Expression of epidermal growth factor receptor and HER-2/neu in normal and neoplastic cervix, vulva, and vagina.

Monoclonal antibodies were used to localize immunohistochemically epidermal growth factor receptor and HER-2/neu in normal and neoplastic frozen tissue samples from the lower genital tract of women. In squamous epithelia of the cervix, vulva, and vagina, epidermal growth factor receptor and HER-2/neu both were expressed most strongly by basal keratinocytes. Expression of both of these cell surface molecules decreased as cells underwent differentiation toward the mucosal surface. In contrast, both epidermal growth factor receptor and HER-2/neu were expressed throughout the entire thickness of the epithelium by undifferentiated squamous cells in squamous metaplasia, raised condyloma, and carcinoma in situ. In 34 squamous cancers of the cervix, vulva, and vagina, all malignant cells were found to have moderate to heavy staining for epidermal growth factor receptor. Staining of 33 of these cancers for HER-2/neu was light, although one patient who presented with distant metastases had heavy staining for HER-2/neu. These data suggest that although overexpression of HER-2/neu in squamous cancers of the lower genital tract is a rare event, it may be associated with aggressive biologic behavior.

Tumor markers in gynecologic neoplasms.

The applications of tumor markers and steroid receptors in gynecologic neoplasms are described. The value of immunohistologic techniques in the histogenetic assessment of gynecologic neoplasms is examined. Approaches to the diagnosis of similar-appearing lesions are presented.

Vulvovaginal polyps. Histologic appearance, ultrastructure, immunocytochemical characteristics, and clinicopathologic correlations.

A clinicopathologic study of 18 vulvovaginal fibroepithelial polyps with a comparison to normal stroma is presented. Twelve cases were analyzed by immunohistochemical methods for the presence of vimentin, desmin, muscle-specific actin, myoglobin, S-100 protein, alpha 1-antitrypsin (AAT), alpha 1-antichymotrypsin (ACHT), cytokeratin (AE1/AE3), and epithelial membrane antigen. Stromal cells reacted with vimentin antiserum in eight cases. Desmin reactivity was detected in five of 12 cases, four of which coexpressed vimentin. Two cases exhibited muscle-specific actin reactivity, and a single case weak AAT reactivity. The stromal cells were unreactive with S-100 protein, epithelial membrane antigen, cytokeratin, ACHT, and myoglobin. Ultrastructurally, the stromal cells of four fibroepithelial polyps resembled fibroblasts and myofibroblasts. Our immunohistochemical and ultrastructural data suggest that the stromal cells of fibroepithelial polyps are a collection of functional fibroblasts that may be capable of differentiating along two or more cell lines. Dramatically elongated cytoplasmic processes extend from both normal vulvovaginal stromal cells and the cells of the polyps. The cell attachments to each other and to bundles of collagen suggest a potential for a physiologic role in tissue contractility, especially in the immediate postpartum state. The common association with pregnancy may represent a local exuberant response to some presently unidentified trophic or stimulating factor.

Análise dos casos de carcinoma de vulva do Hospital de Clínicas de Porto Alegre / Vulvar carcinoma in the Hospital de Clínicas de Porto Alegre: analysis of clinical cases

Apesar de sua raridade, o carcinoma de vulva näo deixa de ter a sua importância devido a sua morbidade. Os autores realizam uma análise retrospectiva de 21 pacientes cadastradas com carcinoma de vulva no Setor de Oncologia Genital do Serviço de Ginecologia e Obstetrícia do Hospital de Clínicas de Porto Alegre. A faixa etária variou de 31 a 83 anos com uma idade média de 60 anos. As lesöes mais encontradas foram as vegetaçöes, a hiperemia, a leucoplasia e o tumor ulcerado. Um terço das pacientes apresentou carcinoma intraepitelial e dois terços, algum tipo de invasäo. O prurido vulvar foi encontrado em 66% das pacientes

Malignant rhabdoid tumor of the vulva: is distinction from epithelioid sarcoma possible? A pathologic and immunohistochemical study.

Epithelioid sarcoma (ES) and malignant rhabdoid tumor (MRT) have heretofore been regarded as two separate clinicopathologic entities. However, they have some histologic similarities, and both represent histogenetic and phenotypic enigmas. This study reports the pathologic and immunohistochemical findings of four vulvar neoplasms occurring in young women that represented diagnostic dilemmas because of their similarity to both ES and MRT. Only one case had the classic histologic features of ES, whereas, in our opinion, the other three cases fulfilled the histologic criteria of MRT, despite the fact that two of the three cases were reported earlier as examples of ES. Neither electron microscopy nor immunohistochemistry has been found to be helpful in separating ES from MRT, mainly because they share several ultrastructural and immunophenotypic features. The behavior of these vulvar tumors--ours and the few published examples of ES--is generally aggressive, more in keeping with MRT than classic ES. We believe that some, if not most, putative ES of the vulva are in fact MRT, a neoplasm with an unfavorable prognosis.

