Inverse modulation of intraepithelial Langerhans' cells and stromal macrophage/dendrocyte populations in human papillomavirus-associated squamous intraepithelial lesions of the cervix

Ninety-four cervical biopsies from normal tissue to high-grade squamous intraepithelial lesion (SILs) were examined for the presence of intraepithelial Langerhans' cells and subpopulations of stromal macrophages/dendrocytes by immunohistochemistry using anti-S100,-L1, -CD68 and -factor XIIIa antibodies. Human papillomavirus (HPV) detection was performed in all cases by using first a mixture of DNA probes for 14 HPV types commonly found in anogenital biopsies at low stringency conditions (T _m -40°C) and by reanalyzing the tissues at high stringency (T _m - 10°C) with HPV 6/11, 16/18 and 31/33/35 biotinylated probe cocktails and individual digoxigenin-labelled probes. SILs and metaplastic tissues were significantly associated with a depletion of S100-positive intraepithelial Langerhans' cells when compared with normal epithelium. In contrast, there was a significant increase in L1-positive stromal macrophages in SIL biopsies compared with normal or metaplastic cervix. A significantly higher density of CD68-positive macrophages was also observed in high-grade SILs compared with normal or metaplastic biopsies and with low-grade SILs. The density of factor XIIIa-positive dendrocytes was found to be higher in SILs compared with metaplastic tissues and in high-grade SILs when compared with normal cervical biopsies. No specific relationship was found between the densities of these cells and the HPV type detected in SILs separated into low grade and high grade. The significance of this inverse modulation of intraepithelial Langerhans' cells and stromal macrophages/dendrocytes in normal and SIL biopsies is discussed in relation to HPV infection and malignant transformation.     

Human papillomavirus-16 E6 variants in cervical squamous intraepithelial lesions from HIV-negative and HIV-positive women.

We studied 48 human papillomavirus (HPV)-16-positive squamous intraepithelial lesions (SILs) from HIV-negative patients (16 low-grade SILs [LSILs]; 32 high-grade SILs [HSILs]) and 13 HPV-16-positive SILs from HIV-positive patients with AIDS (1 LSIL; 12 HSILs). After HPV typing, the entire HPV-16 E6 coding region was amplified and sequenced in all samples. We detected 12 HPV-16 E6 prototypes and 4 variants among the LSILs in HIV-negative patients, and 15 HPV-16 E6 prototypes and 17 HPV-16 variants in the HSIL group. The most prevalent variant of SIL types was European 350G, present in 3 and 13 cases, respectively. In 3 HSILs and no LSILs we found mixed infection by an HPV-16 E6 prototype and a variant. Two variants (1 each in LSIL and HSIL) were of non-European lineage. The only LSIL in HIV-positive patients had an HPV-16 E6 prototype; in the HSILs, we found 8 HPV-16 E6-prototypes, 4 with mixed infection with HPV-31 and 4 variants, all European 350G. The higher proportion of HPV-16 E6 variants in HSIL than in LSIL in HIV-negative patients suggests a greater risk of progression. However, further studies are needed.     

Loss of cytokeratin 14 expression is related to human papillomavirus type and lesion grade in squamous intraepithelial lesions of the cervix

