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Human cervical epithelial changes produced by podophyllin
Human cervical epithelial changes produced by podophyllin HAROLD
A.
KAMINETZKY,
M.D.
Chicago, Illinois
epithelium. McGrew and 14 undertook the study of the histologic and cytologic effects of podophyllin applied to the cervices of mice. We found this substance was capable of inducing changes in the cervix resembling atypical epithelial hyperplasia. Such lesions exfoliated and cells examined by the Papanicolaou technique were often indistinguishable from those shed by squamous carcinoma. Our experiments and those of others 1 2 3 9 who utilized skin as a target organ • • • indicate that neither podophyllin nor its partition products is carcinogenic. In order to test rigorously the potential of podophyllin as a carcinogen, we are currently painting the cervices of 50 mice three times a week for an entire year. To date, after 6 months, we have not produced a single cervical cancer although the animals continue to shed malignant appearing cells.
D Y s P L A s T I c lesions of the cervix constitute a serious clinical problem. They may be found adjacent to in situ or invasive cancer. On the other hand, they may exfoliate malignant appearing cells when there is no cancer. Therefore, treatment has not been standardized and remains empirical. The lesions included u~der the designation, "cervical dysplasia" or "atypical epithelial hyperplasia," may follow variable courses. In some patients they remain unchanged or disappear entirely. In others they become progressively more atypical. It is even thought they may develop into in situ or invasive cancer, but this assumption remains unproved. Moreover, it is impossible to predict the potential of any particular lesion from current morphologic criteria. Finally, it seems possible that abnormal tissues and cells of malignant appearance can be produced by various stimuli unrelated to the process of carcinogenesis. Trichomonas infection and pregnancy are said to be such stimuli. Experiments testing these hypotheses are in process or projected. Following the experiments of King and his co-workers,5-8 Saphir10 applied podophyllin to the cervices of patients prior to hysterectomy. He reported changes resembling carcinoma in situ in the squamous
Material Forty-four women were included in this study. All required hysterectomy for conditions unrelated to cervical disease. The most frequent indications for hysterectomy were chronic pelvic inflammatory disease, prolapse, and leiomyomas. No patient with malignancy or suspicion of malignancy was included in this group. No postmenopausal patients were found suitable for this study.
From the Department of Obstetrics and Gynecology, College of Medicine, University of Illinois. Aided by a grant from the American Cancer Society. Presented at a meeting of the Chicago Gynecological Society, Dec. 18, 1959.
Method The posterior lip of the cervix was selected as the target for podophyllin and biopsy specimens were obtained 3 weeks 1055
1056
Kaminetzky
December, 1960 Am.]. Obst. & Gynec.
No effect 14
c
(/)
.~
0
a.
M il d
12 10 8
Moderate
'+--
0
6
li
4
:J
2
E
z
0
Advanced 2 3 4 5 6 7 8 9 10
12
14
16
18
20 22
24
Number of daily paintings Fig. I. Histologic effect of podophyllin.
before the patient's admission to t!te hospital. The anterior lip provided the control. More than half of the patients had four-quadrant biopsies. A cervical smear and a vaginal smear were also prepared at this time. No patient was included in the study in whom biopsy showed atypia or whose smear con-
tained suspiCious cells. As expected, most specimens had evidence of chronic cervicitis. Painting was begun after the patient was hospitalized. Ten per cent podophyllin in mineral oil was applied to the entire posterior lip including the area of the squamocolumnar junction. An attempt was made
Fig. 2. Marked inflammation after two podophyllin applications. Hypercellularity and some disorganization of the epithelium may represent intense pre-existing cervicitis, but the vacuolization and areas of rather marked nuclear pleomorphism are probably due to podophyllin. ( x250; reduced !4.)
Volume 80 Number6
Cervical changes produced by podophyllin
Fig. 3. Moderate epithelial change observed after seven applications. Basal cell crowding and irregularity reflect increased mitotic activity. Characteristic degeneration vacuoles are noted in all layers. (x250; reduced V4.)
Fig. 4. Dysplasia observed in another patient after seven paintings. Basal and parabasal mitoses are present. There are scattered vacuolated cells. Atypical cells occupy most of the epithelium. (x250; reduced V4.)