Mucin-negative biopsy in extra-mammary Paget's disease. A diagnostic problem.

A case of extra-mammary Paget's disease with a mucin-negative small biopsy is reported. Study of a small series of vulvar Paget's disease demonstrated that areas devoid of mucin in the mm range are frequently found, creating the conditions for diagnostic problems in small biopsies. In this situation, positive immunoreactions for carcinoembryonic antigen and low molecular weight cytokeratins and a negative reaction for S-100 protein serves to differentiate Paget's disease from other pagetoid lesions.

New monoclonal antibodies recognizing epidermal differentiation-associated keratins in formalin-fixed, paraffin-embedded tissue. Keratin 10 expression in carcinoma of the vulva.

Two monoclonal antibodies (MAb) specific for differentiation-related epidermal keratins have been developed. They represent specific molecular probes for different stages of epidermal differentiation. Antibody DE-K10 is chain-specific for cytokeratin polypeptide no. 10 (56.5 kD) expressed in all suprabasal layers of the epidermis. Antibody DE-SCK is specific for modified stratum corneum keratins and thus represents a marker for the terminal step of epidermal differentiation. Since the epitopes identified by both antibodies are preserved in formalin-fixed, paraffin-embedded tissue sections, these antibodies can be used for retrospective studies of differentiation in various pathological processes. We have used antibody DE-K10 to study the cytokeratin 10 expression in 26 stage II or III vulvar squamous cell carcinomas. Preliminary data suggest an increased risk of recurrence in cytokeratin 10 negative tumours.

[Three cases of vulvar bowenoid papulosis: the localization of HPV DNA by in situ hybridization].

Cytological, histological, and molecular biological studies were conducted in 3 cases of vulvar Bowenoid papulosis, using biotinylated HPV DNA probes by in situ hybridization. 1) Cytological findings showed dyskaryotic cells that revealed hyperchromatism with a coarse granular pattern, and a high N/C ratio was observed among the dyskeratotic cells. 2) In 2 cases of Bowenoid papulosis lesions, HPV 16 DNA was detected in the nucleus of the dysplastic cells. 3) In one case of Bowenoid papulosis, a complicated carcinoma in situ of the uterine cervix was observed, and the HPV 16 DNA was found to be positive in both the vulva and cervix.

Histogenesis of extramammary and mammary Paget cells. An immunohistochemical study.

To define the histogenesis of the Paget cell and possibly identify differences in cells from the two sites, six vulvar and 23 mammary specimens from Paget's disease lesions were studied for immunocytochemical antigens. All vulvar and 21 (91%) mammary lesions were strongly reactive for glandular cytokeratin. All lesions showed immunopositive Paget cells with epithelial membrane antigen (EMA). An apocrine antigen, gross cystic disease fluid protein (GCDFP-15), decorated 66.5% and 56.5% of extramammary and mammary lesions, respectively. All vulvar Paget cells stained for carcinoembryonic antigen (CEA), a frequency significantly greater than the 35% in mammary lesions (p = 0.02). However, CEA is expressed by both eccrine and apocrine sweat glands and their tumors. Vulvar Paget cells were negative for lysozyme, casein, lactalbumin, and S100 protein, compared with 9%, 4%, 4%, and 26% in nipple lesions. S100 protein expression is similar to that in mammary ductal carcinoma (32%). The glandular origin of both extramammary and mammary Paget cells is indicated by the presence of glandular cytokeratin, EMA, and CEA. Approximately 60% of all cases in both sites showed evidence of apocrine derivation (GCDFP-15 positivity). Variable antigen expression suggests possible malignant transformation of pluripotent germinative cells able to differentiate in an apocrine or an eccrine direction, or in both.

Buffered formalin is the superior fixative for the detection of HPV DNA by in situ hybridization analysis.

In situ hybridization is used commonly for detection of human papillomavirus (HPV) DNA. There is little information, however, on whether the detection of HPV DNA by in situ hybridization can be affected by the way in which the tissue is fixed. To address this question, the authors compared the hybridization signal using this technique under low stringency conditions for several genital condylomata containing HPV 6 or 11 that were randomly subdivided and fixed in various fixatives for 16 hours. In all cases, the largest proportion of cells with koilocytotic atypia that had detectable HPV DNA was in buffered formalin-fixed tissue (80%), followed by tissue fixed in unbuffered formalin (70%), Hartman's solution (40%), and Bouin's solution (10%). After a high stringency wash, the greatest decrease in the overall hybridization signal was with tissue fixed in Bouin's solution; a minimal decrease was noted with tissue fixed in buffered formalin. Fixation in Bouin's solution for 2 hours gave in situ hybridization results comparable with buffered formalin fixation but with poorer cytologic detail. It is concluded that, of the fixatives studied, buffered formalin is superior for the detection of HPV DNA by in situ hybridization analysis.

