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Fibrinolytic activity in cervical and endometrial neoplasia
Fibrinolytic activity in cervical and endometrial neoplasia JACK
ZELLER,
TAKASHI RALPH New
OKAGAKI, M.
York,
M.D.
New
M.D.,
RICHART,
PH.D
M.D.*
York
Plasminogen activator fibrinolytic activity and nonspecific protease fibrinolytic activity in uterine tissue controls and neofilasias were studied by the histochemical fibrin film method of Kwaan and A&up. The frozen tissue sections of 2 to 3 p thick were mounted on fibrin-plasminogen films and incubated, at 37” C. for 60 minutes for demonstration of plasminogea activator fibrinolytic activity. Plasminogeninactivated control films by incubating at 80” C. for 45 minutes were used for nonspecific protease fibrinolytic activity. Plasminogen activator fibrinolytic activity was found not only in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix, but also in areas of squamous metaplasia of the cervix and invasive endometrial adenoacanthoma. Nonspecific protease fibrinolytic activity was seen in endometrial carcinoma cells and in cervical intraepithelial neoplasia.
I N I N v A s I v E carcinoma of the endometrium and uterine cervix increased amounts of extractable fibrinolytic activity are present compared to normal tissue. This increase has been positively correlated with the size, viability, and invasive growth pattern of malignant epithelial cells.2 One limitation of such pure biochemical studies of tumor extracts is the inability of localizing and distinguishing the enzyme-producing epithelial cells from endothelial cells31 4 with the same enzymatic activity. Since most neoplasms are associated with new vessel forma-
tion it is necessary to differentiate between the fibrinolytic activity of the vessels and that of the neoplastic cells, lest activity in the former be attributed to the latter. In the present study a histochemical examination of uterine tissue was performed to obtain information about the distribution of fibrinolytic activity on a cellular level and to determine whether fibrinolytic activity is found exclusively in invasive carcinoma. The tissues examined included benign cervical squamous metaplasia, dysplasia, and carcinoma in situ (CIS) , as well as invasive carcinoma. Endometrial adenocarcinoma and endometrial adenoacanthoma were also selected to compare enzyme activity in squamous and glandular epithelia.
From the Department of Pathology and Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, and the Obstetrical and Gynecological Service (The Sloane Hospital for Women) of the Presbyterian Hospital. This investigation was supported by United States Public Health Service Fellowshifi No. 5-K03-CA 13975-06 United States Public Health Service Research Grant No. CA 07060-06.
Material
and
methods
A modification of the fibrin slide method of Kwaan and Astrupl was used for the histochemical demonstration and localization of fibrinolytic activity. Glass slides were coated with fibrin-plasminogen film and cryostat cut tissue sections of 2 to 3 p thickness were mounted on it and incubated at
and
*Reprint requests should be addressed to Dr. R. M. Richart, 630 West 168th Street, New York, New York 10032. 1071
1072
Zeller,
Okagaki,
Fie. 1A. Moderate Fig.
and Richart
cervical
Amer.
dvwlasia. I
.
Hematoxylin
and
eosin-stained
section
of block
August J. Obstet.
used
1, 1970 Gynec.
in
1B.
37O C. for 30 minutes. This incubation causes tissue plasminogen activator to convert plasminogen to plasmin which in turn digests the fibrin and produces a clear zone in the film (fibrinolysis) which is distinguishable from adjacent undissolved fibrin. Cells producing fibrinolysis on plasminogenfibrin films have “plasminogen activator fibrinolytic activity.” Cells producing fibrinolysis on fibrin films free of plasminogen have “nonspecific protease fibrinolytic activity.” The amount of fibrinolysis was evaluated by noting the size of the area of dissolved film. The amount of fibrin in each film was kept constant by using the same amount of fibrin-
ogen per slide. The amount of plasminogen was kept constant by using the same source of fibrinogen-plasminogen substrate.* As a positive control, slides were not included in the study unless vessels in the section produced an area of fibrinolysis in the film. A negative control for fibrinolytic activator was its absence in unincubated tissue. Plasminogen-free fibrin films were prepared by heating the coated slides to 80’ C. for 4.5 minutes. The specimens used in the study were obtained fresh from the operating room and
*Borne Fibrinogen, ceutical Company, Lot
Cohn Fraction No. B8512D06.