1057
1058 Kaminetzky
December, 1960 Am. ]. Obst. & Gyncc.
Fig. 5. Squamous cells from intermediate layer of epithelium ( 15 podophyllin applications ) . There is variation in size and shape of cells and nuclei, nuclear and nucleolar enlargement, and binucleation. ( <700: reduced Y~. )
to exclude the anterior lip from contact with the podophyllin; however, in the region of the squamocolumnar junction this was not always possible. Painting was carried out once a day, and new smears were prepared prior to each painting. Hysterectomy was performed at various intervals depending on a number of uncontrolled variables and by whatever route best served the requirements of the underlying pathologic process. Three sections were usually prepared from the posterior lip of the cerv1x and two from the anterior lip.
normality was found in patients seen 6 weeks after hysterectomy. The histologic effects of podophyllin are graphically portrayed in Fig. 1. Suspicious or malignant appearing cells were recovered from 12 of the 44 women. In one the cervix was painted only once, in two it was painted 3 times, in two 5 times, in one 6, in one 7,
Results
Grossly evident inflammation of the posterior lip of the cervix and the adjacent vaginal mucosa was noted after one or two applications of podophyllin. Increased quantities of cervical mucus were produced by most patients. There were no complaints of irritation. At operation the vaginal mucosa was somewhat friable because of the chemical inflammation. On the other hand, this did not cause any problem of cuff healin.g or noticeable postoperative irritation of the peritoneum. No persistent cytologic ab-
Fig. 6. Giant basophilic squamous cell containing a small rosinophilic prematurely cornified cell ; nuclei ar ~ both abnormal. See n in a cervical smear after 25 applications of podophyllin. ( x900; reduced Ys.)
Volume 80 Number r,
in two 10, in one 15, in one 17, and in one 25 times. Curiously, one patient whose cervix was painted 16 times produced only slightly distorted cells. Certainly this was not the rule. Seven patients had mild changes in smears and 4 others showed moderate effect. There were, in addition, 20 patients from whom no atypical cells were recovered, 11 of these having had but a single podophyllin application. On the other hand, there was advanced atypia in the histologic specimens of 3 patients painted only once. In 25 patients histologic atypia was more advanced than the corresponding cytologic change. However, the opposite was true in 7 patients. One patient, whose cervix was painted 17 times and whose smears contained malignant appearing cells, had no identifiable squamous epithelium in the tissue sections examined. All but 3 patients exhibited evidence of the inflammatory component of the podophyllin effect when the posterior lip of the cervix was examined histologically. This consisted of an accumulation of polymorphonuclear leukocytes in the cell layers of the squamous epithelium, edema, and vasodilation in the stroma. The inflammatory cell infiltrate might involve one or all layers of the epithelium as well as the subjacent stroma (Fig. 2). Vacuolization of squamous cells, often seen as a part of nonspecific inflammatory processes, was usually very extensive after one to three applications of podophyllin. At times this feature was found in all cell layers above the basal area. However, vacuolization was not prominent in the areas containing the most advanced atypia. Mitotic figures were frequently seen in the deepest layers of the epithelium. All phases of mitosis were represented, although metaphase and anaphase figures predominated. Multipolar mitoses were not seen. Nuclear enlargement and distortion extending through the basal and intermediate layers were noted in most cervices painted one to four times. Moderate epithelial change usually involved the deepest 3 or 4 cell layers and was
Cervical changes produced by podophyllin 1059
noted after variable numbers of podophyllin applications, usually one to four. The nuclei were large, often paired, and at times filled the entire cell. There was considerable irregularity of the cellular outlines coarsening of the chromatin network, and prominence of the nucleoli (Fig. 3). The most advanced atypia with a few notable exceptions was found in patients whose cervices were painted the greatest number of times. Cells were greatly pleomorphic, and nuclei were considerably distorted. The nuclear chromatin was disposed in coarse clumps. In a number of cells the nuclear membrane could not be made out (Fig. 4). These abnormalities were usually limited to the deeper one half of the epithelium but in some sections occupied almost its entire extent. No evidence of stromal invasion was found in any histologic specimen. After many paintings the posterior lip was usually ulcerated and ragged appearing. Small, irregular sheets of abnormal epithelium were found showing considerable distortion and pleomorphism. These sheets often had little or no connection to the remaining epithelium. This was probably the source of masses of cells found in some of the cytology smears. Atypical or premature cornification was occasionally noted in sections exhibiting all degrees of podophyllin change. Cells of this kind were found in all layers of tl1e epithelium but most often above the basal layer. They were eosinophilic, had densely staining amorphous nuclei, and were at times found within the cytoplasm of other cells, which retained their cytoplasmic basophilia. Whereas significant histologic effects were apparent after one to four paintings with podophyllin, the cytologic effects during this time were not spectacular, and inflammatory features predominated. The smears contained large numbers of polymorphonuclear leukocytes between and within the cells. Many cells were vacuolated and the vacuoles were often filled with inflammatory cells. There was, in addition, moderate cyto-
l 060
Kaminetzky
Fig. 7. A sheet of abnormal squamous cells found applications of podophyllin. (x400; reduced l/:J .)
