Human cell adhesion molecule uvomorulin is differentially expressed in various skin tumors.

The cell adhesion molecule uvomorulin is important in cell recognition processes, both during tissue formation in embryonic development and in the maintenance of adult epithelia. In addition, uvomorulin appears to play a crucial role in carcinogenesis. Therefore, in the present study, the expression of uvomorulin in normal human skin and several benign and malignant proliferative skin lesions was evaluated by immunofluorescence microscopy using affinity purified antibodies. In normal human epidermis, basal and suprabasal keratinocytes showed a strong and homogeneous staining of the cell membrane. In contrast, uvomorulin expression was decreased in squamous cell, as well as in solid basal cell, carcinoma. Interestingly, solid basal cell carcinoma showed a dimorphic staining pattern with a reduced fluorescence of the inner cell layers and normal staining of the peripheral basal cells. In contrast, no such dimorphic staining pattern could be observed in squamous cell carcinoma, in which uvomorulin expression was homogeneously reduced. Decreased expression of uvomorulin was not specific for malignant skin lesions, since it could also be observed in condylomata acuminata. These studies demonstrate that human uvomorulin is differentially expressed in proliferative skin disorders, which may account at least in part for the differences observed in the clinical course between squamous cell and basal cell carcinoma.

Patterns of keratin 19 expression in normal, metaplastic, condylomatous, atrophic, dysplastic, and malignant cervical squamous epithelium.

Keratin 19 (K-19) expression has been strongly correlated with dysplasia in oral epithelium. Expression of K-19 was evaluated by immunoperoxidase staining in formalin-fixed normal ectocervical tissue, normal endocervical tissue, cervical dysplasia, squamous metaplasia, atrophic epithelium, cervical condylomas, and invasive carcinoma to determine if a correlation of K-19 expression with dysplasia was present in the cervical epithelium. Uniform expression of K-19 was seen in endocervical epithelium and in the basal layer of normal ectocervical epithelium in all areas where these epithelia were present. Cervical dysplasia without associated condylomatous changes showed increased expression of K-19 in suprabasal epithelium, corresponding to the level of immature cells. Squamous metaplasia was characterized by scattered cells with increased staining (patch-quilt pattern). There was considerable overlap in the patterns of K-19 expression in dysplastic and metaplastic epithelium. Thus K-19 staining pattern could not be used as a distinctive marker for dysplasia in the cervical epithelium. Atrophic epithelium showed a characteristic uniform but low-level expression of K-19 in suprabasal areas. This pattern may be of diagnostic use in differentiating atrophic lesions from dysplasia. Condylomas showed focal loss of K-19 in the basal layer, suggesting induction of premature differentiation in the basal layer by human papillomavirus infection. Invasive carcinomas showed variable patterns. K-19 is a marker of immature cervical squamous epithelium, with generally distinctive but sometimes overlapping patterns of expression in various diagnostic categories.

Expression of transforming growth factor alpha mRNA in benign and malignant tissues derived from gynecologic patients with various proliferative conditions.

Growth factors are polypeptides involved in the regulation of normal and malignant cell growth. Transforming growth factor alpha (TGF alpha) is one of such protein growth factors that plays an important role in the regulation of mammalian cell growth. In this study, the expression of TGF alpha mRNA was studied in tissue specimens obtained at the time of surgery from patients with benign and malignant gynecologic proliferative conditions. To analyze TGF alpha mRNA expression we utilized the highly sensitive technique denoted Message Amplification Phenotyping which can detect mRNA in single cells. This technique consists of isolating RNA, reverse transcription of total cellular RNA to produce copy DNA, followed by enzymatic amplification of TGF alpha cDNA fragments using specific TGF alpha primers and polymerase chain reaction. The results showed significant levels to TGF alpha mRNA expression in vulvar (100% of the cases positive), cervical (66% positive), and endometrial (66% positive) carcinomas. Moreover, vulvar condylomas produced by human papilloma virus (HPV) showed the highest levels of TGF alpha mRNA expression of all the pathological tissues examined. In contrast, vulvar melanoma, fibrocystic disease of the breast, and certain ovarian tumors showed undetectable TGF alpha mRNA expression. Normal mesodermal tissues such as myometrium, abdominal rectus muscle, and fallopian tubes were negative for TGF alpha mRNA expression. However, TGF alpha mRNA was present in normal cervix and in normal endometrium. The results showed that TGF alpha mRNA expression is frequently associated with various malignant tumors and HPV-induced lesions of epithelial origin, suggesting that TGF alpha mRNA protein product may be a contributory factor in the progression of these pathological tissue alterations. Finally, TGF alpha mRNA expression was not restricted to malignant cells, suggesting that the TGF alpha mRNA protein product may function as a mitogen in the normal human epithelial tissues examined.