I,
Armour
Pharma-
Volume 107 Number
Fibrinolytic
activity in neoplasia
1073
7
Fig. 1B. Fibrin film and zone of fibrinolysis minogen activator fibrinolytic activity.
representative samples quick frozen with Dry Ice and alcohol. Serial time studies indicated that fibrinolytic activity was unchanged after being deep frozen for several months. Most tissues, however, were examined the same day they were received. After freezing the block of tissue, sections 2 to 3 p thick were cut on a cryostat. Thicker sections were also made but were less satisfactory, since the intensity of the nuclear stain usually obscured the more lightly stained fibrin. The following lesions were examined: cervical squamous metaplasia, one case; cervical squamous epithelial dysplasia, 2 cases ; cervical carcinoma in situ, 2 cases;
present at the surface (arrows)
indicating
pIa-
cervical carcinoma in situ with invasive squamous cell carcinoma, one case; endometrial adenoacanthoma, one case; and endometrial adenocarcinoma, one case. Results Plasminogen activator fibrinolytic activity was present focally in the epithelial cells of cervical squamous metaplasia, dysplasia (Figs. 1A and 1B) , carcinoma in situ (Figs. 2A and 2B), and invasive squamous cell carcinoma. It was absent in normal squamous epithelium. The number of foci of plasminogen activator fibrinolytic activity and the size of each had no relationship to the type
1074
Zeller,
Okagaki,
and Richart
Fig, 2A. Carcinoma in situ of the cervix with stained section of block used in Fig. 2B.
of neoplastic cell which was present. The tissue containing both carcinoma in situ and invasive carcinoma on the same slide had larger zones of plasminogen activator fibrinolytic activity in the former than in the fatter (Fig. 3). Plasminogen activator fibrinolytic activity was also present in the squamous portions of the endometrial adenoacanthoma but was absent in the endometrial adenocarcinoma without squamous elements. In contrast to its focal distribution in squamous epithelial lesions plasminogen activator fibrinolytic activity was present in most small venules and capillaries (Fig. 4). The size of the area of lysis varied from vessel to vessel and patient to patient, but in general exceeded that occurring in the epithelial lesions. When areas
Amer.
gland involvement.
Hematoxylin
August J. Obstet.
1, 1970 Gynec.
and eosin-
of the same lesion with or without plasminogen activator fibrinolytic activity were compared histologically, no consistent differences were noted. Necrotic cells did not contain plasminogen activator fibrinolytic activity confirming the observations made using tissue extract assays.l Nonspecific protease fibrinolytic activity was seen both in the epithelium of endometrial adenocarcinoma and in the epithehum of cervica.1 carcinoma in situ (Fig. 5). The distribution was diffuse in both, but the zones of fibrinolysis were small compared to the zones of plasminogen activator fibrinolytic activity seen in squamous epithelium or in vessels. No vessels containing nonspecific protease fibrinolytic activity were noted.
Volume Number
107 1
Fig. lium
Fibrinolytic
2B. Fibrin film preparation. Plasminogen activator fibrinolytic and in a gland replaced by carcinoma in situ (arrows).