plasmic pleomorphism in these early smears. Nuclei were enlarged in some of the specimens but hyperchromatism was rare. After 6 or more paintings, the majority of smears exhibited more advanced abnormalities. The cells appeared parabasal in type and contained large nuclei with moderate condensation of chromatin and rather prominent nucleoli (Fig. 5 ). In general, the most atypical cells were recovered from patients whose cervices were painted many times. Great enlargement of the nuclei, irregularity of the nuclear outlines, clumping of the chromatin, and gross distortions of cytoplasmic outlines characterized such cells (Fig. 6 ) . The smears of 3 patients contained sheets of grossly abnormal cells (Fig. 7). Abnormal naked nuclei were found in smears from 2 patients (Fig. 8 ).
December, 1960 Am . J. Obst. & Gynec.
In
a cervical smt:ar after 14
Study of the histologic sections m sequence according to the number of podophyllin applications suggests a certain train of events. The cells in the superficial layers show the effects of inflammation. They become edematous, are infiltrated by polymorphonuclear leukocytes, and develop vacuoles. These layers which ordinarily contain cells that are dying as they approach the surface do not exhibit the nuclear effects
Comment
Precise controls were impossible for these preliminary experiments. A single biopsy of the posterior lip of the cervix might miss a spontaneous dysplastic lesion. On the other hand, occult spontaneous dysplasia would be unusual in an appreciable percentage of 44 women.
Fig. 8. Naked abnormal nucleus. Seen in a cervical smear after I 0 applications of podophyllin. (x900; reduced 13.)
Volume 80 Number 6
of podophyllin. These layers finally lose con~ nection with the deeper layers of cells. Nuclear effects seem to occur in basal and parabasal layers where cells are growing and reproducing. Repeated application of podophyllin produces generation upon gen~ eration of abnormal cells which do not mature in the normal manner. Finally, a number of layers of abnormal cells are seen. However, even these cells ultimately attempt maturation. This gives a picture resembling spontaneous atypical epithelial hyperplasia. It is probable that the initial layers of cells at the surface must be removed before the altered basal cells can attain a free surface and be exfoliated. This would appear to
REFERENCES
1. Belkin, M.: J. Pharmacal. & Exper. Therap. 93: 18, 1948. 2. Berenblum, I.: J. Nat. Cancer Inst. 11: 839, 1950. 3. Greenspan, E. M., Leiter, J., and Shear, M. J.: J. Nat. Cancer Inst. 10: 1295, 1950. 4. Kaminetzky, H. A., McGrew, E. A., and Phillips, R. L.: Obst. & Gynec. 14: 1, 1959. 5. King, L. S.: J. Nat. Cancer Inst. 8: 215, 1948.
Cervical changes produced by podophyllin 1061
explain the lag of cytologic atypia as compared with the histologic change. Summary
Podophyllin produced atypia in the cervical squamous epithelia of 28 of the 44 women of this study. It produced an abnormal inflammatory response without dysplasia in 12 more women. No appreciable effect was seen in 3 women. Dysplastic lesions produced by podophyllin have many features in common with spontaneously occurring atypical epithelial hyperplasia, including the shedding of malignant appearing cells. There is at present no evidence that podophyllin is carcinogenic.
