of squamous epithelium and the less its tendency to grow as cylindrical, gland-like epithe lium; conversely, the higher up the tissue is situated the greater the tendency PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 281 of the epithelium to grow in a gland-like form into the underlying tissue and the less its tendency to produce squamous epithelium. Near the uterus and in the beginning of the uterus the islands of squamous epithelium become less and less frequent. These differences in the tendency to grow in a certain way find expression, also, in the behavior of the normal surface epithelium during the sexual cycle and after injections of estrogen. While in the vagina proper there is a tendency of the squamous epithelium to keratinize, in the cervix this tendency is more and more replaced by a tendency to hyalinize. While in the lower part of the cervix the squamous epithelium which forms rhyth mically is still able to undergo keratinization, farther up towards the uterus
FIG. 7. STRAND OF TUMOR TISSUE IN LUMEN OF A VESSEL this ability to keratinize is replaced by hyalinization, and still farther up we may find instead of hyalinization a vacuolar degeneration of the upper cell layers. This progressive change in the chemical nature of the epithelium in the direction from vagina to uterus leads to a still further differentiation. As stated, in the beginning of the uterus or in the uterine cervix the glands or gland-like processes may undergo hyalinization whenever they are abnormally stimulated, but this hyaline material may liquefy and become converted into a viscid liquid material, so that here, instead of pearls, cysts develop. These potentialities, however, do not end abruptly at the beginning of the uterus; even the uterine epithelium and its glandular structures, if they are stimulated 282 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB by injections of estrogen, retain this ability, although in a weakened degree. They are able to produce a low squamous epithelium whose upper layer hyal inizes, and in the gland-like invaginations this hyaline substance tends to liquefy.
DISCUSSION From the data here presented we may conclude that in mice an increase takes place, with advancing age, in the average depth of the processes which reach from the surface epithelium of the vagina and cervix into the underly ing connective tissue. If estrogen is injected into mice over long periods of time the formation of epithelial processes and their gradual lengthening pro ceed, on the average, at a more rapid rate and reach, therefore, a higher degree of intensity at an earlier period of life. These conclusions apply, however, only so far as averages of groups of mice and averages of the changes in the vaginal-cervical tract are concerned. In different areas of vagina and cervix the processes may be unequally developed in the same animal and there are, furthermore, some indications that different individual mice may react with advancing age and under the influence of estrogen unequally. Whether dif ferent strains of mice differ in regard to this tendency towards process forma tion remains still to be seen. In mice treated with estrogen more precancerous or carcinoma-like proc esses developed than in control mice. The transition from normal to abnor mal processes takes place step by step; and there is no sharp separation be tween the points where the condition may still be considered as within the range of normality and where the stage of abnormality has been reached. These data suggest that in normal, control animals, not injected with estrogen, it is essentially the follicular hormone given off intermittently during the sexu ally active life which is responsible for the formation and the deepening of the epithelial processes extending into the connective tissue and for their ulti mate transformation into carcinoma-like proliferations. Under such circum stances we have a condition analogous to that which we have previously ob served in the case of mammary gland carcinoma in mice. As in the case of the mammary gland proliferations-normal and carcinomatous-so also in the case .of the epithelial processes in vagina and cervix, the definite proof for the conclusion that ovarian hormones are the agent responsible for their development should be furnished by subjecting the animals to early ovari ectomy, thus removing the source of the intermittent stimulus. We have begun to study process formation in vagina and cervix in ovariectomized mice and so far there are indications that removal of the ovaries diminishes this tendency. It is, then, in all probability a physiological process, occurring in the course of normal life, which with advancing age may lead to patholog ical changes. These changes can be accelerated and intensified, however, by intensifying experimentally the spontaneous hormone action. In the case of mammary gland carcinoma it could be shown that the effects of the normal hormones or of the hormones experimentally increased in quan tity were greatly influenced by the hereditarily determined substratum on which the hormones acted. There exist hereditary strain differences ami perhaps also individual differences of reactivity to the stimulation. In cer- PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 283 tain strains of mice the mammary gland seems to be sensitized to the action of the hormones. As far