activity
activity
in neoplasia
is present
1075
in epithe-
Comment Plasminogen activator fibrinolytic activity was present in a wide variety of squamous epithelia, varying from benign squamous metaplasia to invasive carcinoma of the cervix. Among the neoplastic lesions, the amount of plasminogen activator fibrinolytic activity did not correlate with the degree of malignancy. Plasminogen activator fibrinolytic activity was not present in endocervical glands, in the gland cells of endometrial adenocarcinoma, or in normal cervical epithelium, but was present in all types of cervical squamous epithelial neoplasia, squamous metaplasia of the endocervix, the squamous portion of an endometrial adeno-
acanthoma, and in small new blood vessels lined by endothelium. These findings suggest that two attributes are necessary for cellular plasminogen activator fibrinolytic activity to occur-the cell must be epithelial and there must be active tissue growth. It is possible that the focal and apparently random distribution of plasminogen activator fibrinolytic activity in squamous cells of similar morphology might be accounted for by differences in local growth rates occurring at a cellular level. In a study of human vaginal epithelial smears, Tympanidis, King, and Astrup5 found a similar random distribution of plasminogen activator fibrinolytic activity “re-
Fig. 3. Carcinoma in situ of the cervix associated with invasive squamous cell carcinoma with multiple large foci of plasminogen activator fibrinolytic activity (arrows). Other areas (not seen) of invasive carcinoma had less plasminogen activator fibrinolytic activity. Smaller foci (arrows) of activity in invasive carcinoma are also seen in the same photomicrograph.
Fig. 4. Extensive plasminogen activator fibrinolytic activity of vascular origin with diffusion into stroma. Small foci of plasminogen activator fibrinolytic activity was also present in areas of carcinoma in situ not seen in this picture. Note that every vessel has activity, but of different amounts.
Volume Number
107 7
Fibrinolytic
Fig. 5. Carcinoma is present.
Fibrin
in situ with film, pretreated
gland involvement. Nonspecific by heat, has no plasminogen.
lated preferably to young cells of the vaginal epithelium.” In contrast to the random and focal plasminogen activator fibrinolytic activity in the squamous epithelia, there was relatively uniform vascular plasminogen activator fibrinolytic activity in all the tissues examined. The sum total of the vascular plasminogen activator fibrinolytic activity often exceeded that of the squamous epithelium, depending upon the ratio of epithelial cells and vessels in different specimens or parts of the same specimen. These differences underscore the limitations of purely biochemical studies2 of the activity of this enzyme. If epithelial and vas-
proteolytic
activity
in
fibrinolytic
neoplasia
1077
activity
cular components cannot be distinguished, activity in one may be attributed to the other. In view of this problem the positive correlation between plasminogen activator fibrinolytic activity and endometrial adenocarcinoma reported by Schmidt-MatthiesenZ must be re-examined. The vascularization of malignancy, rather than the malignant glandular cells themselves, could have produced the increased plasminogen activator fibrinolytic activity. Schmidt-Matthiesen also reported that large amounts of nonspecific protease fibrinolytic activity were found in endometrial carcinoma. We have confirmed this finding histochemically. It should be noted
1078
Zeller,
Okagaki,
and
Richart
here that, unlike plasminogen activator fibrinolytic activity, the vessels make no contribution to this type of nonspecific fibrinolysis. Our observations concerning the absence of plasminogen activator fibrinolytic activity and the presence of nonspecific protease fibrinolytic activity in endometrial carcinoma confirm those of Weiss and Beller.” We differ from these authors concerning plasminogen activator fibrinolytic activity in invasive cervical carcinoma, however, since they found high activity exclusively in the
REFERENCES 1. Kwaan, H. C., and Astrup, T.: Lab. Invest. 17:140, 1967. 2. Schmidt-Matthiesen, H.: Bibl. Gynaec., Suppl. 44, 1967. 3. Todd, A. S.: J. Path. Bact. 78: 281, 1959.
Amer.
August J. Obstet.
1, 1970 Gynec.
invasive fingers of carcinoma and in metastases and we noted such activity in all neoplastic lesions examined. This may be due, in part, to differences in technique. In our studies the thin 2 to 3 p sections permitted good transmission of light, and one could easily observe fibrinolysis through the overlying tissue. The use of monochromatic and metachromatic stains without counterstains also afforded views of fibrin or its lysis not otherwise obtained. The authors are indebted to Mrs. Ida Nathan for
photomicrography.
4. 5.
Todd, A. S.: J. Clin. Path. 17: 324, 1964. Tympanidis, K., King, A. E., and Astrup, T.: AMER. J. OBSTET. GYNEC. 100: 185, 1968. Weiss, G., and Belier, F. K.: AMER. J. OBSTET. GYNEC. 103: 1023, 1969.
6.
