Path. Res. Pract. 187,36-43 (1991)
Immunohistochemical Studies on Uterine Tumors I. Invasive Squamous Cell Carcinomas of the Cervix and Their Precursors G. Dallenbach-Hellweg and G. Lang Institut fur Pathologie, Mannheim, FRG
SUMMARY 40 invasive carcinomas and 80 preinvasive lesions of the uterine cervix were studied immunohistochemically; 40 benign lesions served as controls. On histological and immunohistochemical examination, invasive and preinvasive carcinomas were subdivided in the squamous (large cell, ectocervical) type and the reserve cell (small, large or clear cell, endocervical) type. Immunohistochemically, 100% of the invasive and preinvasive squamous cell carcinomas were positive with anticytokeratins 13, 14, 16 and negative with anticytokeratin 8 and anti-CEA. Most of the invasive and preinvasive reserve cell carcinomas showed a coexpression ofcytokeratins 13,14,16,8 and CEA. The subdivision ofinvasive carcinomas ofthe ecto- and endocervix into squamous cell and reserve cell types made by means of their structural differences is substantiated and re-evaluated by their immunohistochemical reactions. Both types of carcinomas retain the complex pattern of cytokeratins shown by their cells oforigin. The reserve cell carcinomas, in addition, acquire a coexpression for CEA that indicates malignant transformation. The subdivision is of clinical importance because both types of carcinomas vary in their mode and speed of invasion and spread and in their association with HPV infection. Introduction Cell typing with molecular markers has become increasingly important for the classification of tumors and for our understanding of their histogenetic development. A number of molecular markers have been described that are specific for certain cell types or lines of differentiation. As components of the cytoskeleton, besides microfilaments and microtubules, the intermediate filaments constitute a large proportion of the total cytoplasmic material. Among these, the cytokeratins are a complex family of at least 20 polypeptide chains that vary in their pH values and in their molecular weights. The types of polypeptide chains found in the cells of a tissue are specific for that tissue 21 • Regarding the carcinomas of the uterus, the following questions seemed to be important: 1. What expression or coexpression of intermediate filaments can help recognize the directions of differentia0344-0338/91/0187-0036$3.5 0/0
tion that an epithelial cell might take, and at which stage of differentiation is such a recognition possible? 2. Is the composition of the cytoskeleton of a cell undergoing carcinogenesis maintained or does it change? 3. Can uterine carcinomas that appear alike histologically but arise from different epithelia be immunohistochemically differentiated from one another from their histogenesis? Since prediction of patient survival in squamous cell carcinomas of the uterine cervix often proves difficult on histological examination 8 , valuable additional information could possibly be gained by evaluating the intermediate filament composition of the tumor cells. Furthermore, such studies may help to explain why cervical cancer has changed in clinical and morphological behaviour, and why its incidence has proportionally increased in young women 14 • © 1991 by Gustav Fischet Verlag, Stuttgatt
Immunohistochemistry on Uterine Tumors· 37 Material and Methods
The immunohistochemical observations are summarized in Table 1. The normal, papillomatous and dysplastic squamous epithelium of the ectocervix as well as the carcinoma in situ and the invasive large cell squamous carcinoma arising from it react positively with anticytokeratins 13/14/16, but negatively with anticytokeratin 8, antivimentin and anti-CEA (Fig. 1). The metaplastic squamous epithelium of the endocervix reacts similarly. Only the basal layer of the squamous ectocervical epithelium focally shows a coexpression of cytokeratin 8 in addition to 13/14/16. The reserve cell hyperplasia of the endocervical mucosa is in roughly 90% of the cases examined positively for cytokeratin 13/14/16 and for cytokeratin 8. The carcinoma in situ of the reserve cell type that originates from this hyperplasia gives the same reactions and, in addition, is positive for CEA. The invasive squamous cell carcinoma of the reserve cell type is, independent of its grade of differentiation, strongly positive for cytokeratin 8 (Figs. 2-4) and weakly positive or even negative with anticytokeratin 13/14/16. As in the preinvasive reserve cell carcinoma, and contrary to the squamous cell type of the ectocervix, the invasive reserve cell carcinoma shows a distinct coexpression of CEA (Fig. 2). - A very similar expression of intermediate filaments is observed in the clear cell carcinomas of the endocervix. 20 of the invasive carcinomas and 40 of the preinvasive lesions were tested for their content of genomic HPV by in-situ-hybridization: of 6 squamous type carcinomas all 6 were negative, of 14 reserve cell carcinomas 6 were negative, 7 were positive for HPV 16, one for HPV 18. Of the 35 carcinomas in situ of reserve cell type 24 were positive for HPV 16 (Fig. 5), 11 were negative, 3 carcinomas in situ of squamous type were negative, 2 koilocytic dysplasias were positive for HPV 6 and 11. The koilocytic dysplasias and carcinomas in situ presumably induced by human papilloma viruses (HPV) express patterns of cytoskeletal filaments that correspond to those shown by their tissue of origin. In a corresponding study (in preparation), we could not detect a transformation of intermediate filaments following HPV -infection27 : the expression of cytokeratins is the same as in the equivalent nonkoilocytic preinvasive lesions. As has been previously reported 28 , the HP virus can infect not only the squamous