6. King, L. S., and Cauldwell, E. W.: J. Nat. Cancer Inst. 10: 131, 1949. 7. King, L. S., and Sullivan, M.: Science 104: 244, 1946. 8. King, L. S., and Sullivan, M.: Arch. Path. 43: 374, 1947. 9. Leiter, J., Downing, V., Hartwell, J. L., and Shear, M. J.: J. Nat. Cancer Inst. 10: 1273, 1950. 10. Saphir, 0., and Leventhal, M. L.: Am. J. Path. 34: 555, 1958.
Discussion
DR. ELIZABETH A. McGREw, Chicago, Illinois. We must learn to distinguish between the rather common epithelial "dysplasias" of the cervix and preinvasive carcinoma; which of these morphologically similar lesions are reversible and which should be considered carcinoma in situ, capable of progressing inevitably to invasive carcinoma. Dr. Ronald Greene brought this difference to our attention some years ago by pointing out that preinvasive cervical carcinoma during pregnancy does not represent a peculiar epithelial abnormality of pregnancy and that those lesions which disappear are not preinvasive carcinoma. He did not say exactly what they were or establish criteria for differentiating them from carcinoma in situ except to illustrate them in excellent photomicrographs. People working in this field have come to recognize some relative criteria for making this distinction, such as the greater thickness of the epithelium in carcinoma in situ and the failure of the cells to mature
and reorient themselves as they reach the surface. There are still many borderline lesions which are resistant to interpretation, however. In Dr. Kaminetzky's work we have seen marked epithelial abnormality produced chemically by a noncarcinogenic substance. The cells of this epithelium looked malignant, both in smears and in sections; yet, as Dr. Kaminetzky indicated, when daily podophyllin painting of the mouse cervix is continued for several months and then discontinued, vaginal smears become normal in a few days and none of the animals develop carcinoma. He has, then, produced changes in cells which mimic malignancy, yet are temporary and reversible, thus corresponding to spontaneous cervical epithelial dysplasia. Apparently the production of these changes depends on the antimitotic action of podophyllin. It prevents the formation of the mitotic spindle so that the chromosomes cannot separate. These cells are blocked in the metaphase but survive
1062 Kaminetzky
sufficiently to continue synthesis of nucleic acids. Their nuclei may then be reconstituted, but they have an excessive amount of nucleic acids so that they arc hyp('rchromatic and unusually large. Some of the carcinogenic agents also han~ mitotic effects; the difference is that these cause permanent mutations. I think that this work gives us some insight into the pathogenesis of malignancy. It also adds to the battery of tools with which to study the reversible as compared with the truly neoplastic epithelial abnormalities. DR. S. R. LAsH, Chicago, Illinois. In this experimental work carried out over a very short period the morphologic changes we have seen in cast-off cells are almost impossible to tell from those seen in carcinoma cells. The prominent nuclei, the chromatin, the marked irregularity and enlargement of the nuclei are morphologic criteria used to determine malignancy. These were all short-term experiments. To say that this chemical is not carcinogenic is impossible in this short period of time. I think if the experiment were carried out over a long period of time such conclusions could be made.
December, 1960 Am. J. Ob;t. & Gynec.
The experiments in mice will lead to conclusions on the cff<•ct in mice only.
DR. C!L\RLES E. GALLOWAY, Evanston, Illinois. There is another substance that produces carcinoma-like l(•sions in the cervix, and that is carbon dioxide snow. If carbon dioxide snow is put on the cPrvix, it will undergo change that looks very malignant both grossly and microscopically during the healing stagt'. DR. KAMINETZKY (Closing). Dr. Lash indicated the need for long-term studies of the effect of podophyllin on human cervical epithelium. This would be very useful, but it poses a number of practical problnns in finding and keeping patients on such a program. For the moment the objectives of the human studies arc limited to those possible in the preoperative patient who is already hospitalized. In response to Dr. Galloway, I have not applied carbon dioxide snow to the cervix. Tissue injury is often followed by compensatory hyperplasia. We have felt it unwise to follow the course of the podophyllin treated patient with serial biopsies for the same reason. It seemed possible that we might add another kind of tissue injury to the effect of the drug.