ZELLER,
TAKASHI RALPH New
OKAGAKI, M.
York,
M.D.
New
M.D.,
RICHART,
PH.D
M.D.*
York
Plasminogen activator fibrinolytic activity and nonspecific protease fibrinolytic activity in uterine tissue controls and neofilasias were studied by the histochemical fibrin film method of Kwaan and A&up. The frozen tissue sections of 2 to 3 p thick were mounted on fibrin-plasminogen films and incubated, at 37” C. for 60 minutes for demonstration of plasminogea activator fibrinolytic activity. Plasminogeninactivated control films by incubating at 80” C. for 45 minutes were used for nonspecific protease fibrinolytic activity. Plasminogen activator fibrinolytic activity was found not only in intraepithelial neoplasia and invasive squamous cell carcinoma of the cervix, but also in areas of squamous metaplasia of the cervix and invasive endometrial adenoacanthoma. Nonspecific protease fibrinolytic activity was seen in endometrial carcinoma cells and in cervical intraepithelial neoplasia.
I N I N v A s I v E carcinoma of the endometrium and uterine cervix increased amounts of extractable fibrinolytic activity are present compared to normal tissue. This increase has been positively correlated with the size, viability, and invasive growth pattern of malignant epithelial cells.2 One limitation of such pure biochemical studies of tumor extracts is the inability of localizing and distinguishing the enzyme-producing epithelial cells from endothelial cells31 4 with the same enzymatic activity. Since most neoplasms are associated with new vessel forma-
tion it is necessary to differentiate between the fibrinolytic activity of the vessels and that of the neoplastic cells, lest activity in the former be attributed to the latter. In the present study a histochemical examination of uterine tissue was performed to obtain information about the distribution of fibrinolytic activity on a cellular level and to determine whether fibrinolytic activity is found exclusively in invasive carcinoma. The tissues examined included benign cervical squamous metaplasia, dysplasia, and carcinoma in situ (CIS) , as well as invasive carcinoma. Endometrial adenocarcinoma and endometrial adenoacanthoma were also selected to compare enzyme activity in squamous and glandular epithelia.
From the Department of Pathology and Obstetrics and Gynecology, College of Physicians and Surgeons, Columbia University, and the Obstetrical and Gynecological Service (The Sloane Hospital for Women) of the Presbyterian Hospital. This investigation was supported by United States Public Health Service Fellowshifi No. 5-K03-CA 13975-06 United States Public Health Service Research Grant No. CA 07060-06.
Material
and
methods
A modification of the fibrin slide method of Kwaan and Astrupl was used for the histochemical demonstration and localization of fibrinolytic activity. Glass slides were coated with fibrin-plasminogen film and cryostat cut tissue sections of 2 to 3 p thickness were mounted on it and incubated at
and
*Reprint requests should be addressed to Dr. R. M. Richart, 630 West 168th Street, New York, New York 10032. 1071
1072
Zeller,
Okagaki,
Fie. 1A. Moderate Fig.
and Richart
cervical
Amer.
dvwlasia. I
.
Hematoxylin
and
eosin-stained
section
of block
August J. Obstet.
used
1, 1970 Gynec.
in
1B.
37O C. for 30 minutes. This incubation causes tissue plasminogen activator to convert plasminogen to plasmin which in turn digests the fibrin and produces a clear zone in the film (fibrinolysis) which is distinguishable from adjacent undissolved fibrin. Cells producing fibrinolysis on plasminogenfibrin films have “plasminogen activator fibrinolytic activity.” Cells producing fibrinolysis on fibrin films free of plasminogen have “nonspecific protease fibrinolytic activity.” The amount of fibrinolysis was evaluated by noting the size of the area of dissolved film. The amount of fibrin in each film was kept constant by using the same amount of fibrin-
ogen per slide. The amount of plasminogen was kept constant by using the same source of fibrinogen-plasminogen substrate.* As a positive control, slides were not included in the study unless vessels in the section produced an area of fibrinolysis in the film. A negative control for fibrinolytic activator was its absence in unincubated tissue. Plasminogen-free fibrin films were prepared by heating the coated slides to 80’ C. for 4.5 minutes. The specimens used in the study were obtained fresh from the operating room and
*Borne Fibrinogen, ceutical Company, Lot
Cohn Fraction No. B8512D06.