as the abnormal proliferations of vagina and cervix are concerned, we have not yet found a corresponding hereditary factor; the sharp strain differences noted in the case of the mammary gland were lacking in the case of vagina and cervix. However, we cannot yet exclude the possi bility that in regard to the latter tissue hereditary strain differences do exist, but are much weaker and therefore much less evident than in the mammary gland. If, then, the proliferations in vagina and cervix are essentially due to stimulation by hormones, it might be expected that they would proceed at the same rate throughout the genital tract and that a precancerous or carci noma-like condition would be attained in the whole vagina and cervix at about the same time. In a limited way this may occur; we do actually find in some instances a generalized proliferation of the epithelial processes over wide areas and in a number of adjoining areas the epithelial processes may become carci noma-like. But, as a rule, this carcinoma-like proliferation starts at a single point or at several points unconnectedly in an apparently fortuitous manner. We have here to deal with the same problem as in the case of mammary gland, where, also, the stimuli apparently affected the whole tissue equally, but with very unequal effects in different areas. Evidently variable factors enter into the interactions between hormones and substratum and cause un equal results in various places. In the case of the mammary gland these fortuitous factors are as yet unknown. In the case of vagina and cervix there is good reason for assuming that a mechanical factor, namely the long continued rubbing off of the keratin and of the upper epithelial layers from the surface epithelium, is largely responsible for this inequality of prolifera tive action. This is probably the reason why the portio is so often the site of precancerous or cancer-like proliferations in the mouse. In women, also, the portio is frequently affected by malignant proliferations; it may be that here, too, a similar mechanical factor plays a certain role. In the end, how ever, the longer the stimulation lasts, the more general becomes the abnormal proliferation. In one of our oldest mice the carcinoma-like proliferation was generalized in the vagina, cervix, and beginning of uterus. In a similar man ner we have noted a gradually increasing generalization of carcinomatous proliferation in the mammary gland. It is this cumulative action of stimula tion by hormones and by other factors, with advancing age of an organism, which is largely responsible for the increase of cancerous changes in old age, as we have pointed out on previous occasions. Certain other changes, such as hyalinization of connective tissue, characteristic of old age in some organs and observed by us in vagina and portio, tend to act injuriously rather than favorably on carcinomatous growths; but it is conceivable that indirectly these may also help to induce cancerous growth by causing injury to the over lying epithelium, thus leading to regenerative growth processes which may gradually progress to cancerous proliferation. We have seen that, as a result of rhythmic stimulation by hormones ex tending over long periods of time, a gradual downgrowth of the epithelium into the connective tissue takes place, which may eventuate in the production of precancerous or carcinoma-like tissue alterations. We have seen further- 284 v. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB more that the last stages in this process may be aided by long continued stim ulation of a mechanical nature. It may then be concluded that under the influence of estrogen two kinds of growth of the epithelium covering the sur face of the vagina and cervix are found. In the first place there occurs at the time of proestrus in mice the well known transformation of the two-cell layered epithelium covering the vagina and the lower part of the cervix into squamous epithelium with keratin. This is a rhythmic process corresponding to a certain phase in the rhythmically recurring sexual cycle. But there oc curs, in the second place, a growth of the epithelium in a direction parallel to the surface of the epithelium. It seems that at first this leads to a wavy condition of the epithelium; then, gradually, the lower points in the epithelial wave extend further downward into the connective tissue and develop into processes which, in the course of time, become longer and ultimately may become irregular and carcinoma-like. As pointed out above, under the influence of these intense stimulations leading to carcinoma-like growths morphogenetic potentialities of the tissues are revealed which, under ordinary conditions, would remain hidden. Thus we find that the squamous epithelium of the vagina has the potentiality to grow in the form of gland-like processes and that, on the other hand, the cylin drical epithelium of the uterine cervix has the power to produce squamous hyalinizing epithelium, but that a gradation exists in the ability of the epi thelium to give origin to these various structures. As in the case of the guinea pig, so in the case of the mouse we find that the transition between adjoining epithelia is gradual as far as the inherited potentialities are concerned, even if under ordinary conditions the transition seems to be sharp. There is justi fication for assuming that graded