160 specimens of uterine cervices obtained by hysterectomy or conization were examined histologically and immunohsitochemically. Formalin-fixed paraffin slides were routinely stained with Hematoxylin and Eosin (H + E) and Hematoxylin-Picric-acidAcid-fuchsin (van Gieson). Immunohistochemical studies were performed on freshly frozen cryostat sections and on formalinfixed paraffin slides with the APAAP immuno-alkaline-phosphotase technique? after applying various monoclonal antibodies: the broad-spectrum cytokeratin antibody KL 1 (Dianova, Hamburg, Germany3!); Ks 13.1 against cytokeratin 13 and to a minor degree cytokeratin 14 and 16 (Progen, Heidelberg, Germany20)j CAM 5.2 against cytokeratin 8 (Becton Dickinson, Heidelberg, Germany!9); V 9 against Vimentin (Dakopatts, Hamburg, Germany); CM 010 against CEA, purified from non-specific crossreacting antigen (NCA) (Ortho Diagnostics, Neckargemiind, Germany29). For in situ-hybridization we applied DNA probes for HPV 6/11, 16/18 and 18 (Ortho Diagnostics, Neckargemiind, Germany) to paraffin slides. Results Of the 160 cervical specimens studied by routine histological methods, 40 were invasive carcinomas, 80 were preinvasive lesions (10 dysplasias, 70 carcinomas in situ), and 40 benign lesions (metaplasias or regenerative epithelium) served as controls. On histological and immunohistochemical examination, the preinvasive and invasive carcinomas, according to their location and cellular appearence, were subdivided into a) the squamous (large cell) type with or without keratinization, located at or near the squamo-columnar junction (20 cases: 10 invasive carcinomas and 10 in situ lesions), and b) the reserve cell (small or large cell) type located within the endocervical mucosa (95 cases: 25 invasive carcinomas and 70 in situ lesions). c) the clear cell type of endocervical carcinoma (5 cases). 21 invasive adenocarcinomas and adeno-squamous carcinomas of the endocervix are described in a separate paper 12 .
Table 1. Immunohistochemical results on invasive and in situ carcinomas of the uterine cervix Diagnosis
invasive Carcinoma squamous cell type reserve cell type clear cell type in situ lesions (CIN) squamous cell type reserve cell type
average age
Number of cases
Cytokeratin 13.14.16
Cytokeratin 8
56 40 41
10 25 5
10 23 3
2 2
24 5
35 35
10 70
10 67
3
66
+
+
10
CEA
Vimentin
+
+
1
25 3
10 4
70
10 2 10
10 25 5 10 70
Fig. 1. Invasive carcinoma of the ectocervix, squamous cell type.
55-year-old patient. - a: Immunohistochemical reaction with anticytokeratin 13: virtually all tumor cells stain positively. - b: Negative reaction with anticytokeratin 8. - c: Negative reaction with anti-CEA (x 140).
Fig. 2. Invasive carcinoma of the endocervix, reserve cell type. 38-year-old patient. Most of the tumor cells show a coexpression of cytokeratin 13 (a), 8 (b) and CEA (c). Some basally located undifferentiated cells express cytokeratin 8 only (b) and are negative with anticytokeratin 13 (a). (a + b: x 350, c: x 140).
40 . G. Dallenbach-Hellweg and G. Lang
glands, then the cytokeratin pattern this type of carcinoma shows is maintained from benign reserve cell origin to its endstage carcinoma. The basal cell layer of the ectocervical squamous epithelium as well as the reserve cell layer of the endocervical columnar epithelium are bipotential, as shown by their coexpression of various cytokeratins. This fact may explain the occasional observation of gland formation from the basal layer of the ectocervical epithelium. It may also explain why the reserve cells of the endocervix may vary greatly in developing preinvasive and invasive carcinomas, being once small-celled solid, largecelled with or without keratinization, clear-celled, or producing mucin or glands 32 . Transitional stages between squamoid reserve cell carcinomas and adeno-squamous carcinomas may also be seen 26 • Consequently, the carcinomatous transformation of the squamous epithelium of the ectocervix does not induce a loss or change in intermediate filaments. Likewise, preinvasive and invasive carcinomas of the reserve cell type also retain the complex pattern of cytokeratins shown by the cells from which they derive. In addition, however, reserve cell carcinomas, like the endocervical adenocarcinomas,
Fig. 3. Reserve cell carcinoma of the endocervix with a distinct expression of cytokeratin 8 in poorly differentiated reserve cells as well as in a beginning gland formation (on the left) (x 350).
epithelium of the ectocervix, but also, and much more frequently, the reserve cells of the endocervix. Discussion We noted previously that the various types of invasive carcinomas of the cervix showed differences in their expression of cytokeratins 9,22. Whereas some of the large-cell squamous carcinomas produce the same cytokeratins as the squamous epithelium of the ectocervix (cytokeratins 5,6, 13, 14, 17 and 19) others, and most of the small-cell carcinomas (poorly differentiated forms) express as well cytokeratins 7,8 and 18. These differences appeared to be an exception to the rule for the female genital tract that malignant tumors maintain their expression of specific intermediate filaments regardless of the degree of differentiation. Anticytokeratin 8 (CAM 5.2) was even thought to be a useful marker for indicating malignant transformation in cervical cancer 25 . If one assumes, however, that the small-cell carcinoma of the cervix arises from the reserve cells of the endocervical
Fig. 4. Undifferentiated small cell carcinoma of the endocervix. Positive reaction with anticytokeratin 8 (x 350).