I,
Armour
Pharma-
Volume 107 Number
Fibrinolytic
activity in neoplasia
1073
7
Fig. 1B. Fibrin film and zone of fibrinolysis minogen activator fibrinolytic activity.
representative samples quick frozen with Dry Ice and alcohol. Serial time studies indicated that fibrinolytic activity was unchanged after being deep frozen for several months. Most tissues, however, were examined the same day they were received. After freezing the block of tissue, sections 2 to 3 p thick were cut on a cryostat. Thicker sections were also made but were less satisfactory, since the intensity of the nuclear stain usually obscured the more lightly stained fibrin. The following lesions were examined: cervical squamous metaplasia, one case; cervical squamous epithelial dysplasia, 2 cases ; cervical carcinoma in situ, 2 cases;
present at the surface (arrows)
indicating
pIa-
cervical carcinoma in situ with invasive squamous cell carcinoma, one case; endometrial adenoacanthoma, one case; and endometrial adenocarcinoma, one case. Results Plasminogen activator fibrinolytic activity was present focally in the epithelial cells of cervical squamous metaplasia, dysplasia (Figs. 1A and 1B) , carcinoma in situ (Figs. 2A and 2B), and invasive squamous cell carcinoma. It was absent in normal squamous epithelium. The number of foci of plasminogen activator fibrinolytic activity and the size of each had no relationship to the type
1074
Zeller,
Okagaki,
and Richart
Fig, 2A. Carcinoma in situ of the cervix with stained section of block used in Fig. 2B.
of neoplastic cell which was present. The tissue containing both carcinoma in situ and invasive carcinoma on the same slide had larger zones of plasminogen activator fibrinolytic activity in the former than in the fatter (Fig. 3). Plasminogen activator fibrinolytic activity was also present in the squamous portions of the endometrial adenoacanthoma but was absent in the endometrial adenocarcinoma without squamous elements. In contrast to its focal distribution in squamous epithelial lesions plasminogen activator fibrinolytic activity was present in most small venules and capillaries (Fig. 4). The size of the area of lysis varied from vessel to vessel and patient to patient, but in general exceeded that occurring in the epithelial lesions. When areas
Amer.
gland involvement.
Hematoxylin
August J. Obstet.
1, 1970 Gynec.
and eosin-
of the same lesion with or without plasminogen activator fibrinolytic activity were compared histologically, no consistent differences were noted. Necrotic cells did not contain plasminogen activator fibrinolytic activity confirming the observations made using tissue extract assays.l Nonspecific protease fibrinolytic activity was seen both in the epithelium of endometrial adenocarcinoma and in the epithehum of cervica.1 carcinoma in situ (Fig. 5). The distribution was diffuse in both, but the zones of fibrinolysis were small compared to the zones of plasminogen activator fibrinolytic activity seen in squamous epithelium or in vessels. No vessels containing nonspecific protease fibrinolytic activity were noted.
Volume Number
107 1
Fig. lium
Fibrinolytic
2B. Fibrin film preparation. Plasminogen activator fibrinolytic and in a gland replaced by carcinoma in situ (arrows).