Detection with the polymerase chain reaction of human papillomavirus DNA in condylomata acuminata treated in vitro with liquid nitrogen, trichloroacetic acid, and podophyllin.

BACKGROUND: The mechanisms of action for local treatments used against condylomata acuminata are unknown, but most are believed to cause physical destruction of infected tissue. OBJECTIVE: Our purpose was to determine whether liquid nitrogen, trichloroacetic acid (TCA), and podophyllin damage HPV DNA found in condylomata acuminata. METHODS: Fourteen genital warts were excised from 14 patients and divided. One part was treated with liquid nitrogen, the second and third parts were treated with TCA and podophyllin, respectively, and the remainder served as a control. DNA was then extracted from tissue by proteolytic digestion and amplified by the polymerase chain reaction. Dot blots were performed with the use of radiolabeled consensus and HPV type-specific probes. RESULTS: HPV DNA was amplified and detected in 100% of untreated specimens, in 92% of specimens treated with liquid nitrogen, and in 15% and 7% of specimens treated with podophyllin and TCA, respectively. CONCLUSION: TCA and podophyllin damage HPV DNA more effectively than does liquid nitrogen.

Expression of high-affinity laminin receptor mRNA correlates with cell proliferation rather than invasion in human papillomavirus-associated cervical neoplasms.

Induction of the expression of the Mr 67,000 high-affinity laminin receptor gene has been postulated as playing a role in the progression of human tumors to invasive cancers. We tested this hypothesis by examining histopathological sections of a large number of epithelial lesions of the genital tract associated with human papillomaviruses. In situ hybridization was performed with a riboprobe generated from a laminin receptor complementary DNA. Laminin receptor mRNA was expressed primarily in the less differentiated cells in normal squamous tissues and in a spectrum of squamous neoplasms. There was no net induction of mRNA per cell in intraepithelial or invasive squamous neoplasms relative to normal tissue. In contrast, laminin receptor mRNA was not expressed at a detectable level in normal glands of the uterine cervix but was dramatically induced in morphologically abnormal, human papillomavirus-positive glands, irrespective of the genotype of human papillomaviruses present. The induction occurred before any evidence of invasion, and there was no further increase during the transition from adenocarcinoma in situ to invasive carcinoma. We conclude that induction of high-affinity laminin receptor gene expression is associated with the development of malignancies of cervical glandular epithelia, but the increased expression appears to correlate with the proliferative rather than the invasive properties of these cells.

Immunohistochemical analysis of estrogen receptors, progesterone receptors, Ki-67 antigen, and human papillomavirus DNA in normal and neoplastic epithelium of the uterine cervix.

To investigate the relationship between the sex steroid receptor (estrogen receptor [ER] and progesterone receptor [PR]) status and the cell proliferation kinetics during the menstrual cycle in normal and neoplastic epithelium of the uterine cervix, immunohistochemical localization of ER, PR, and cell proliferation-associated antigen, Ki-67, was investigated in 35 normal cervical specimens, 3 condylomas, 26 cervical intraepithelial neoplasia (CIN) samples, and 22 invasive squamous carcinoma samples. The presence of human papillomavirus (HPV) DNA was also studied. In the normal cervix, basal cells were usually ER positive, PR negative, and Ki-67 negative throughout the menstrual cycle. Parabasal cells were ER positive and PR negative in the follicular phase, but ER negative and PR positive, and Ki-67 positive in the luteal phase, and Ki-67-positive cells increased in number in the luteal phase. In contrast, PR positivity was observed in the cells of condyloma (2 of 2 cases), CIN (19 of 26 cases), and invasive squamous carcinoma (13 of 22 cases) irrespective of the menstrual phase. Moreover, most neoplastic cells containing HPV DNA type 16/18 were ER negative, whereas several lesions containing HPV DNA type 31/33/35 were weakly ER positive. Many Ki-67-labeled cells were observed in the neoplastic lesions. These results suggest that reduced ER expression and increased PR expression are associated with the proliferation of normal cervical squamous epithelium, and this proliferation-related receptor status, which is probably induced by HPV infection, is usually expressed in neoplastic cervical squamous cells.