Immunohistochemistry on Uterine Tumors· 41
acquire a coexpression for CEA that indicates malignant transformation. The subdivision of invasive carcinomas of the ecto-and endocervix into squamous cell and reserve cell types", made by means of their structural differences, is accordingly substantiated by immunohistochemical studies, but at the same time re-evaluated: not all invasive carcinomas derived from the reserve cells are of the small cell type as
Fig. 5. Carcinoma in situ of reserve cell type, developing from koilocytic dysplasia of the endocervical mucosa. - a: Hematoxylin-eosin stain (x 140). - b: in-situ-hybridization with DNA probe 16 (x 140).
previously supposed on behalf of cytological criteria visible in routine histological sections. The immunohistochemical detection of intermediate filaments has shown that a high proportion of large cell or clear cell carcinomas also arises from reserve cells. Accordingly, and as suggested by previous reviews 5,6 approximately 80% of invasive squamous carcinomas of the cervix are derived from reserve cells, and only about 20% originate from
42 . G. DaUenbach-Hellweg and G. Lang
carcinomas with coexistence of a neuroendocrine and an transformed ectocervical epithelium. If one re-evaluates the statistical results according to the immunohistochemepidermoid portion may also occur 16 . ical findings, the average age of patients with a reserve cell The clear-cell carcinomas as well most likely arise from type squamous carcinoma is 40 years in contrast to that of reserve cells, which presumably through faulty differentiation remain at an intermediate stage of development women with an ectocervical type (56 years). Furthermore, the great majority of carcinomas derived from the reserve between incomplete keratinization and the secretion of cells are apparently associated with HPV-infection of types mucus. This assumption is supported by the fact that 16 or 18, whereas most ectocervical type carcinomas are clear-cell carcinomas irregularly express cytokeratins and CEA in both mucus-secreting and squamous epithelia. HPV-negative. From these studies the customary subdivision of squaOur results allow the following conclusions: The large cell squamous epithelial carcinomas may originate from mous carcinomas of the cervix into the keratinizing and the ectocervical mucosa, genetically determined to pro- . non-keratinizing types seems outmoded and should be replaced by a subdivision into the squamous epithelial and duce a squamous epithelium, or from reserve cells of the reserve cell types. It must be admitted, however, that mixed endocervical mucosa, genetically determined to produce squamous as well as mucinous and glandular epithelium. forms of these two types of carcinomas may develop, On the other hand, small-cell carcinomas of the cervix which then show positive immunohistochemical staining for the respective intermediate filaments 9,15,22. One must usually arise from either undifferentiated bipotential assume that the stem cells of both epithelia undergo a reserve cells or those genetically determined to form carcinomatous change at the same time, or that they alter glands, rarely from the basal cells of the ectocervix. Both their direction of differentiation during the course of types of carcinoma may keratinize. Furthermore, the small-cell type derived from reserve cells can produce carcinogenesis. To clarify that assumption precisely, larger series of cases must be studied. monocellular mucus or form glands. The histogenetic distinction between the two types of cervical carcinomas is Since, according to most authors, apparently none of the conventional histologic parameters such as degree of not always possible on routine histological sections, but keratinization, nuclear pleomorphism, number of mitoses, may require immunohistochemical detection of intermecell size, inflammatory or desmoplastic stroma reaction diate filaments. can reliably predict patient survivaI 8,23, the differentiation Differences between these two cell types and their of these two types of carcinomas by identifying their carcinomas exist in their response to hormonal stimulation and in their mode of invasion and spread: Estrogens have histogenetic origins may become of special clinical importance. been shown to stimulate proliferation of the squamous epithelium3,18 and may promote its carcinogenesis 13 . In The distinction is equally important in the preinvasive contrast, gestagens stimulate the reserve cells 4 and may stage of severe dysplasia or carcinoma in situ: up to 90% of promote their carcinogenesis 10, 11. The squamous cell type precancerous lesions positive for cytokeratin 8 and 18 27 shows early net-like stromal infiltration with early invaand derived from reserve cells are associated with HPVsion of lymphatic channels and consequent early extrauinfection, roughly 50% contain genomically integrated terine spread. In the reserve cell type, in contrast, a plump HPV type 16, 20% type 18, and the remaining 20% infiltration predominates which remains confined to the various other types 17 . Types 16 and 18 are known for their uterine cervix until the carcinomatous growth has largely rapid progression to invasive carcinoma, whereas the majority of preinvasive lesions