Cell kinetic analyses and expression of carcinoembryonic antigen,carbohydrate antigen 19-9 and DU-PAN-2 in hyperplastic,pre-neoplastic and neoplastic lesions of intrahepatic bile ducts in livers with hepatoliths

Summary We evaluated cell proliferative activity and expression of carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA 19-9) and DU-PAN-2 in various bile duct lesions in livers with hepatoliths, using histochemical and immunohistochemical methods. Histologically, the bile duct lesions were divisible into hyperplasia, dysplasia, adenocarcinoma in situ and invasive adenocarcinoma. All cases showed mucosal hyperplasia in stone-bearing bile ducts. Livers with invasive adenocarcinoma frequently contained adenocarcinoma in situ and dysplasia, and livers with adenocarcinoma in situ occasionally harboured dysplasia. Proliferating cell nuclear antigen (PCNA) labelling index was low in hyperplasia (mean±SD=20.5±8.7%), intermediate in dysplasia (35.4±15.9%), and high in adenocarcinoma in situ (46.4±9.3%). The mean number of argyrophilic nucleolar organizer regions (AgNORs) was low in hyperplasia (1.52), intermediate in dysplasia (2.26) and high in adenocarcinoma in situ (2.69). There was a significant positive correlation between PCNA labelling index and AgNORs count. CEA was expressed on invasive adenocarcinoma cells and adenocarcinoma in situ cells in most cases and on dysplastic cells in about a half, while CEA was never present in hyperplastic epithelia. Expression of CA 19-9 was low in adenocarcinoma, intermediate in dysplasia and rather high in hyperplasia. There was no significant difference in DU-PAN-2 expression among these bile duct lesions. These data suggest that cell replicative activity is low in hyperplasia, intermediate in dysplasia and high in adenocarcinoma in situ, and that CEA appears in the following order: dysplasia, adenocarcinoma in situ, invasive adenocarcinoma. We suggest that carcinogenesis in biliary epithelia in livers with stones is a multi-step process through hyperplasia, dysplasia and adenocarcinoma in situ to invasive adenocarcinoma.     

Abklärung zervikaler Dysplasien mittels DNA-Bild-Zytometrie

Dysplastic epithelia represent potentially precancerous conditions in which the risk of progression to cancer is unknown in the individual case. The positive predictive value of mild and moderate dysplasias of the uterine cervix is only about 13%. Using DNA image cytometry on restained, conventional Papsmears the cytometric equivalent of chromosomal aneuploidy can be detected as marker for neoplastic transformation of cells. The identification of DNA aneuploidy in dysplastic squamous epithelia can increase the predictive value for malignant transformation to over 90%. DNA aneuploidy qualifies squamous intraepithelial lesions as high grade (H-SIL) which have to be treated whereas lack of DNA aneuploidy characterizes low grade squamous intraepithelial lesions (L-SIL) which have only to be controlled. The methodology is meanwhile internationally standardized concerning performance and diagnostic interpretation.     

Expression of the CD 15 antigen is a marker of cellular differentiation in cervical intra-epithelial neoplasia (CIN)

The CD 15 antigen (3-fucosyl N-acetyllactosamine), present on the outer cell membrane of cervical squamous epithelial cells, is recognized by the monoclonal antibody MC2, which is similar to several commercially available antibodies. Staining sections of cervical biopsies with MC2 clearly demonstrates the zone of supra-basal differentiated cells in the normal squamous epithelium. Staining with MC2 also demonstrates the diminished proportion of this zone occurring with grades of CIN, reflecting progressive de-differentiation of the epithelium. In immature squamous metaplastic epithelium, absence of cytoplasmic differentiation is reflected by lack of staining. As expression of the CD 15 antigen by cervical squamous cells mirrors cytoplasmic maturity and is a marker of cellular differentiation, staining colposcopic biopsies with MC2 may aid the routine histopathological grading of CIN. A comparison is made between the staining pattern observed using MC2 with that of two commercially available antibodies (Leu M1 and Dako M1), and a possible role for the CD 15 antigen in cellular adhesion in squamous mucosae is discussed.     