biochemical conditions correspond to these graded structural conditions, and that the latter depend on the former. This is indicated by the fact that in passing from the vagina to the cervix and then to the beginning of the uterus, the keratin is replaced by the development of a morphologically related substance, hyaline, and that still further in the direction towards the uterus this hyaline substance may become changed in such a way that it undergoes liquefaction and gives origin to a gelatinous ma terial, filling cysts, which latter take the place of the hyaline pearls character istic of squamous-cell carcinoma. In a similar way the epithelial processes reaching down into the connective tissue in vagina and in vaginal cervix and intermediate cervix become transformed into glands in the uterine cervix and in the beginning of the uterus. These gradations in chemical constitution and structure of the vagina cervix-uterus tract are already indicated in the normal mouse; but they are more evident under experimental stimulation by large amounts of estrogen, especially if the latter induce carcinoma-like changes in the vagina, cervix and beginning of the uterus, such as those observed in mouse 131-79 of the" Old Buffalo" strain. These experimentally produced changes reveal very clearly the existence of a graded system of morphogenic potentialities which are greater than could be foreseen on the basis of the normal structure. In a similar manner we were able, as stated above, to show that in the guinea-pig stimulation by hormones furnishes proof of the existence in the vagina, in the different portions of the cervix, and in the uterus, of gradations in histological PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 285 structure and in biochemical constitution, and of corresponding gradations in responsiveness to the action of two different hormones. We may then conclude that mammary gland and vagina and cervix of mice behave very similarly as far as the manner of transformation of normal into carcinomatous or carcinoma-like cells is concerned. In both cases this transformation takes place under the influence of hormones acting as stimuli, and in both cases this process is not general but localized, at least in the earlier stages. With progressing stimulation, wider and wider areas are induced to proliferate to such an extent that the border of the normal is passed and the abnormal begins, so that in the end we can foresee a state where the prolifera tion becomes general. The abnormal proliferation is connected by steady, graded transitions with progressive states of stimulation which are still within the range of normal. This development seems to be incompatible with the assumption of somatic mutations as the immediate cause of cancerous transformation. It is not easily conceivable that with the cumulative effect of persistent stimulation multiple epithelial cells in various places in vagina and cervix would undergo within a very short time, almost simultaneously, chromosomal or gene muta tions of such a kind as would force the affected cell to assume cancerous; growth. Our observations are much more in harmony with the interpretation that, as a result of intense stimulation, there occur in the cells metabolic changes of a nature that would lead to the continued new production of a substance which acts as a growth activator. We may assume that all cells capable of proliferation are genetically endowed with this potential ability, but that, in accordance with genetic factors transmitted by germ cells, stimuli effect this change with unequal readiness. The genetic constitution of the germ cells determines the specific behavior of various tissues and organs in response to certain stimuli. It can therefore be readily understood that if in identical twins inherited peculiarities in a given organ or tissue tend to concen trate stimuli in an organ or tissue, or to make this organ or tissue specifically reactive to certain stimuli, the carcinomatous transformation might be readily accomplished in both individuals. The assumption of a somatic mutation is not necessary to explain such an effect. However, it must again be stated that, while in the case of mammary gland carcinoma of mice the significance of hereditary factors has been established, this has not yet been done in the case of carcinoma-like proliferations in vagina and cervix; if hereditary condi tions are active here also, as they may very well be, they are not identical with those affecting mammary gland carcinoma. Furthermore, the constant growth stimulation must necessarily lead to an increase in the number of cells in the stimulated tissue; but there is no reason for assuming that this is the essential factor leading to cancer formation; it is merely a by-product of the same stimulation which induces the cells to assume cancerous growth. The cancerous tissue formation may, therefore, occur also in cases when the amount of newly formed tissue has remained as yet very limited. There is another aspect of these investigations which is to be considered, namely their bearing on the question as to whether all cancerous tissue growth is ultimately due to the action of a virus. The investigations of Peyton Rous (5) and his collaborators have furnished proof that a virus may induce an 286 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND LEO LOEB epithelial tissue