derived from the ectocervix replaced the cervical wall. Apparent discrepancies in cytokeratin expression of are either HPV-negative or positive for HPV 6 or 11, slowly progressive or even reversible. Hence, if southern invasive ectocervical carcinomas reported in previous publications2,33 may possibly be explained by failing to blot or in-situ-hybridization cannot be performed, the subclassify those carcinomas into squamous cell and immunohistochemical detection of the cell or origin may reserve cell types. The variations in the percentage of be helpful in judging prognosis and deciding the clinical management. cytokeratin 8-positive and negative carcinomas found by these investigators (from 28%, Singapore25 to 70% 1 and 80%, London 2 ) may well be because of geographic or racial differences. Perhaps differences in laboratory techReferences niques are also involved. Variations in the coexpression of cytokeratin 8 in metaplasias and precancerous conditions 1 Angus B, Kiberu S, Purvis J, Wilkinson L, Horne CHW arising from the squamous epithelium and the reserve cells (1988) Cytokeratins in cervical dysplasia and neoplasia: A of the ecto- and endocervix have previously been comparative study of immunohistochemical staining using monoobserved 24 . Moreover, some of the poorly differentiated clonal antibodies NCL-5D3, CAM 5.2, and PKK 1. JPathol155: small-cell carcinomas (" carcinoids", "neuroendocrine 71-75 2 Bobrow LG, Makin CA, Law S, Bodmer WF (1986) Exprestumors"), unless specific neuroendocrine granules could sion of low molecular weight cytokeratin proteins in cervical be demonstrated, may have originated in the reserve cells neoplasia. J Path 148: 135-140 of the endocervix, for in their series of cases Ulich et al. 30 3 Boquoi E, Staisch KJ (1968) Vaginalzytologische Untersufound that these tumors gave a positive reaction for chungen zur Dosis-Wirkungsbeziehung konjugierrer Oestrogene cytokeratin 8 and CEA. On the other hand, a dual or even (Presomen) bei Frauen in der Postmenopause. Geburrsh u Frauentriple differentiation of reserve cell-derived small-cell heilk 28: 1132-1142
Immunohistochemistry on Uterine Tumors· 43 4 Candy, Abell MR (1968) Progestogen-induced adenomatous hyperplasia of the uterine cervix. ]AMA 2023: 323-326 5 Christopherson WM, Nealon N, Gray LA (1979) Noninvasive precursor lesions of adenocarcinoma and mixed adenosquamous carcinoma of the cervix uteri. Cancer 44: 975-983 6 Clement PB, Scully RE (1982) Carcinoma of the cervix: Histologic types. Sem Oncol 9: 251-264 7 Cordell]L, Falini B, Erber W et al. (1984) Immunoenzymatic labelling of monoclonal antibodies using immune complexes of alkaline phosphates and monoclonal anti-alkaline phosphatase (APAAP). ] Histochem Cytochem 32: 2198 Crissman ]D, Budhraja M, Aron BS, Cummings G (1987) Histopathologic prognostic factors in stage II and III squamous cell carcinoma of the uterine cervix. Int ] Gynecol Pathol 6: 97-103 9 Czernobilsky B, Moll R, Franke WW, Dallenbach-Hellweg G, Hohlweg-Majert P (1984) Intermediate filaments of normal and neoplastic tissues of the female genital tract with emphasis on problems of differential tumor diagnosis. Path Res Pract 179: 31-37 10 Dallenbach-Hellweg G (1972) Therapieschaden in der Gynakologie. Morphologische Beobachtungen uber die Auswirkungen weiblicher Sexualhormone und von Stoffen mit ahnlicher Wirkung (Referat). Verh Dtsch Ges Path 56: 252-283 11 Dallenbach-Hellweg G (1981) Strukturformen der Carcinogenese im Portio-Cervix-Bereich unter dem Einflu~ exogener Hormone. Pathologe 3: 13-24 12 Dallenbach-Hellweg G, Lang G (1990) Immunohistochemical studies on uterine tumors: II. Invasive adeno-carcinomas of the endocervix and their precursors (in prep) 13 Dunn TB, Green AW (1963) Cysts of the epididymis, cancer of the cervix, granular cell myoblastoma, and other lesions after estrogen injection in newborn mice. ] Nat Cancer Inst 31: 425-455 14 Elliott PM, Tattersall MHN, Coppleson M, Russell P, Wong F, Coates AS, Solomon H], Bannatyne PM, Atkinson KH, Murray ]C (1989) Changing character of cervical cancer in young women. Brit Med] 298: 288-290 15 Franke WW, Moll R, Achtstaetter T, Kuhn C (1986) Cell typing of epithelia and carcinomas of the female genital tract using cytoskeletal proteins as markers. Bambury Report 21: Viral Etiology Cervical Cancer: 121-147 16 Gersell D], Mazoujian G, Mutch DG, Rudloff MA (1988) Small-cell undifferentiated carcinoma of the cervix. A clinicopathologic, ultrastructural, and immunocytochemical study of 15 cases. Am] Surg Path 12: 684-698 17 Gissmann L (1984) Papillomaviruses and their association with cancer in animals and in man. Cancer Surv 3: 162-181 18 Hammond D (1967) Potency of quinestrol as reflected by vaginal cytologic maturation. Intern] Fertil12: 164-169 19 Makin CA, Bobrow lG, Bodmer WF (1984) Monoclonal antibodies to cytokeratin for use in routine histopathology.] Clin Pathol 37: 975-983 20 Moll R, Achtstatter T, Becht E, Balcarova-Stander ], Ittensohn M, Franke WW (1988) Cytokeratins in normal and