Karyometry of pseudostratified, metaplastic and dysplastic nasal epithelium by morphometry and stereology. 2. Automated image analysis (IBAS) of the basal layer of nickel workers

A mathematical model for the stereological analysis of parallel-oriented, spheroidal nuclei has been applied to the basal layers of respiratory, metaplastic and dysplastic nasal epithelia. Nuclear profiles seen on tissue sections were measured with an automatic image analyser (IBAS). Nuclear profile area distributions were used to assess possible polyploidies. The result are compared to the histopathological grading, consisting of pseudostratified (respiratory), cuboidal, mixed cuboidal/squamous, squamous, and dysplastic epithelia. The estimated nuclear axis lengths, volumes, surface areas and volume/surface ratios are, in the great majority of cases, significantly smaller in pseudostratified or cuboidal epithelium than in squamous metaplasia and dysplasia. Correspondingly, the numerical density of the nuclei is lower in the latter cases, in which is also noted a smaller nuclear eccentricity. No significant difference is found between the nuclei of squamous and dysplastic epithelia. There is a clear correlation between the mean profile areas and the stereologically estimated volumes of the nuclei. When classifying the mean profile areas into four classes, a progressive shift from the smaller towards the larger size classes is observed when passing from pseudostratified to dysplastic epithelia, through the different metaplastic stages. The nuclear profile area plots often show several peaks, even in some pseudostratified and cuboidal epithelia, probably reflecting polyploidy. There is a marked tendency towards larger profile areas in squamous metaplasia and dysplasia. One histopathologically typical dysplasia showed only small-sized nuclei and another had a notable contribution of those in its profile area plot, in contrast with all the other dysplasias and squamous metaplasias, which displayed practically no small nuclei. The possible implications of the existence of "small-sized-nuclei-dysplasias" are discussed. The literature dealing with nuclear volumes or DNA quantities in putative preneoplastic situations in reviewed. Our method adds some valuable objective criteria to those used in classical histopathological grading. It should be possible to apply this method to other epithelial tissues.     

Pseudovascular squamous cell carcinoma of the uterine cervix: A lesion that may simulate an angiosarcoma

A case of pseudovascular squamous cell carcinoma in the uterine cervix of a 64-year-old woman was examined. Histologically, the lesion consisted of atypical, large, non-keratinizing squamous cells that exhibited not only acantholytic changes but also pseudovascular changes. Immunohistochemically, these tumor cells were positive for cytokeratin, epithelial membrane antigen and carcinoembryonic antigen, but none of them were positive for Factor VIII-related antigen or CD34. To our knowledge, pseudovascular carcinoma of the uterine cervix has not been described in the literature. Pathologists should be aware of this unusual form of cervical squamous cell carcinoma, particularly in the differential diagnosis of angiosarcoma.     

Co-expression of p16(INK4A) and laminin 5 gamma2 by microinvasive and superficial squamous cell carcinomas in vivo and by migrating wound and senescent keratinocytes in culture

The high frequency of mutation, deletion, and promoter silencing of the gene encoding p16(INK4A) (p16) in premalignant dysplasias and squamous cell carcinomas (SCC) of epidermis and oral epithelium classifies p16 as a tumor suppressor. However, the point during neoplastic progression at which this protein is expressed and presumably impedes formation of an SCC is unknown. Induction of p16 has been found to be responsible for the senescence arrest of normal human keratinocytes in culture, suggesting the possibility that excessive or spatially abnormal cell growth in vivo triggers p16 expression. We examined 73 skin and oral mucosal biopsy specimens immunohistochemically to test this hypothesis. p16 was not detectable in benign hyperplastic lesions, but instead was expressed heterogeneously in some dysplastic and carcinoma in situ lesions and consistently at areas of microinvasion and at superficial margins of advanced SCCs. p16-positive cells in these regions coexpressed the gamma2 chain of laminin 5, identified previously as a marker of invasion in some carcinomas. Normal keratinocytes undergoing senescence arrest in culture proved to coordinately express p16 and gamma2 and this was frequently associated with increased directional motility. Keratinocytes at the edges of wounds made in confluent early passage cultures also coexpressed p16 and gamma2, accompanying migration to fill the wound. These results have identified the point during neoplastic progression in stratified squamous epithelial at which the tumor suppressor p16 is expressed and suggest that normal epithelia may use the same mechanism to generate non-dividing, motile cells for wound repair.     

A scanning electron microscopic study of the dysplastic epithelia adjacent to oral squamous cell carcinoma

By light microscopy, the dysplastic oral epithelia due to the neoplastic processes are similar to epithelial changes due to the inflammatory processes. Scanning electron microscopy may elucidate the different surface changes between the two. The aim of this study was to examine the surface appearances of the dysplastic oral epithelia adjacent to oral squamous cell carcinoma to see if there are any surface changes. A total of 2 specimens, one specimen from each patient with oral squamous cell carcinoma, were used for this study. Each specimen was divided in two. One half was prepared for light microscopy and the other half was prepared for scanning electron microscopy. Light microscopically, the epithelia showed mild dysplasia. By scanning electron microscopy, the keratinized cells showed irregular microridges surrounding pits, which were variable and irregular in size and shape, and the nonkeratinized cells showed parallel microridges with irregularly widened intervals between each microridge. Irregular, broad, and partly swollen microridges and irregular short, stubby surface projections were also seen. The oral epithelia adjacent to oral squamous cell carcinoma showed mild dysplasia light microscopically but appeared abnormal by scanning electron microscopy. The abnormal epithelial cells showed pleomorphism, irregular and disoriented microridges, and abnormal surface microstructures.     