Verrucous carcinoma of the vulva. A clinicopathologic and immunohistochemical study of five cases.

The clinicopathologic features of five cases of verrucous carcinoma of the vulva and their staining pattern with antikeratin monoclonal antibodies AE1 and AE3 were compared with those of conventional squamous cell carcinoma. Two patients had local recurrences but none died of the tumor. AE1 and AE3 antibodies stained the entire epithelial thickness in both verrucous and squamous cell carcinoma, but in the former the positivity was uniform and homogeneous everywhere, while in squamous cell carcinoma the positivity was extremely disorganized and patchy. The pattern of expression of monoclonal antibodies AE1 and AE3 confirms that verrucous carcinoma is an extremely well-differentiated squamous neoplasm in contrast to squamous cell carcinoma, which is heterogeneous from a viewpoint of differentiation.

An immunoperoxidase study of laminin and type IV collagen distribution in carcinoma of the cervix and vulva.

Basement membrane immunostaining was performed on pepsin-digested, paraffin-embedded blocks of 29 squamous cell carcinomas of the cervix (invasive and in situ) and 13 of the vulva, using polyclonal rabbit antibodies to human laminin and type IV collagen, both staining identically. Laminin with varying defectiveness surrounded invasive foci, whereas adjacent carcinoma in situ or normal epithelium had intact laminin. The amount of laminin usually reflected the degree of tumor differentiation. Absence of laminin around totally keratinized or necrotic tumor nests indicated its dependency on viable cells. New buds from established invasive tumor nests were often more laminin-defective than the parent nest and suggested a cyclic invasive process, with laminin loss during a growth surge followed by laminin reformation during quiescence. In cases of questionable early stromal invasion, deficient laminin could sway the decision toward making a positive diagnosis. The tendency of laminin gaps and tumor buds to contain large malignant cells with pleomorphic nuclei supports the concept of a change in tumor cell metabolism during active invasion. Laminin also appeared around metastatic tumor within lymph nodes. The relationship of inflammation to tumor laminin defectiveness varied.

Human papillomavirus DNA in adenosquamous carcinoma and squamous cell carcinoma of the vulva.

The tissues from 16 cases of adenosquamous carcinoma (pseudoglandular squamous cell carcinoma or adenoacanthoma of the sweat glands of Lever) and 26 cases of invasive squamous cell carcinoma of the vulva were studied for the presence of human papillomavirus (HPV) genomes using Southern blot hybridization on fresh tissues. Types 1, 2, 3, 4, 5, 6, 16, and 18 HPV DNA probes and in situ hybridization were used on formalin-fixed paraffin sections using type 2, 6, 16, and 18 HPV DNA probes. Only one case of adenosquamous carcinoma contained an undetermined type of HPV DNA, whereas five cases of squamous cell carcinoma contained HPV DNA. Three of these five cases contained type 16, one type 6 HPV, and two an undetermined type. These results demonstrate HPV DNA associations with malignancy of the vulva that are similar to those observed elsewhere in the genital tract.

Condylomatous carcinoma of the vulva with special reference to human papillomavirus DNA.

Nine cases of condylomatous carcinoma (squamous cell carcinoma arising in condyloma acuminatum) of the vulva were studied for their clinical history, histopathology, and presence of human papillomavirus (HPV) DNA. Condylomatous carcinoma occurred primarily in an elderly population with a mean age of 70 years. There was an antecedent history of vulvar condyloma in 77%, with a median of nine months before the documentation of an invasive lesion. The disease had a good prognosis, with few recurrences and no metastasis or deaths from the disease. Human papillomavirus DNA was demonstrated to be present in 55% of these tumors by either filter or in situ hybridization techniques. Both HPV 6 and HPV 16 DNA were identified in an equal number of cases.

Immunologic characteristics of keratins in extramammary Paget's disease.