malignant transitional epithelium: Maintenance of expression of urothelial differentiation features in transitional cell carcinomas and bladder carcinoma cell lines. Am] Pathol132: 123-144 21 Moll R, Franke WW, Schiller Dl, Geiger B, Krepler R (1982) The catalog' of human cytokeratin polypeptides: patterns of expression of specific cytokeratins in normal epithelia, tumors and cultured cells. Cell 31: 11-24 22 Moll R, Levy R, Czernobilsky B, Hohlweg-Majert P, Dallenbach-Hellweg G, Franke WW (1983) Cytokeratins of normal epithelia and some neoplasms of the female genital tract. Lab Invest 49: 559-609 21 Narbaix E, Dewandeleer S, Habba Cl, liegeois Ph, Willems T, Rahier ], Donnez] (1989) Nucleolar organizer regions in the normal and carcinomatous epithelium of the uterine cervix: A morphometric study. Int] Gynecol Pathol 8: 237-245 24 Perts ]]G, Moesker 0, Kenemans P, Vooijs GP, Ramackers FCS (1985) Expression of cytokeratins in early neoplastic lesions of the uterine cervix. Int] Gynec Pathol 4: 300-313 25 Raju GC (1988) Expression of the cytokeratin marker CAM 5.2 in cervical neoplasia. Histopathology 12: 437-443 26 Rollason TP, Cullimore], Bradgate MG (1989) A suggested columnar cell morphological equivalent of squamous carcinoma in situ with early stromal invasion. Int ] Gynecol Pathol 8: 230-236 27 Syrjanen S, Cintorino M, Armellini D, Del Vecchio MT, leoncini P, Bugnoli M, Pallini V, Silvestri S, Tosi P, Mantyjarvi R, Syrjanen K (1988) Expression of cytokeratin polypeptides in human papillomavirus (HPV) lesions of the uterine cervix: 1. Relationship to grade of CIN and HPV-type. Int] Gynecol Pathol 7: 23-38 28 Tase T, Okagaki T, Clark BA, Manias DA, Ostrow RS, Twiggs lB, Faras A] (1988) Human papillomavirus types and localization in adenocarcinoma and adenosquamous carcinoma of the uterine cervix: A study by in situ DNA hybridization. Cancer Research 48: 993-998 29 Tsutsumi Y, Nagura H, Watanabe K (1984) Immunohistochemical observations of carcinoembryonic antigen (CEA) and CEA-related substances in normal and neoplastic pancreas. Am] Clin Pathol 82: 535-542 10 Ulich TR, Liao SY, Layfield l, Romansky S, Cheng l, Lewin K] (1986) Endocrine and tumor differentiation markers in poorly differentiated small-cell carcinoids of the cervix and vagina. Arch Pathol Lab Med 110: 1054-1057 11 Viae], Reano A, Brochier], Staquet M], Thivolet] (1983) Reactivity pattern of a monoclonal antikeratin antibody (Kl 1).] Invest Dermatol 81: 351-354 12 Weikel W, Wagner R, Moll R (1987) Characterization of subcolumnar reserve cells and other epithelia of human uterine cervix. Virchows Arch (B) 54: 98-110 .B Wells M, Brown l]R, Jackson P (1986) Letter; Low molecular weight cytokeratin protein in cervical neoplasia.] Path 150: 69
Received August 14, 1989 . Accepted in revised form April 6, 1990
Key words: Uterine carcinomas - Cervix carcinoma - Reserve cell carcinoma - Clear cell carcinoma - Preinvasive lesions Prof. Dr. med. G. Dallenbach-Hellweg, Institut fur Pathologie, A 2, 2, 6800 Mannheim 1, West Germany
Immunohistochemical Studies on Uterine Tumors I. Invasive Squamous Cell Carcinomas of the Cervix and Their Precursors G. Dallenbach-Hellweg and G. Lang Institut fur Pathologie, Mannheim, FRG
SUMMARY 40 invasive carcinomas and 80 preinvasive lesions of the uterine cervix were studied immunohistochemically; 40 benign lesions served as controls. On histological and immunohistochemical examination, invasive and preinvasive carcinomas were subdivided in the squamous (large cell, ectocervical) type and the reserve cell (small, large or clear cell, endocervical) type. Immunohistochemically, 100% of the invasive and preinvasive squamous cell carcinomas were positive with anticytokeratins 13, 14, 16 and negative with anticytokeratin 8 and anti-CEA. Most of the invasive and preinvasive reserve cell carcinomas showed a coexpression ofcytokeratins 13,14,16,8 and CEA. The subdivision ofinvasive carcinomas ofthe ecto- and endocervix into squamous cell and reserve cell types made by means of their structural differences is substantiated and re-evaluated by their immunohistochemical reactions. Both types of carcinomas retain the complex pattern of cytokeratins shown by their cells oforigin. The reserve cell carcinomas, in addition, acquire a coexpression for CEA that indicates malignant transformation. The subdivision is of clinical importance because both types of carcinomas vary in their mode and speed of invasion and spread and in their association with HPV infection. Introduction Cell typing with molecular markers has become increasingly important for the classification of tumors and for our understanding of their histogenetic development. A number of molecular markers have been described that are specific for certain cell types or lines of differentiation. As components of the cytoskeleton, besides microfilaments and microtubules, the intermediate filaments constitute a large proportion of the total cytoplasmic material. Among these, the cytokeratins are a complex family of at least 20 polypeptide chains that vary in their pH values and in their molecular weights. The types of polypeptide chains found in the cells of a tissue are specific for that tissue 21 • Regarding the carcinomas of the uterus, the following questions seemed to be important: 1. What expression or coexpression of intermediate filaments can help recognize the directions of differentia0344-0338/91/0187-0036$3.5 0/0