Ber-EP4 immunoreactivity depends on the germ layer origin and maturity of the squamous epithelium

AIM: To map the expression of Ber-EP4 in well-differentiated squamous epithelia, metaplastic squamous epithelia and dysplastic squamous epithelia of different origins. METHODS AND RESULTS: Squamous epithelium of different origin was stained using a standard immunohistochemistry method applied to paraffin sections. We found that normal squamous epithelium of the oral cavity, oesophagus, uterine cervix, vagina, anal canal, and branchial cysts are Ber-Ep4-negative, as are the mature squamous metaplasia of bronchial mucosa, urinary bladder mucosa and uterine cervical mucosa. In contrast, immature squamous metaplasia of bronchial mucosa, or uterine cervical mucosa, and squamous dysplasia of oral mucosa of endodermal origin, or uterine cervical mucosa in most cases expressed Ber-EP4. CONCLUSION: Squamous epithelia of ectodermal origin never express Ber-EP4, whether normal, hyperplastic, dysplastic or neoplastic. In contrast, squamous epithelium of endodermal origin sometimes contains the target glycoproteins of Ber-EP4 when immature, metaplastic, dysplastic or neoplastic. The results indicate that the differences in expression of Ber-EP4 in squamous epithelium depend primarily on germ layer origin, and on the maturity of the epithelium.     

Concurrent p53 expression in bronchial dysplasias and squamous cell lung carcinomas.

We analyzed the p53 protein immunohistochemically in bronchial dysplasias or squamous cell carcinomas in situ and in squamous cell lung carcinomas occurring in the same patients. The polyclonal antibody used (CM-1) is directed against the wild-type p53 protein, but also recognizes the mutated p53 in formalin-fixed and paraffin-embedded sections. To study the integrity of basement membranes (BMs) and the possible invasion of the dysplastic epithelium, immunostainings for the BM proteins laminin and type IV collagen were used. Nine of the 17 dysplasias showed p53 protein expression (53%); it was significantly more often seen in severe dysplasias and carcinomas in situ than in mild or moderate dysplasias (P = 0.04). The p53 antigenicity was generally located in the basal part of the epithelium. The BMs beneath mildly dysplastic epithelia were continuous. In contrast, those under moderately or severely dysplastic epithelia showed occasional disruptions. p53 protein expression was also found in dysplastic epithelium above a continuous BM suggesting an ominous process before signs of invasion. Twelve of the 17 squamous cell carcinomas showed p53 protein expression (71%). There was a significant concurrent p53 expression in bronchial dysplasias and their related squamous cell carcinomas (P = 0.009), so that all nine cases of p53 positive bronchial dysplasia also showed p53 positivity in the associated squamous cell carcinomas. These findings indicate that p53 protein expression is possible in premalignant bronchial lesions, and suggests that the p53 expression could, at least in some cases, be an early event in the development of a squamous cell carcinoma of the lung.     

p16 Alterations and retinoblastoma protein expression in squamous cell carcinoma and neighboring dysplasia from the upper aerodigestive tract

The progression potential of preinvasive epithelial lesions is usually evaluated by assessing the degree of histologic dysplasia. We examined p16, retinoblastoma protein (pRb), and proliferating cell nuclear antigen (PCNA) immunophenotypes in 57 cases of previously untreated squamous cell carcinoma (SCC) of the upper digestive tract and in the neighboring normal and dysplastic epithelia. Tissue samples were examined for homozygous deletion of exon 2 of the p16 gene using polymerase chain reaction (PCR) analysis. The PCNA index increased with increasing grade of dysplasia. The pRb protein was expressed in 89% of the samples of SCCs and in the neighboring dysplasias and carcinoma in situ (CIS). In cases with a lack of pRb expression, corresponding preinvasive lesions were also negative. Lack of p16 expression was found in 82% of SCCs. The prevalence of p16 expression decreased with increasing grade of dysplasia. Molecular analysis of the p16 gene showed homozygous deletion in 37% of SCCs, 33% of CIS, and 15% of the samples of normal epithelia. Our data indicate that inactivation of p16 may play an important role in early head and neck carcinogenesis, whereas the mutation of Rb may be an infrequent event. The p16 immunophenotype might be a biomarker for an increased risk of progression in squamous dysplasia.     