to become carcinomatous. There remains the problem as to the manner in which this effect is accomplished; in particular, whether it is the result of a long-continued growth stimulation induced by the virus. If this should be so, the virus would in principle act like other growth-stimulating factors which bring about transformation of a normal tissue, and after this critical stage had once been reached a further action of the virus would be no longer necessary. If, however, the continued cooperation of the virus in the cancerous growth should be required, then the action of the virus would differ in principle from that of other types of growth stimuli. There is, furthermore, the question as to whether the cooperation of a virus is required in all cases of cancerous transformation or only in certain cases. The observations here recorded show the gradual, step-by-step transi tion of normal into cancerous or carcinoma-like tissues as the result of long continued action of specific growth stimuli. There is good reason for assum ing that if these stimuli were allowed to act over a still longer time, the carci nomatous change would become general in a given organ or tissue. If the mediation of a virus were required, it would be necessary to assume that as soon as a certain quantity of stimulation had a chance to exert its cumulative effect on a unit of a reactive tissue within the time required to bring about a certain degree of proliferation, this tissue would then be invaded by the virus and from this point on would no longer grow in a normal but in a cancerous manner as long as the virus remained active within the cells. It would be necessary to assume, furthermore, that all members of a hereditarily predis posed strain would thus become affected in the course of time and that a virus is present in all these individuals which, however, remains inactive until a certain intensity of tissue stimulation has been achieved. Whenever this point has been reached different organs, such as the vagina, cervix and mam mary gland, would be invaded and spurred on to proliferation by one or several viruses. The alternative view would be that the constant growth stim ulation would gradually bring about an increased production of an intracellular growth factor, which would constantly be newly produced in a manner com parable to an autocatalytic process. At present we can merely indicate these questions, which can be answered only by further investigations. We have designated the tissue changes observed by us as precancerous or carcinoma-like rather than as cancerous. To use the latter term would imply the continued invasive growth of the stimulated tissues after cessation of the action of the stimuli. In the case of analogous growth processes in the mam mary gland, experience has shown that such a continuation actually occurs; in vagina and cervix it is much more difficult to prove the existence of such a condition. But it is at least very probable that in certain of the animals stud ied by us, such a continued growth would have taken place; and, as we stated on previous occasions, there can be little doubt that ultimately a degree of stimulation is reached when a retrogression no longer occurs. The question as to whether some of these proliferative processes should be designated as carcinomatous rather than as carcinoma-like is therefore not one of principle. Lastly, it may be asked whether the observations here recorded are limited to the species used in our experiments, the mouse, or whether they have a more PROLIFERATIVE CHANGES IN VAGINA AND CERVIX 01" MICE 287 general application. There is no reason why we should not extend our con clusions to other animals and in particular to the human species. In women, portio and cervix are common seats of carcinoma; it is in the portio that, in the mouse, the precancerous or carcinoma-like changes are found most fre quently. In the mouse we attributed this condition to the stimulation by estrogen acting over long-continued periods of time and often in combination with mechanical stimuli of friction. It is probable that in women, also, the effect of estrogen, together with other stimuli, which need not be identical with those affecting the mice in our experiments, is responsible for this ab normal proliferation. However, cancerous changes in the vaginal wall proper seem to be more rare in the human species than in mice exposed to the action of estrogen. In nine monkeys Overholser and Edgar Allen (6) exposed the portio to the action of estrogen alone, or to the combined action of estrogen and lutein hormone. In all but one of these repeated mechanical trauma tisms were added to the hormone action. Essentially the changes observed consisted in the transformation of the cylindrical cervical epithelium into squamous epithelium and the subsequent downgrowth of the squamous epi thelium underneath the cylindrical epithelium of the gland, which was thus raised up. It seems, however, that in one case squamous-cell nests were pro duced which invaded the underlying connective tissue. The relatively slight effects produced in these experiments can perhaps be accounted for by the short time, usually from thirty to sixty days, during which the hormones were applied and by the relatively small doses of estrogen used in animals which are so much heavier and live longer than mice. Non-carcinomatous changes similar