[Argyrophilic nucleoproteins of the cervical epithelium in HPV infection and intraepithelial neoplasia]. / Studio delle nucleoproteine argirofile dell'epitelio cervicale nelle infezioni da HPV e nelle neoplasie intraepiteliali.

50 colposcopic biopsies of cervical epithelium were studied, using a silver colloid technique. These comprised 28 cases of human papillomavirus infection of the cervix, 8 cases of cervical intraepithelial neoplasia (CIN) I, 8 cases of CIN II, 6 cases of CIN III. The AgNOR mean number of the basal and parabasal cells of the cervical epithelium was significantly different in virus infected cells and in CIN. Different patterns of AgNOR distribution were observed: they were single and compact in virus infection without dysplasia whereas they appeared small and often loosely arranged in dysplastic lesions. Our data suggest that this simple technique is diagnostically useful in the evaluation of borderline lesions.

Estrogen and progesterone receptors in cervical human papillomavirus related lesions.

According to recent studies showing that human papillomavirus (HPV) infections can be influenced by sex steroid hormones, we performed estrogen (ER) and progesterone (PgR) receptor assays in fresh frozen biopsies of genital-HPV-related lesions. Seventy-three women with normal cervix, condyloma, low- and high-grade CIN and squamous carcinoma were evaluated in comparison with 15 persons with vulvar and 9 with penile papillomavirus-associated lesions. HPV genotypes were determined by dot-blot hybridization. Non-cervical lesions did not express HR. Condyloma on squamous metaplasia of the cervix and high-grade CIN expressed high levels of HR, particularly PgR (mean 4,086 and 4,518 fmoles/g tissue, respectively). Cervical squamous carcinoma expressed very low concentrations of PgR in a limited number of cases. High levels of PgR were correlated with high-grade CIN (p less than 0.05), HPV16-18-associated lesions (p less than 0.01) and ER were correlated to HPV6-11-related lesions (p less than 0.01). The levels were independent of age, cycle stage and oral contraception. Morphological localization of PgR, using an immunocytochemical method using a monoclonal antibody (MAb) (PR-ICA), showed intense homogeneous staining in the nuclei of the stromal fibroblasts underlying dysplastic epithelium and condyloma on squamous metaplasia. These results suggest that, under in vivo conditions, sex steroid hormones, particularly progesterone, may act indirectly on HPV-infected epithelial cells and be implicated as co-factors in HPV-related cervical neoplasia. They could explain the relative predisposition to malignant transformation of the cervix as compared with vulvar and penile mucosa.

Detection of human papillomavirus types 6 and 11 E4 gene products in condylomata acuminatum.