tion that an epithelial cell might take, and at which stage of differentiation is such a recognition possible? 2. Is the composition of the cytoskeleton of a cell undergoing carcinogenesis maintained or does it change? 3. Can uterine carcinomas that appear alike histologically but arise from different epithelia be immunohistochemically differentiated from one another from their histogenesis? Since prediction of patient survival in squamous cell carcinomas of the uterine cervix often proves difficult on histological examination 8 , valuable additional information could possibly be gained by evaluating the intermediate filament composition of the tumor cells. Furthermore, such studies may help to explain why cervical cancer has changed in clinical and morphological behaviour, and why its incidence has proportionally increased in young women 14 • © 1991 by Gustav Fischet Verlag, Stuttgatt
Immunohistochemistry on Uterine Tumors· 37 Material and Methods
The immunohistochemical observations are summarized in Table 1. The normal, papillomatous and dysplastic squamous epithelium of the ectocervix as well as the carcinoma in situ and the invasive large cell squamous carcinoma arising from it react positively with anticytokeratins 13/14/16, but negatively with anticytokeratin 8, antivimentin and anti-CEA (Fig. 1). The metaplastic squamous epithelium of the endocervix reacts similarly. Only the basal layer of the squamous ectocervical epithelium focally shows a coexpression of cytokeratin 8 in addition to 13/14/16. The reserve cell hyperplasia of the endocervical mucosa is in roughly 90% of the cases examined positively for cytokeratin 13/14/16 and for cytokeratin 8. The carcinoma in situ of the reserve cell type that originates from this hyperplasia gives the same reactions and, in addition, is positive for CEA. The invasive squamous cell carcinoma of the reserve cell type is, independent of its grade of differentiation, strongly positive for cytokeratin 8 (Figs. 2-4) and weakly positive or even negative with anticytokeratin 13/14/16. As in the preinvasive reserve cell carcinoma, and contrary to the squamous cell type of the ectocervix, the invasive reserve cell carcinoma shows a distinct coexpression of CEA (Fig. 2). - A very similar expression of intermediate filaments is observed in the clear cell carcinomas of the endocervix. 20 of the invasive carcinomas and 40 of the preinvasive lesions were tested for their content of genomic HPV by in-situ-hybridization: of 6 squamous type carcinomas all 6 were negative, of 14 reserve cell carcinomas 6 were negative, 7 were positive for HPV 16, one for HPV 18. Of the 35 carcinomas in situ of reserve cell type 24 were positive for HPV 16 (Fig. 5), 11 were negative, 3 carcinomas in situ of squamous type were negative, 2 koilocytic dysplasias were positive for HPV 6 and 11. The koilocytic dysplasias and carcinomas in situ presumably induced by human papilloma viruses (HPV) express patterns of cytoskeletal filaments that correspond to those shown by their tissue of origin. In a corresponding study (in preparation), we could not detect a transformation of intermediate filaments following HPV -infection27 : the expression of cytokeratins is the same as in the equivalent nonkoilocytic preinvasive lesions. As has been previously reported 28 , the HP virus can infect not only the squamous
160 specimens of uterine cervices obtained by hysterectomy or conization were examined histologically and immunohsitochemically. Formalin-fixed paraffin slides were routinely stained with Hematoxylin and Eosin (H + E) and Hematoxylin-Picric-acidAcid-fuchsin (van Gieson). Immunohistochemical studies were performed on freshly frozen cryostat sections and on formalinfixed paraffin slides with the APAAP immuno-alkaline-phosphotase technique? after applying various monoclonal antibodies: the broad-spectrum cytokeratin antibody KL 1 (Dianova, Hamburg, Germany3!); Ks 13.1 against cytokeratin 13 and to a minor degree cytokeratin 14 and 16 (Progen, Heidelberg, Germany20)j CAM 5.2 against cytokeratin 8 (Becton Dickinson, Heidelberg, Germany!9); V 9 against Vimentin (Dakopatts, Hamburg, Germany); CM 010 against CEA, purified from non-specific crossreacting antigen (NCA) (Ortho Diagnostics, Neckargemiind, Germany29). For in situ-hybridization we applied DNA probes for HPV 6/11, 16/18 and 18 (Ortho Diagnostics, Neckargemiind, Germany) to paraffin slides. Results Of the 160 cervical specimens studied by routine histological methods, 40 were invasive carcinomas, 80 were preinvasive lesions (10 dysplasias, 70 carcinomas in situ), and 40 benign lesions (metaplasias or regenerative epithelium) served as controls. On histological and immunohistochemical examination, the preinvasive and invasive carcinomas, according to their location and cellular appearence, were subdivided into a) the squamous (large cell) type with or without keratinization, located at or near the squamo-columnar junction (20 cases: 10 invasive carcinomas and 10 in situ lesions), and b) the reserve cell (small or large cell) type located within the endocervical mucosa (95 cases: 25 invasive carcinomas and 70 in situ lesions). c) the clear cell type of endocervical carcinoma (5 cases). 21 invasive adenocarcinomas and adeno-squamous carcinomas of the endocervix are described in a separate paper 12 .