A.
KAMINETZKY,
M.D.
Chicago, Illinois
epithelium. McGrew and 14 undertook the study of the histologic and cytologic effects of podophyllin applied to the cervices of mice. We found this substance was capable of inducing changes in the cervix resembling atypical epithelial hyperplasia. Such lesions exfoliated and cells examined by the Papanicolaou technique were often indistinguishable from those shed by squamous carcinoma. Our experiments and those of others 1 2 3 9 who utilized skin as a target organ • • • indicate that neither podophyllin nor its partition products is carcinogenic. In order to test rigorously the potential of podophyllin as a carcinogen, we are currently painting the cervices of 50 mice three times a week for an entire year. To date, after 6 months, we have not produced a single cervical cancer although the animals continue to shed malignant appearing cells.
D Y s P L A s T I c lesions of the cervix constitute a serious clinical problem. They may be found adjacent to in situ or invasive cancer. On the other hand, they may exfoliate malignant appearing cells when there is no cancer. Therefore, treatment has not been standardized and remains empirical. The lesions included u~der the designation, "cervical dysplasia" or "atypical epithelial hyperplasia," may follow variable courses. In some patients they remain unchanged or disappear entirely. In others they become progressively more atypical. It is even thought they may develop into in situ or invasive cancer, but this assumption remains unproved. Moreover, it is impossible to predict the potential of any particular lesion from current morphologic criteria. Finally, it seems possible that abnormal tissues and cells of malignant appearance can be produced by various stimuli unrelated to the process of carcinogenesis. Trichomonas infection and pregnancy are said to be such stimuli. Experiments testing these hypotheses are in process or projected. Following the experiments of King and his co-workers,5-8 Saphir10 applied podophyllin to the cervices of patients prior to hysterectomy. He reported changes resembling carcinoma in situ in the squamous
Material Forty-four women were included in this study. All required hysterectomy for conditions unrelated to cervical disease. The most frequent indications for hysterectomy were chronic pelvic inflammatory disease, prolapse, and leiomyomas. No patient with malignancy or suspicion of malignancy was included in this group. No postmenopausal patients were found suitable for this study.
From the Department of Obstetrics and Gynecology, College of Medicine, University of Illinois. Aided by a grant from the American Cancer Society. Presented at a meeting of the Chicago Gynecological Society, Dec. 18, 1959.
Method The posterior lip of the cervix was selected as the target for podophyllin and biopsy specimens were obtained 3 weeks 1055
1056
Kaminetzky
December, 1960 Am.]. Obst. & Gynec.
No effect 14
c
(/)
.~
0
a.
M il d
12 10 8
Moderate
'+--
0
6
li
4
:J
2
E
z
0
Advanced 2 3 4 5 6 7 8 9 10
12
14
16
18
20 22
24
Number of daily paintings Fig. I. Histologic effect of podophyllin.
before the patient's admission to t!te hospital. The anterior lip provided the control. More than half of the patients had four-quadrant biopsies. A cervical smear and a vaginal smear were also prepared at this time. No patient was included in the study in whom biopsy showed atypia or whose smear con-
tained suspiCious cells. As expected, most specimens had evidence of chronic cervicitis. Painting was begun after the patient was hospitalized. Ten per cent podophyllin in mineral oil was applied to the entire posterior lip including the area of the squamocolumnar junction. An attempt was made
Fig. 2. Marked inflammation after two podophyllin applications. Hypercellularity and some disorganization of the epithelium may represent intense pre-existing cervicitis, but the vacuolization and areas of rather marked nuclear pleomorphism are probably due to podophyllin. ( x250; reduced !4.)
Volume 80 Number6
Cervical changes produced by podophyllin
Fig. 3. Moderate epithelial change observed after seven applications. Basal cell crowding and irregularity reflect increased mitotic activity. Characteristic degeneration vacuoles are noted in all layers. (x250; reduced V4.)
Fig. 4. Dysplasia observed in another patient after seven paintings. Basal and parabasal mitoses are present. There are scattered vacuolated cells. Atypical cells occupy most of the epithelium. (x250; reduced V4.)