Polyclonal antiserum to an Escherichia coli-produced beta-galactosidase/E4 fusion protein of human papillomavirus type 6b (antiserum 256), and affinity purified HPV 11 anti-E4 antibodies were tested for reactivity in Western blots with bacterially expressed trpE/E4 fusion proteins of HPV types 6b, 11, 16, and 18. To further characterize the affinity purified anti-E4 antibodies, a dot-immunobinding assay was performed using overlapping synthetic HPV 11 E1E4 peptides as antigens. Protein extracts of condylomata acuminatum from 18 patients containing HPV type 6 or 11 DNA sequences were tested in Western blots using antiserum 256 or affinity purified HPV 11 anti-E4 antibodies. In the Western blots of the trpE proteins, antiserum 256 identified the HPV types 6b and 11 fusion proteins; the affinity purified HPV 11 anti-E4 antibodies identified only the HPV 11 fusion protein. In the dot-immunobinding assay, three HPV 11 peptides were recognized, each containing a shared 8 amino acid sequence that differs significantly from the corresponding sequences of HPV types 6b, 16, or 18. In the Western blots of protein extracts from 18 condylomata acuminatum samples shown to contain HPV types 6 or 11 DNA, putative E4 gene products were identified in six samples by antiserum 256. The affinity purified HPV 11 anti-E4 antibodies identified putative E4 gene products in one of these same six lesions, which was shown to contain HPV 11 sequences by the Southern blot method. All six samples containing E4 gene products were from women. Three of these women were pregnant, one had serum antibodies to the human immunodeficiency virus, and one was a renal transplant recipient receiving glucocorticoids.(ABSTRACT TRUNCATED AT 400 WORDS)

Human papillomavirus segregation patterns in genital and nongenital warts in prepubertal children and adults.

This study compared the segregation patterns of human papillomavirus (HPV) in genital and nongenital warts in prepubertal children and adults. HPV 2 was detected in most nongenital warts in children and adults, whereas neither HPV 6 or 11 was detected at nongenital sites in either group with the use of in situ or Southern blot hybridization analyses. Of nine genital tract lesions in children. HPV 2 was detected in two and HPV 6 or 11 in six. More than 90% of cases of regional tract condylomata in adults contained HPV 6 or 11. HPV 2 was not detected in any of 99 genital tract lesions in adults. It is concluded that HPV 6/11 cannot proliferate at nongenital cutaneous sites and HPV 2 can proliferate in the genital tract of children but not adults. Thus, the detection of HPV 6 or 11 in a genital wart in a child implies, assuming cutaneous transmission, infection from a genital site, whereas the detection of HPV 2 presumes nongenital transmission.

Cytokeratin pattern in normal and HPV infected oral mucosa in women with genital HPV infections.

The distribution of cytokeratins Nos. 19 (CK 19), 14, 16 and 17 (CK2-27), and 8 and 18 (CK 60-61) in 96 oral mucosal biopsies taken from women with genital HPV infections were studied by immunohistochemistry, using polyclonal antibody CK 19, as well as monoclonal antibodies CK 2-27 and CK 60-61. White staining of the buccal mucosa after acetic acid application, which recently was shown to be affected mostly by smoking and age, could not be explained by differences in cytokeratin pattern. In HPV DNA-positive biopsies, the staining with CK 19 antibody in the basal cell layer was more intense than in HPV DNA-negative biopsies. The staining with CK 2-27 antibody was seen in 76% and 91% of the basal and superficial layers, respectively, even though these low molecular weight cytokeratins should be found mainly from the basal and parabasal cells. CK 60-61 staining was almost similar to that seen recently in normal genital mucosa. When trying to distinguish oral HPV infections from normal mucosa, CK 2-27 and CK 60-61 stainings were of no diagnostic value. The more efficient expression of CK 19 in HPV DNA-positive samples suggests that viral infection might accelerate the production of low molecular weight cytoskeletal protein. This could be interpreted as evidence that HPV might disturb the keratinocyte differentiation in the basal cells. As a result of the present study, CK 19 staining in oral mucosa needs to be further studied in regard to viral infections, because it may help to better understand the interaction between a virus and a host cell.

Estrogen receptor localization in normal and neoplastic epithelium of the uterine cervix.

To investigate the estrogen receptor (ER) status of cells during carcinogenesis of the uterine cervix, the immunohistochemical reactivity for a monoclonal anti-ER antibody (H 222) was studied in 26 normal cervical specimens, 21 cases of cervical intraepithelial neoplasia (CIN), and 21 cases of invasive cervical carcinoma. In addition, the presence of human papillomavirus (HPV) DNA (types 6/11, 16/18, or 31/33/35) was analyzed by in situ hybridization. In the normal cervix, basal cells of the squamous epithelium, metaplastic cells, and endocervical glandular cells were ER positive. In contrast, neoplastic cells of CIN (17 of 21 cases) and invasive carcinoma (19 of 21 cases) were ER negative. The remaining four cases of CIN and two cases of invasive carcinoma were focally ER positive. The HPV DNA analysis revealed that HPV DNA in ER-negative cases was either types 16/18 or undetectable, but all ER-positive neoplasms contained HPV DNA types 31/33/35. These results suggest that most neoplastic cells in CIN and invasive cervical carcinoma lose their ER expression and that this may be related to the HPV DNA types which they possess.