to those noted by Overholser and Allen were seen by Engle and Smith (7) in the cervix of monkeys. Zucker man (8), also, found similar effects and in one case he could show that these changes were reversible and had therefore not yet reached the stage of typical carcinoma. Lipschutz (9) quite recently observed a tissue change, corresponding to grade 3 in our mice, in the cervix of a guinea-pig in which about thirty-two months previously the greater part of both ovaries had been extirpated. This investigator assumes that as the result of the diminution in the amount of functioning ovarian tissue there took place-presumably by way of the an terior pituitary gland-an increase in the production of folliculin which was responsible for the proliferative changes in the cervical epithelium. We would then have to deal in this case with a condition analogous to that observed by us in mice. We may refer, also, to the experiments of Perry and Ginzton (10), who, in three mice painted for a long time with a combination of 1: 2: 5: 6 dibenzanthracene and theelin, noted the development of proliferations in the uterine tissue resembling those graded by us as 3 and 3-4; they did not ob serve such changes in mice injected with dibenzanthracene alone. However, painting with the latter substance did induce estrous changes in the vagina and led to the development of mammary carcinoma. Attempts by previous investigators to cause cancerous growth in inner organs by painting the skin with estrogen had been unsuccessful. 288 V. SUNTZEFF, E. L. BURNS, MARIAN MOSKOP AND I.EO LOEB
SUMMARY (1) With advancing age the average depth of the epithelial processes ex tending from the surface epithelium into the underlying connective tissue in the vagina and cervix of mice increases. If an estrogenic substance is in jected into these animals at regular intervals over long periods of time these processes reach a greater depth than in non-injected controls. Large doses of estrogenic substance dissolved in oil are more effective than smaller doses, or than equally large doses of estrogen in watery solutions. (2) In a much larger number of mice injected with estrogenic substance over long periods of time these processes gradually, and through continuous growth, pass into a precancerous and into a carcinoma-like growth, than in older, non-injected mice. In the control non-injected mice such a change was observed in only one animal out of 128 (= 0.870), while in the mice injected with estrogenic substance this change occurred in 2S mice out of a total of 234 (= 10.7%). (3) We have to deal in vagina and cervix with a condition analogous to that seen in the mammary gland, where so-called spontaneous carcinoma also develops under the influence of hormones which stimulate the growth of this gland even in normal life. Ovariectomy greatly diminishes the proliferation of the mammary gland. It still remains to be determined whether ovari ectomy also diminishes the production of epithelial processes in the connective tissue in the vagina and cervix. However, the hereditary factors which in teract with the stimulating factors in the production of mammary gland carci noma are not active, or are not active in the same degree, in vagina and cervix. Further investigations must show how far hereditary conditions cooperate with hormone action in the case of the precancerous or carcinoma-like growth in vagina and cervix. (4) There are indications that, in addition to the hormones, mechanical factors, leading to partial erosion of the surface epithelium, may participate in the development of these abnormal growth processes in vagina and cervix. These secondary factors are, presumably, at least partly responsible for the fact that the development of precancerous or carcinoma-like proliferations does not proceed equally over all parts of vagina and cervix, but is localized in certain areas. As stimulation continues, however, the proliferation not only becomes intensified in localized areas, but also becomes more and more gener alized in a way similar to that noted in the case of the mammary gland. (S) Under the influence of intense stimulation leading in the end to carcinoma-like growth processes in the vagina and cervix of mice, morpho logical potentalities of the tissues may be revealed which under ordinary conditions would remain hidden. These observations confirm the conclusion that, as in the guinea-pig, so also in the mouse the transitions in the epithelial tissue of vagina, cervix, and uterus are gradual, representing a continuous series, even when under relatively static conditions they appear to be sharp. There are, furthermore, indications that these structural gradations are based upon or associated with specific gradations of a biochemical nature. (6) The study of the changes which take place in vagina and cervix with advancing age and under the influence of long continued, experimentally pro- PROLIFERATIVE CHANGES IN VAGINA AND CERVIX OF MICE 289 duced stimulations by hormones, which ultimately may lead to the production of carcinoma-like proliferations, is not compatible with the view that somatic mutations in the stimulated tissues are the immediate cause of their cancerous transformation. The observations in vagina and cervix are in accordance with our previously recorded findings in the mammary glands of mice.
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