Six cases of extramammary Paget's disease were immunohistochemically investigated with several antikeratin monoclonal antibodies. Paget cells and surrounding epidermal keratinocytes were equally stained with an antikeratin monoclonal antibody, HKN-4, which recognizes a broad spectrum of keratins. However, Paget cells were clearly distinguished from the surrounding epidermal keratinocytes by HKN-2, which does not react with keratins of secretory cells but does react with keratins of ductal and myoepithelial cells of sweat glands and with epidermis and hair tissue of the normal skin. The HKN-2 did not bind to Paget cells, but the surrounding keratinocytes were positive. CK7, LE41, RGE53, and LP2K, which recognize simple epithelium-type keratins 7 (molecular weight [MW], 54,000; type II), 8 (MW, 52,500; type II), 18 (MW, 45,000; type I), and 19 (MW, 40,000; type I), respectively, stained Paget cells but not the surrounding keratinocytes. Two cases of Merkel cell carcinoma, examined as controls, showed positivity to LE41 and RGE53 but not to CK7 and LP2K. Since in the normal skin the secretory cells of sweat glands showed the same keratin expression as that of Paget cells, Paget cells of extramammary Paget's disease may be derived from or differentiate to the secretory cells of sweat glands.

Squamous cell carcinoma antigen for monitoring cervical cancer.

The tumour-associated antigen was determined in the plasma of patients with squamous cell carcinoma (SCC) of the uterine cervix by radioimmunoassay. Setting a limit of 2 ng/ml, levels were abnormal in 13.4% of healthy controls, in 14% of patients with carcinoma in situ and in 62% of patients with invasive cervical SCC. The incidence of elevated SCC antigen levels and the absolute antigen plasma concentration were dependent upon the tumour load, increasing significantly with advanced stage disease. Abnormal SCC antigen values in operable cervical cancer declined to normal within one week after radical hysterectomy with pelvic lymphadenectomy. In cases of radiotherapy antigen values took 4-6 weeks after the start of treatment to return to normal. The success of both treatment modalities was announced by an early rise in the SCC antigen in the initial phase of therapy, followed by normalisation. After successful primary treatment and a complete remission during further follow-up SCC antigen in plasma was only increased in 3.8% of the cases. Retrospective evaluations in ten patients with progressive disease showed the reappearance of abnormal SCC titers and further increase preceeding the clinically detectable relapse or progression, with a median interval of 8 weeks. The present study indicates that SCC antigen determination is not useful for the early diagnosis of cervical cancer, but it is a potential means for monitoring the efficacy of individual anticancer therapy of SCC of the uterine cervix and for detecting recurrent disease.

Immunohistochemical study of granular cell tumour.

Immunoperoxidase studies were performed on 8 granular cell tumours using various intermediate filament proteins, as well as lysozyme, S-100 protein, and lectins. All the lesions gave negative results to cytokeratin, vimentin, desmin, myoglobin, neurofilament protein, glial fibrillary acidic protein and lysozyme. One was positive for alpha-1-antitrypsin and alpha-1-antichymotrypsin. S-100 protein and lectins (Concanavalin ensiformis and Triticum vulgaris) were uniformly positive in all the lesions. S-100 protein positivity would indicate that granular cell tumours are of neural or neuroectodermal origin, although the cell type involved is not clear. There is no obvious explanation for the lectin-binding properties of granular tumour cells. It is hoped that further studies will evaluate the usefulness of lectin histochemistry in defining the nature of granular cell tumours.

Presence of tumor-associated antigens in epidermal growth factor receptors from different human carcinomas.

The epidermal growth factor (EGF) receptor in two colon carcinoma lines and in one vulval carcinoma line tested contains carbohydrate determinants that are recognized by monoclonal antibodies to tumor-associated antigens. These antibodies are directed to sialylated Lea and to difucosylated structures of the Y type. Cell lines which react with these antibodies express these antigens on their surface glycolipids and glycoproteins, including the EGF receptor. These unusual carbohydrates are absent in EGF receptors from normal untransformed cells, and from tumor cells which do not express these specific antigens. Although EGF receptor represents only 0.1-2% of total plasma membrane proteins of antigen-positive carcinomas, it accounts for 20-80% of total protein-associated sialylated Lea/Y type of nonsecreted carbohydrates present in these cells. The results of cell-binding, immunoprecipitation, and Western blot analyses of the antigen-positive carcinomas indicate that sialylated Lea/Y type of antigenicity is intrinsic to the EGF receptors of these cells, and that the antigen is present in receptors from both over-expressing and normal-expressing carcinomas.

A nuclear deoxyribonucleic acid analysis of normal and abnormal vulvar epithelium.

Vulvar and groin skin from 21 women with clinical squamous cell carcinoma of the vulva was compared with that from nine patients with vulvar intraepithelial neoplasia. Feulgen-stained nuclear densitometry showed a constancy of Feulgen-deoxyribonucleic acid (DNA) density in normal epithelium and all subepithelial connective tissue nuclei. At three sites--tumor edge, opposite labia, and perineum--there were significant differences in nuclear chromatin content between the two groups and also as compared with normal epithelium.