1057
1058 Kaminetzky
December, 1960 Am. ]. Obst. & Gyncc.
Fig. 5. Squamous cells from intermediate layer of epithelium ( 15 podophyllin applications ) . There is variation in size and shape of cells and nuclei, nuclear and nucleolar enlargement, and binucleation. ( <700: reduced Y~. )
to exclude the anterior lip from contact with the podophyllin; however, in the region of the squamocolumnar junction this was not always possible. Painting was carried out once a day, and new smears were prepared prior to each painting. Hysterectomy was performed at various intervals depending on a number of uncontrolled variables and by whatever route best served the requirements of the underlying pathologic process. Three sections were usually prepared from the posterior lip of the cerv1x and two from the anterior lip.
normality was found in patients seen 6 weeks after hysterectomy. The histologic effects of podophyllin are graphically portrayed in Fig. 1. Suspicious or malignant appearing cells were recovered from 12 of the 44 women. In one the cervix was painted only once, in two it was painted 3 times, in two 5 times, in one 6, in one 7,
Results
Grossly evident inflammation of the posterior lip of the cervix and the adjacent vaginal mucosa was noted after one or two applications of podophyllin. Increased quantities of cervical mucus were produced by most patients. There were no complaints of irritation. At operation the vaginal mucosa was somewhat friable because of the chemical inflammation. On the other hand, this did not cause any problem of cuff healin.g or noticeable postoperative irritation of the peritoneum. No persistent cytologic ab-
Fig. 6. Giant basophilic squamous cell containing a small rosinophilic prematurely cornified cell ; nuclei ar ~ both abnormal. See n in a cervical smear after 25 applications of podophyllin. ( x900; reduced Ys.)
Volume 80 Number r,
in two 10, in one 15, in one 17, and in one 25 times. Curiously, one patient whose cervix was painted 16 times produced only slightly distorted cells. Certainly this was not the rule. Seven patients had mild changes in smears and 4 others showed moderate effect. There were, in addition, 20 patients from whom no atypical cells were recovered, 11 of these having had but a single podophyllin application. On the other hand, there was advanced atypia in the histologic specimens of 3 patients painted only once. In 25 patients histologic atypia was more advanced than the corresponding cytologic change. However, the opposite was true in 7 patients. One patient, whose cervix was painted 17 times and whose smears contained malignant appearing cells, had no identifiable squamous epithelium in the tissue sections examined. All but 3 patients exhibited evidence of the inflammatory component of the podophyllin effect when the posterior lip of the cervix was examined histologically. This consisted of an accumulation of polymorphonuclear leukocytes in the cell layers of the squamous epithelium, edema, and vasodilation in the stroma. The inflammatory cell infiltrate might involve one or all layers of the epithelium as well as the subjacent stroma (Fig. 2). Vacuolization of squamous cells, often seen as a part of nonspecific inflammatory processes, was usually very extensive after one to three applications of podophyllin. At times this feature was found in all cell layers above the basal area. However, vacuolization was not prominent in the areas containing the most advanced atypia. Mitotic figures were frequently seen in the deepest layers of the epithelium. All phases of mitosis were represented, although metaphase and anaphase figures predominated. Multipolar mitoses were not seen. Nuclear enlargement and distortion extending through the basal and intermediate layers were noted in most cervices painted one to four times. Moderate epithelial change usually involved the deepest 3 or 4 cell layers and was
Cervical changes produced by podophyllin 1059
noted after variable numbers of podophyllin applications, usually one to four. The nuclei were large, often paired, and at times filled the entire cell. There was considerable irregularity of the cellular outlines coarsening of the chromatin network, and prominence of the nucleoli (Fig. 3). The most advanced atypia with a few notable exceptions was found in patients whose cervices were painted the greatest number of times. Cells were greatly pleomorphic, and nuclei were considerably distorted. The nuclear chromatin was disposed in coarse clumps. In a number of cells the nuclear membrane could not be made out (Fig. 4). These abnormalities were usually limited to the deeper one half of the epithelium but in some sections occupied almost its entire extent. No evidence of stromal invasion was found in any histologic specimen. After many paintings the posterior lip was usually ulcerated and ragged appearing. Small, irregular sheets of abnormal epithelium were found showing considerable distortion and pleomorphism. These sheets often had little or no connection to the remaining epithelium. This was probably the source of masses of cells found in some of the cytology smears. Atypical or premature cornification was occasionally noted in sections exhibiting all degrees of podophyllin change. Cells of this kind were found in all layers of tl1e epithelium but most often above the basal layer. They were eosinophilic, had densely staining amorphous nuclei, and were at times found within the cytoplasm of other cells, which retained their cytoplasmic basophilia. Whereas significant histologic effects were apparent after one to four paintings with podophyllin, the cytologic effects during this time were not spectacular, and inflammatory features predominated. The smears contained large numbers of polymorphonuclear leukocytes between and within the cells. Many cells were vacuolated and the vacuoles were often filled with inflammatory cells. There was, in addition, moderate cyto-
l 060
Kaminetzky
Fig. 7. A sheet of abnormal squamous cells found applications of podophyllin. (x400; reduced l/:J .)