Variations of mitotic index in normal and dysplastic squamous epithelium of the uterine cervix as a function of endometrial maturation.

Cervical intraepithelial neoplasia is a premalignant (dysplastic) lesion that is characterized by abnormal cellular proliferation, maturation and nuclear atypia. The intraepithelial distribution, density, and nature (typical or atypical) of mitotic figures are routinely utilized diagnostic criteria to grade dysplasia and to distinguish high-grade dysplasia from potential histologic mimics such as transitional metaplasia, atrophy or immature squamous metaplasia. In this study, we evaluated the total mitotic indices of the cervical epithelia in hysterectomy specimens from patients with and without dysplastic lesions and investigated a possible relationship between mitotic index and hormonal status, using the endometrial maturation phase as a surrogate indicator of the latter. Two hundred seventy-four cervices from hysterectomy specimens (135 cases without dysplasia, 33, 35 and 71 cases with grades 1, 2 and 3 cervical intraepithelial neoplasia, respectively) were analyzed. A cervical mitotic index (total mitotic figures/10 high-power fields in the most proliferative area) was determined for each case. The endometrium in each case was classified into atrophic, early proliferative, late proliferative and secretory. For all three dysplasia grades, cases in the proliferative endometrium group always had a higher average mitotic index than those in the secretory and atrophic endometrium groups; this observation also held true for the benign cases. Furthermore, in all three dysplasia grades, the average mitotic index was always lowest in the atrophic endometrium group. Although the mitotic index showed expected patterns of increases with increasing dysplasia grades for most of the endometrial phases, this was not a universal finding. Notably, the average mitotic index for our cervical intraepithelial neoplasia 1 cases with late proliferative endometrium was higher than our cervical intraepithelial neoplasia 2 cases with secretory and atrophic endometrium. It is concluded that hormonal status, as reflected in endometrial maturation, can significantly affect the mitotic index of dysplastic squamous epithelium of the uterine cervix. Our findings confirm that the pathologic grading of dysplasia, especially in equivocal cases such as in metaplastic squamous epithelium, should not be solely dependent on the finding mitoses in the cervical squamous epithelium. The full composite of histopathologic features should form the basis for this determination.     

EXPRESSION OF CD44 SPLICE VARIANTS IN SQUAMOUS EPITHELIA AND SQUAMOUS CELL CARCINOMAS OF THE HEAD AND NECK

Splice variants of the adhesion molecule CD44 have been described as essential for the lymphatic spread of rat tumour cells and are claimed to be involved in the metastatic spread of several human tumours. Immunohistochemistry has been used to analyse the expression pattern of CD44 standard (CD44s) and variant (CD44v) isoforms in normal and dysplastic squamous epithelia, as well as in primary and metastatic squamous cell carcinomas (SCCs), which spread predominantly by way of the lymphatic system. Frozen sections of squamous epithelia and of squamous cell carcinomas were stained with a panel of monoclonal antibodies recognizing epitopes of CD44s as well as of the variant exons v5, v6, v7, v7–v8, and v10. The stratum basale and stratum suprabasale of squamous epithelia stained with all antibodies; the stratum spinosum stained with anti-CD44v5, anti-CD44v6, anti-CD44v7–8 and anti-CD44v10; the lower layers of the stratum corneum stained with anti-CD44v5. This expression profile was seen in epithelia of the lip, the tongue, the gingiva, the hard palate, the floor of the mouth, the buccal mucosa, and the pharynx. The same pattern of expression was also noted in dysplastic epithelia, but expression of the variant exons v7, v8, and v10 was significantly downregulated in primary squamous cell carcinomas and was not detected at all in the majority of metastasis-derived specimens. Expression of CD44v5 and CD44v6, on the other hand, was mainly unaltered. Thus, epithelial cell layers representing different stages of differentiation express distinct sets of CD44 variant isoforms, where especially exons v8–v10 might be required for the maintenance of the structural integrity of squamous epithelium. Downregulation of these exons on tumour cells could indicate that they are irrelevant for tumour progression or may even hamper infiltration of surrounding tissue or of lymphatics.     