Table 1. Immunohistochemical results on invasive and in situ carcinomas of the uterine cervix Diagnosis
invasive Carcinoma squamous cell type reserve cell type clear cell type in situ lesions (CIN) squamous cell type reserve cell type
average age
Number of cases
Cytokeratin 13.14.16
Cytokeratin 8
56 40 41
10 25 5
10 23 3
2 2
24 5
35 35
10 70
10 67
3
66
+
+
10
CEA
Vimentin
+
+
1
25 3
10 4
70
10 2 10
10 25 5 10 70
Fig. 1. Invasive carcinoma of the ectocervix, squamous cell type.
55-year-old patient. - a: Immunohistochemical reaction with anticytokeratin 13: virtually all tumor cells stain positively. - b: Negative reaction with anticytokeratin 8. - c: Negative reaction with anti-CEA (x 140).
Fig. 2. Invasive carcinoma of the endocervix, reserve cell type. 38-year-old patient. Most of the tumor cells show a coexpression of cytokeratin 13 (a), 8 (b) and CEA (c). Some basally located undifferentiated cells express cytokeratin 8 only (b) and are negative with anticytokeratin 13 (a). (a + b: x 350, c: x 140).
40 . G. Dallenbach-Hellweg and G. Lang
glands, then the cytokeratin pattern this type of carcinoma shows is maintained from benign reserve cell origin to its endstage carcinoma. The basal cell layer of the ectocervical squamous epithelium as well as the reserve cell layer of the endocervical columnar epithelium are bipotential, as shown by their coexpression of various cytokeratins. This fact may explain the occasional observation of gland formation from the basal layer of the ectocervical epithelium. It may also explain why the reserve cells of the endocervix may vary greatly in developing preinvasive and invasive carcinomas, being once small-celled solid, largecelled with or without keratinization, clear-celled, or producing mucin or glands 32 . Transitional stages between squamoid reserve cell carcinomas and adeno-squamous carcinomas may also be seen 26 • Consequently, the carcinomatous transformation of the squamous epithelium of the ectocervix does not induce a loss or change in intermediate filaments. Likewise, preinvasive and invasive carcinomas of the reserve cell type also retain the complex pattern of cytokeratins shown by the cells from which they derive. In addition, however, reserve cell carcinomas, like the endocervical adenocarcinomas,
Fig. 3. Reserve cell carcinoma of the endocervix with a distinct expression of cytokeratin 8 in poorly differentiated reserve cells as well as in a beginning gland formation (on the left) (x 350).
epithelium of the ectocervix, but also, and much more frequently, the reserve cells of the endocervix. Discussion We noted previously that the various types of invasive carcinomas of the cervix showed differences in their expression of cytokeratins 9,22. Whereas some of the large-cell squamous carcinomas produce the same cytokeratins as the squamous epithelium of the ectocervix (cytokeratins 5,6, 13, 14, 17 and 19) others, and most of the small-cell carcinomas (poorly differentiated forms) express as well cytokeratins 7,8 and 18. These differences appeared to be an exception to the rule for the female genital tract that malignant tumors maintain their expression of specific intermediate filaments regardless of the degree of differentiation. Anticytokeratin 8 (CAM 5.2) was even thought to be a useful marker for indicating malignant transformation in cervical cancer 25 . If one assumes, however, that the small-cell carcinoma of the cervix arises from the reserve cells of the endocervical
Fig. 4. Undifferentiated small cell carcinoma of the endocervix. Positive reaction with anticytokeratin 8 (x 350).
Immunohistochemistry on Uterine Tumors· 41
acquire a coexpression for CEA that indicates malignant transformation. The subdivision of invasive carcinomas of the ecto-and endocervix into squamous cell and reserve cell types", made by means of their structural differences, is accordingly substantiated by immunohistochemical studies, but at the same time re-evaluated: not all invasive carcinomas derived from the reserve cells are of the small cell type as
Fig. 5. Carcinoma in situ of reserve cell type, developing from koilocytic dysplasia of the endocervical mucosa. - a: Hematoxylin-eosin stain (x 140). - b: in-situ-hybridization with DNA probe 16 (x 140).