plasmic pleomorphism in these early smears. Nuclei were enlarged in some of the specimens but hyperchromatism was rare. After 6 or more paintings, the majority of smears exhibited more advanced abnormalities. The cells appeared parabasal in type and contained large nuclei with moderate condensation of chromatin and rather prominent nucleoli (Fig. 5 ). In general, the most atypical cells were recovered from patients whose cervices were painted many times. Great enlargement of the nuclei, irregularity of the nuclear outlines, clumping of the chromatin, and gross distortions of cytoplasmic outlines characterized such cells (Fig. 6 ) . The smears of 3 patients contained sheets of grossly abnormal cells (Fig. 7). Abnormal naked nuclei were found in smears from 2 patients (Fig. 8 ).
December, 1960 Am . J. Obst. & Gynec.
In
a cervical smt:ar after 14
Study of the histologic sections m sequence according to the number of podophyllin applications suggests a certain train of events. The cells in the superficial layers show the effects of inflammation. They become edematous, are infiltrated by polymorphonuclear leukocytes, and develop vacuoles. These layers which ordinarily contain cells that are dying as they approach the surface do not exhibit the nuclear effects
Comment
Precise controls were impossible for these preliminary experiments. A single biopsy of the posterior lip of the cervix might miss a spontaneous dysplastic lesion. On the other hand, occult spontaneous dysplasia would be unusual in an appreciable percentage of 44 women.
Fig. 8. Naked abnormal nucleus. Seen in a cervical smear after I 0 applications of podophyllin. (x900; reduced 13.)
Volume 80 Number 6
of podophyllin. These layers finally lose con~ nection with the deeper layers of cells. Nuclear effects seem to occur in basal and parabasal layers where cells are growing and reproducing. Repeated application of podophyllin produces generation upon gen~ eration of abnormal cells which do not mature in the normal manner. Finally, a number of layers of abnormal cells are seen. However, even these cells ultimately attempt maturation. This gives a picture resembling spontaneous atypical epithelial hyperplasia. It is probable that the initial layers of cells at the surface must be removed before the altered basal cells can attain a free surface and be exfoliated. This would appear to
REFERENCES
1. Belkin, M.: J. Pharmacal. & Exper. Therap. 93: 18, 1948. 2. Berenblum, I.: J. Nat. Cancer Inst. 11: 839, 1950. 3. Greenspan, E. M., Leiter, J., and Shear, M. J.: J. Nat. Cancer Inst. 10: 1295, 1950. 4. Kaminetzky, H. A., McGrew, E. A., and Phillips, R. L.: Obst. & Gynec. 14: 1, 1959. 5. King, L. S.: J. Nat. Cancer Inst. 8: 215, 1948.
Cervical changes produced by podophyllin 1061
explain the lag of cytologic atypia as compared with the histologic change. Summary
Podophyllin produced atypia in the cervical squamous epithelia of 28 of the 44 women of this study. It produced an abnormal inflammatory response without dysplasia in 12 more women. No appreciable effect was seen in 3 women. Dysplastic lesions produced by podophyllin have many features in common with spontaneously occurring atypical epithelial hyperplasia, including the shedding of malignant appearing cells. There is at present no evidence that podophyllin is carcinogenic.