activity
activity
in neoplasia
is present
1075
in epithe-
Comment Plasminogen activator fibrinolytic activity was present in a wide variety of squamous epithelia, varying from benign squamous metaplasia to invasive carcinoma of the cervix. Among the neoplastic lesions, the amount of plasminogen activator fibrinolytic activity did not correlate with the degree of malignancy. Plasminogen activator fibrinolytic activity was not present in endocervical glands, in the gland cells of endometrial adenocarcinoma, or in normal cervical epithelium, but was present in all types of cervical squamous epithelial neoplasia, squamous metaplasia of the endocervix, the squamous portion of an endometrial adeno-
acanthoma, and in small new blood vessels lined by endothelium. These findings suggest that two attributes are necessary for cellular plasminogen activator fibrinolytic activity to occur-the cell must be epithelial and there must be active tissue growth. It is possible that the focal and apparently random distribution of plasminogen activator fibrinolytic activity in squamous cells of similar morphology might be accounted for by differences in local growth rates occurring at a cellular level. In a study of human vaginal epithelial smears, Tympanidis, King, and Astrup5 found a similar random distribution of plasminogen activator fibrinolytic activity “re-
Fig. 3. Carcinoma in situ of the cervix associated with invasive squamous cell carcinoma with multiple large foci of plasminogen activator fibrinolytic activity (arrows). Other areas (not seen) of invasive carcinoma had less plasminogen activator fibrinolytic activity. Smaller foci (arrows) of activity in invasive carcinoma are also seen in the same photomicrograph.
Fig. 4. Extensive plasminogen activator fibrinolytic activity of vascular origin with diffusion into stroma. Small foci of plasminogen activator fibrinolytic activity was also present in areas of carcinoma in situ not seen in this picture. Note that every vessel has activity, but of different amounts.
Volume Number
107 7
Fibrinolytic
Fig. 5. Carcinoma is present.
Fibrin
in situ with film, pretreated
gland involvement. Nonspecific by heat, has no plasminogen.
lated preferably to young cells of the vaginal epithelium.” In contrast to the random and focal plasminogen activator fibrinolytic activity in the squamous epithelia, there was relatively uniform vascular plasminogen activator fibrinolytic activity in all the tissues examined. The sum total of the vascular plasminogen activator fibrinolytic activity often exceeded that of the squamous epithelium, depending upon the ratio of epithelial cells and vessels in different specimens or parts of the same specimen. These differences underscore the limitations of purely biochemical studies2 of the activity of this enzyme. If epithelial and vas-
proteolytic
activity
in
fibrinolytic
neoplasia
1077
activity
cular components cannot be distinguished, activity in one may be attributed to the other. In view of this problem the positive correlation between plasminogen activator fibrinolytic activity and endometrial adenocarcinoma reported by Schmidt-MatthiesenZ must be re-examined. The vascularization of malignancy, rather than the malignant glandular cells themselves, could have produced the increased plasminogen activator fibrinolytic activity. Schmidt-Matthiesen also reported that large amounts of nonspecific protease fibrinolytic activity were found in endometrial carcinoma. We have confirmed this finding histochemically. It should be noted
1078
Zeller,
Okagaki,
and
Richart
here that, unlike plasminogen activator fibrinolytic activity, the vessels make no contribution to this type of nonspecific fibrinolysis. Our observations concerning the absence of plasminogen activator fibrinolytic activity and the presence of nonspecific protease fibrinolytic activity in endometrial carcinoma confirm those of Weiss and Beller.” We differ from these authors concerning plasminogen activator fibrinolytic activity in invasive cervical carcinoma, however, since they found high activity exclusively in the
REFERENCES 1. Kwaan, H. C., and Astrup, T.: Lab. Invest. 17:140, 1967. 2. Schmidt-Matthiesen, H.: Bibl. Gynaec., Suppl. 44, 1967. 3. Todd, A. S.: J. Path. Bact. 78: 281, 1959.
Amer.
August J. Obstet.
1, 1970 Gynec.
invasive fingers of carcinoma and in metastases and we noted such activity in all neoplastic lesions examined. This may be due, in part, to differences in technique. In our studies the thin 2 to 3 p sections permitted good transmission of light, and one could easily observe fibrinolysis through the overlying tissue. The use of monochromatic and metachromatic stains without counterstains also afforded views of fibrin or its lysis not otherwise obtained. The authors are indebted to Mrs. Ida Nathan for
photomicrography.
4. 5.
Todd, A. S.: J. Clin. Path. 17: 324, 1964. Tympanidis, K., King, A. E., and Astrup, T.: AMER. J. OBSTET. GYNEC. 100: 185, 1968. Weiss, G., and Belier, F. K.: AMER. J. OBSTET. GYNEC. 103: 1023, 1969.
6.