previously supposed on behalf of cytological criteria visible in routine histological sections. The immunohistochemical detection of intermediate filaments has shown that a high proportion of large cell or clear cell carcinomas also arises from reserve cells. Accordingly, and as suggested by previous reviews 5,6 approximately 80% of invasive squamous carcinomas of the cervix are derived from reserve cells, and only about 20% originate from
42 . G. DaUenbach-Hellweg and G. Lang
carcinomas with coexistence of a neuroendocrine and an transformed ectocervical epithelium. If one re-evaluates the statistical results according to the immunohistochemepidermoid portion may also occur 16 . ical findings, the average age of patients with a reserve cell The clear-cell carcinomas as well most likely arise from type squamous carcinoma is 40 years in contrast to that of reserve cells, which presumably through faulty differentiation remain at an intermediate stage of development women with an ectocervical type (56 years). Furthermore, the great majority of carcinomas derived from the reserve between incomplete keratinization and the secretion of cells are apparently associated with HPV-infection of types mucus. This assumption is supported by the fact that 16 or 18, whereas most ectocervical type carcinomas are clear-cell carcinomas irregularly express cytokeratins and CEA in both mucus-secreting and squamous epithelia. HPV-negative. From these studies the customary subdivision of squaOur results allow the following conclusions: The large cell squamous epithelial carcinomas may originate from mous carcinomas of the cervix into the keratinizing and the ectocervical mucosa, genetically determined to pro- . non-keratinizing types seems outmoded and should be replaced by a subdivision into the squamous epithelial and duce a squamous epithelium, or from reserve cells of the reserve cell types. It must be admitted, however, that mixed endocervical mucosa, genetically determined to produce squamous as well as mucinous and glandular epithelium. forms of these two types of carcinomas may develop, On the other hand, small-cell carcinomas of the cervix which then show positive immunohistochemical staining for the respective intermediate filaments 9,15,22. One must usually arise from either undifferentiated bipotential assume that the stem cells of both epithelia undergo a reserve cells or those genetically determined to form carcinomatous change at the same time, or that they alter glands, rarely from the basal cells of the ectocervix. Both their direction of differentiation during the course of types of carcinoma may keratinize. Furthermore, the small-cell type derived from reserve cells can produce carcinogenesis. To clarify that assumption precisely, larger series of cases must be studied. monocellular mucus or form glands. The histogenetic distinction between the two types of cervical carcinomas is Since, according to most authors, apparently none of the conventional histologic parameters such as degree of not always possible on routine histological sections, but keratinization, nuclear pleomorphism, number of mitoses, may require immunohistochemical detection of intermecell size, inflammatory or desmoplastic stroma reaction diate filaments. can reliably predict patient survivaI 8,23, the differentiation Differences between these two cell types and their of these two types of carcinomas by identifying their carcinomas exist in their response to hormonal stimulation and in their mode of invasion and spread: Estrogens have histogenetic origins may become of special clinical importance. been shown to stimulate proliferation of the squamous epithelium3,18 and may promote its carcinogenesis 13 . In The distinction is equally important in the preinvasive contrast, gestagens stimulate the reserve cells 4 and may stage of severe dysplasia or carcinoma in situ: up to 90% of promote their carcinogenesis 10, 11. The squamous cell type precancerous lesions positive for cytokeratin 8 and 18 27 shows early net-like stromal infiltration with early invaand derived from reserve cells are associated with HPVsion of lymphatic channels and consequent early extrauinfection, roughly 50% contain genomically integrated terine spread. In the reserve cell type, in contrast, a plump HPV type 16, 20% type 18, and the remaining 20% infiltration predominates which remains confined to the various other types 17 . Types 16 and 18 are known for their uterine cervix until the carcinomatous growth has largely rapid progression to invasive carcinoma, whereas the majority of preinvasive lesions derived from the ectocervix replaced the cervical wall. Apparent discrepancies in cytokeratin expression of are either HPV-negative or positive for HPV 6 or 11, slowly progressive or even reversible. Hence, if southern invasive ectocervical carcinomas reported in previous publications2,33 may possibly be explained by failing to blot or in-situ-hybridization cannot be performed, the subclassify those carcinomas into squamous cell and immunohistochemical detection of the cell or origin may reserve cell types. The variations in the percentage of be helpful in judging prognosis and deciding the clinical management. cytokeratin 8-positive and negative carcinomas found by these investigators (from 28%, Singapore25 to 70% 1 and 80%, London 2 ) may well be because of geographic or racial differences. Perhaps differences in laboratory techReferences niques are also involved. Variations in the coexpression of cytokeratin 8 in metaplasias and precancerous conditions 1 Angus B, Kiberu S, Purvis J, Wilkinson L, Horne CHW arising from the squamous epithelium and the reserve cells (1988) Cytokeratins in cervical dysplasia and neoplasia: A of the ecto- and endocervix have previously been comparative study of immunohistochemical staining using monoobserved 24 . Moreover, some of the poorly differentiated clonal antibodies NCL-5D3, CAM 5.2, and PKK 1. JPathol155: small-cell carcinomas (" carcinoids", "neuroendocrine 71-75 2 Bobrow LG, Makin CA, Law S, Bodmer WF (1986) Exprestumors"), unless specific neuroendocrine granules could sion of low molecular weight cytokeratin proteins in cervical be demonstrated, may have originated in the reserve cells neoplasia. J Path 148: 135-140 of the endocervix, for in their series of cases Ulich et al. 30 3 Boquoi E, Staisch KJ (1968) Vaginalzytologische Untersufound that these tumors gave a positive reaction for chungen zur Dosis-Wirkungsbeziehung konjugierrer Oestrogene cytokeratin 8 and CEA. On the other hand, a dual or even (Presomen) bei Frauen in der Postmenopause. Geburrsh u Frauentriple differentiation of reserve cell-derived small-cell heilk 28: 1132-1142
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Received August 14, 1989 . Accepted in revised form April 6, 1990
Key words: Uterine carcinomas - Cervix carcinoma - Reserve cell carcinoma - Clear cell carcinoma - Preinvasive lesions Prof. Dr. med. G. Dallenbach-Hellweg, Institut fur Pathologie, A 2, 2, 6800 Mannheim 1, West Germany