6. King, L. S., and Cauldwell, E. W.: J. Nat. Cancer Inst. 10: 131, 1949. 7. King, L. S., and Sullivan, M.: Science 104: 244, 1946. 8. King, L. S., and Sullivan, M.: Arch. Path. 43: 374, 1947. 9. Leiter, J., Downing, V., Hartwell, J. L., and Shear, M. J.: J. Nat. Cancer Inst. 10: 1273, 1950. 10. Saphir, 0., and Leventhal, M. L.: Am. J. Path. 34: 555, 1958.
Discussion
DR. ELIZABETH A. McGREw, Chicago, Illinois. We must learn to distinguish between the rather common epithelial "dysplasias" of the cervix and preinvasive carcinoma; which of these morphologically similar lesions are reversible and which should be considered carcinoma in situ, capable of progressing inevitably to invasive carcinoma. Dr. Ronald Greene brought this difference to our attention some years ago by pointing out that preinvasive cervical carcinoma during pregnancy does not represent a peculiar epithelial abnormality of pregnancy and that those lesions which disappear are not preinvasive carcinoma. He did not say exactly what they were or establish criteria for differentiating them from carcinoma in situ except to illustrate them in excellent photomicrographs. People working in this field have come to recognize some relative criteria for making this distinction, such as the greater thickness of the epithelium in carcinoma in situ and the failure of the cells to mature
and reorient themselves as they reach the surface. There are still many borderline lesions which are resistant to interpretation, however. In Dr. Kaminetzky's work we have seen marked epithelial abnormality produced chemically by a noncarcinogenic substance. The cells of this epithelium looked malignant, both in smears and in sections; yet, as Dr. Kaminetzky indicated, when daily podophyllin painting of the mouse cervix is continued for several months and then discontinued, vaginal smears become normal in a few days and none of the animals develop carcinoma. He has, then, produced changes in cells which mimic malignancy, yet are temporary and reversible, thus corresponding to spontaneous cervical epithelial dysplasia. Apparently the production of these changes depends on the antimitotic action of podophyllin. It prevents the formation of the mitotic spindle so that the chromosomes cannot separate. These cells are blocked in the metaphase but survive
1062 Kaminetzky
sufficiently to continue synthesis of nucleic acids. Their nuclei may then be reconstituted, but they have an excessive amount of nucleic acids so that they arc hyp('rchromatic and unusually large. Some of the carcinogenic agents also han~ mitotic effects; the difference is that these cause permanent mutations. I think that this work gives us some insight into the pathogenesis of malignancy. It also adds to the battery of tools with which to study the reversible as compared with the truly neoplastic epithelial abnormalities. DR. S. R. LAsH, Chicago, Illinois. In this experimental work carried out over a very short period the morphologic changes we have seen in cast-off cells are almost impossible to tell from those seen in carcinoma cells. The prominent nuclei, the chromatin, the marked irregularity and enlargement of the nuclei are morphologic criteria used to determine malignancy. These were all short-term experiments. To say that this chemical is not carcinogenic is impossible in this short period of time. I think if the experiment were carried out over a long period of time such conclusions could be made.
December, 1960 Am. J. Ob;t. & Gynec.
The experiments in mice will lead to conclusions on the cff<•ct in mice only.
DR. C!L\RLES E. GALLOWAY, Evanston, Illinois. There is another substance that produces carcinoma-like l(•sions in the cervix, and that is carbon dioxide snow. If carbon dioxide snow is put on the cPrvix, it will undergo change that looks very malignant both grossly and microscopically during the healing stagt'. DR. KAMINETZKY (Closing). Dr. Lash indicated the need for long-term studies of the effect of podophyllin on human cervical epithelium. This would be very useful, but it poses a number of practical problnns in finding and keeping patients on such a program. For the moment the objectives of the human studies arc limited to those possible in the preoperative patient who is already hospitalized. In response to Dr. Galloway, I have not applied carbon dioxide snow to the cervix. Tissue injury is often followed by compensatory hyperplasia. We have felt it unwise to follow the course of the podophyllin treated patient with serial biopsies for the same reason. It seemed possible that we might add another kind of tissue injury to the effect of the drug.