Malpighian epithelia and papillomavirus infections]

Human papillomaviruses (HPV) are a large group of DNA viruses, with over 60 types identified to date, which can cause the development of benign tumors in the skin and mucosal squamous epithelia. Most of these tumors regress spontaneously but some, especially in the mucosal membranes, become malignant. HPV types with a high risk for inducing malignancies (e.g. 16 and 18) are the subject of increasing interest. HPVs are both host-specific and tissue-specific: some types preferentially infect specific epithelia, giving rise to lesions with distinct topographic characteristics. HPVs are difficult to study because they do not replicate in available in vitro models. In vivo, HPVs replicate well in epithelial cells undergoing terminal differentiation, e.g. in keratinized cells. Some 40 different types have been reported in epidermal keratinocytes, the most common being types 1 and 2 which produce large amounts of viral antigens and viral particles. In contrast, HPVs replicate poorly in the weakly keratinized squamous epithelia which line the digestive, respiratory, and genital tracts. Junctional epithelia, e.g. on the uterine cervix, are especially prone to HPV infection. The most prevalent HPV types in benign genital lesions are types 6 and 11, whose characteristic features include extrachromosomal DNA and production of only small amounts of viral antigens. The profound nuclear and cytoplasmic changes induced by HPVs lead to the formation of ko?locytes which are found mainly in the granular layer of epithelia and have been especially well described in the uterine cervix and vagina. HPV epithelial tumors are squamous cell carcinomas that often harbor HPV types 16 and 18; this is especially true of cervical intraepithelial neoplasias.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunohistochemical detection of Ca antigen in normal, dysplastic and neoplastic squamous epithelia of the human uterine cervix.

Immunohistochemical staining was performed on biopsies and cytological samples from normal, dysplastic and neoplastic squamous epithelia using the monoclonal Ca 1 antibody. The results of staining 92 biopsies and 20 cytological samples are described and it is reported that positive staining with Ca 1 antibody was detected in normal, dysplastic and neoplastic epithelia. The role of the Ca 1 antibody in the study of cervical cancer is discussed.     

Skin calcium-binding protein in squamous metaplasia of human uterine cervix

The distribution of skin calcium-binding protein in squamous cell metaplasia of human endocervix, in normal human skin, and in ovarian cancer was determined by the immunofluorescence technique. A rabbit antiserum specific to rat SCaBP was characterized by Ouchterlony immunodiffusion and by immunoprecipitation of 125I-labeled SCaBP. The specificity of antibody labeling was demonstrated by using preimmune rabbit serum and SCaBP antiserum competitively absorbed with purified SCaBP. In normal human skin SCaBP was found exclusively in the basal layer cell cytoplasm. This protein was not detected in normal columnar epithelium of endocervix. Epithelial tissues in the zone of transition between the cylindrical epithelium of the endocervical mucosa and the stratified squamous epithelium of the exocervix were obtained from 14 patients with a wide variety of squamous cell metaplasia. In the early stage of metaplasia SCaBP was detected exclusively in the cytoplasm of reserve undifferentiated cells. In the terminal stage of metaplasia the SCaBP was present only in the basal cell layer. SCaBP was found in several layers of dysplastic tissue, and this distribution appeared to be related to the loss of normal maturation of the epithelium. SCaBP was also present in squamous cell carcinoma of endocervix especially in the least differentiated regions of the tumor. No SCaBP was detected in any ovarian cancer cells. These findings are potentially useful as a means of early detection of squamous metaplasia and of distinguishing premalignant anaplastic lesions from those that are benign and nonproliferative. In addition, the presence of SCaBP in tumors derived from metaplastic epithelia and its absence in the ovarian cancer indicate that immunohistochemical search for this protein might be of value in tumors in which an epidermoid origin is a possibility.     

Tissue engineering of oral dysplasia

Keratinocytes and fibroblasts isolated from dysplastic oral lesions were combined to provide a renewable source of epithelia. A dysplasia-scoring index was devised to compare the architectural and cytological features and used together with robust immunophenotyping to show that the engineered epithelia showed most of the characteristics of the clinical lesions. The strains of dysplastic oral keratinocytes with an extended or immortal lifespan provided a reproducible resource of epithelia showing mild (DOK), moderate (POE9n) or severe (D20) dysplasia when maintained under defined conditions. The dysplasia score was influenced by growth conditions, with KGF polarizing proliferation to the basal layer and reducing the severity of dysplasia. When compared to the normal counterparts, dysplasia-associated fibroblasts expressed MMP9, secreted more HGF, increased the dysplasia score for epithelia generated with mortal dysplastic keratinocytes and induced morphological changes in normal keratinocytes, highlighting the role of the microenvironment in determining the phenotype of dysplastic epithelia.