Detection of human papillomavirus DNA in human cervical lesions by tissue in situ nucleic acid hybridization.

Cervical cancer is one of the most common female cancers in Taiwan. Certain types of human papillomavirus (HPV) are frequently detected in the epithelial precancerous and cancerous lesions of the cervix. By the use of tissue in situ hybridization, we investigated the relationship of various types of HPV (group I, HPV-6 & 11, group II, HPV-16 & 18, group III, HPV-31, 33 & 35) with cervical condyloma, carcinoma as well as precancerous lesions. Group I HPV DNAs were mainly found in cervical condylomatous lesions (2/2) of the cervix and cervical intraepithelial neoplasia I (CIN I) (2/4), but were only occasionally found in CIN II (1/4), CIN III (1/9) or non-keratinized squamous cell carcinoma (1/15). HPV DNAs of groups II and III were mainly detected in lesions of CIN III (5/9) and invasive squamous cell carcinoma (large cell, keratinized type: 4/7; large cell, non-keratinized type: 11/15). HPV DNA sequences were invariably detectable only in the cell nuclei of condyloma or dysplastic epithelium or invasive carcinoma. However, they could not only be detected in the upper layer dysplastic cells and koilocytes but also in the well and poorly differentiated cervical cancer cells. The distribution of HPV DNA positive cells in the carcinomas fell into four different patterns: (1) upper zone and non-invasive regions of the carcinoma (11/22, 50%), (2) basal zone and invasive regions (2/22, 9%), (3) randomly scattered (7/22, 32%), and (4) extensively distributed over the whole tumor lesions (2/22, 9%). Thus, our results are consistent with a strong correlation between the presence of HPV-16, 18, 31, 33 and 35 and malignant conversion of cervical epithelial cells.

Differential detection of human papillomavirus DNA type 6 and 11 amplified by polymerase chain reaction.

We have developed a method for differential detection of human papillomavirus type 6 and 11 with selective amplification of a segment of the viral DNAs by the polymerase chain reaction. The target sequence for amplification was a 134-bp (bp 206-339) in the E6 open reading frame of HPV 6 and 11 DNAs. DNA extracted from a paraffin-section or a minute fresh biopsy specimen of condyloma acuminatum was amplified and hybridized with an oligonucleotide probe specific for HPV 6 or 11 DNA. The method was applied to analyse 5 cases of genital condyloma acuminatum, and as a result, HPV 6 and 11 DNAs were detected in two and one case, respectively. This method is useful for differential diagnosis of HPV 6 and 11 infection.

[Preliminary study of the possibility of a viral etiology in cervical cancer]. / Preliminarna istrazivanja o mogucoj virusnoj etiologiji raka grlica materice.

The authors examined 10 patients with cervical cancer and 5 with condyloma acuminatum. The sera were collected pre- and postoperatively and checked up to the presence of C-reactive protein (CRP), while the method of the complement fixation reaction (CFR) was used to determine the presence of antibodies against herpes adeno and EB virus. The technique of indirect immunofluorescence was used to determine the presence of the mentioned viruses in the maliguant tissue. Eight preoperatively obtained sera contained antibodies against herpes virus, the titre ranging from 1:8 to 1:64. The presence of antibodies against other viruses was insignificant. The antibody titre against herpes virus decreased in 6 postoperatively obtained samples, the highest titre being 1:16. The presence of herpes virus was determined by the immunofluorescence method in 7 out of 10 samples of the maliguant tissue. Adenovirus was found in 1 sample while EB virus was not found. The presence of the investigated viruses was not evidenced in the cases of condylomas either in the tissue or as sera antibodies.