In a recent study of low-grade cervical squamous intraepithelial lesions (SILs), we reported that infection with both low- and high-risk human papillomaviruses (HPVs) upregulated cyclin A, B, E, and Ki67 expression in basal and suprabasal cells. In view of the intricate link between cell cycle exit, proliferation, and differentiation, we examined the morphologic distribution of cytokeratins 13 and 14 and involucrin expression in 49 low-grade SILs infected with HPV types 6, 11, 16, 18, 31, 33, 39, 42, 43, 44, 45, 51, 52, 56, 58, and 66; 2 lesions contained both low- and high-risk HPVs. The findings were compared with 30 high-grade SILs infected with HPV types 16, 31, 33, 51, 58, 66, and 67; 3 of these were infected with 2 different HPVs. In low-grade lesions, the differentiation markers were expressed normally, showing that differentiation proceeds despite upregulation of cell cycle--associated proteins. Loss of involucrin (3 of 33) and cytokeratin 13 (8 of 33) expression occurred only in the high-grade lesions and was therefore related to lesion grade. Loss of cytokeratin 14 expression was also significantly more frequent in high-grade than in low-grade lesions (19 of 33 v 12 of 51; P < .01). In addition, cytokeratin 14 expression was significantly less frequent in the intermediate and superficial layers of low-grade SILs infected with high-risk HPVs than in those infected with low-risk HPVs (3 of 27 v 14 of 24; P < .001). These findings are consistent with in vitro data and suggest that abnormalities of both cell cycle control and squamous differentiation are important in HPV-associated neoplastic transformation.     

Correlation of T-helper secretory differentiation and types of antigen-presenting cells in squamous intraepithelial lesions of the uterine cervix

This study addressed the notion that the progression of cervical cancer is associated with a T-helper 2 (TH2) immunodeviation by analysing cytokine expression in 60 cervical biopsy specimens, spanning the spectrum from normal cervical tissue to high-grade squamous intraepithelial lesions (SILs). The biopsies were analysed by immunohistochemistry for the expression of TH1 [interleukin-2 (IL2), interferon gamma (IFNγ)] and of TH2-type cytokines (IL4, IL6). Positive cells were usually observed in the subepithelial connective tissue, where most CD4+ cells were also detected. The density of IL2+ cells was significantly lower in high-grade SILs than in normal tissues taken either from the ectocervix or from the transformation zone. In contrast, significantly higher densities of IL4+ cells and, to a lesser degree, IL6+ cells were found in SIL biopsies compared with histologically normal tissues taken from the adjacent ectocervical region. A significantly higher IL4+/CD4+ cell ratio was also found in high-grade SILs (82 per cent) than in normal cervical biopsies taken from the transformation zone of healthy women showing squamous metaplasia (27 per cent). The elevated density of TH2+ cells in SIL biopsies was associated with both the expression of HLA-DR by keratinocytes and a diminished number of intraepithelial Langerhans' cells (CD1a+). In conclusion, the increased TH2+/CD4+ cell ratio in SIL biopsies suggest the presence, during cervical carcinogenesis, of a TH2 immunodeviation that could participate in the immunoescape of preneoplastic cervical keratinocytes. © 1998 John Wiley & Sons, Ltd.     

E-cadherin-dependent adhesion of dendritic and Langerhans cells to keratinocytes is defective in cervical human papillomavirus-associated (pre)neoplastic lesions

Although human papillomavirus (HPV) DNA is detected in the majority of squamous intraepithelial lesions (SILs) and squamous cell carcinomas (SCCs) of the uterine cervix, the persistence and progression of cervical lesions suggest that viral antigens are not adequately presented to the immune system. This hypothesis is reinforced by the observation that most SILs show quantitative and functional alterations of Langerhans cells (LCs). The aim of this study was to determine whether modulation of E-cadherin-mediated homophilic and heterotypic interactions between keratinocytes and LCs is involved in these abnormalities of LCs in (pre)neoplastic cervical epithelium. Cell membrane expression of E-cadherin and the density of CD1a+ LCs were low in the epithelium of SILs and SCC biopsy specimens, compared with normal exocervical epithelium. Dendritic cells (DCs) and LCs generated in vitro were randomly distributed throughout the full thickness of organotypic cultures of E-cadherin- HPV-transformed cells. In contrast, these cells rapidly adhered to the keratinocyte cell layers when HPV-transformed cells transfected with E-cadherin were used. These data suggest that the E-cadherin-mediated contact between keratinocytes and LCs is potentially important for initiating or maintaining the immune response during chronic HPV infection.     

Altered expression of desmosomal components in high-grade squamous intraepithelial lesions of the cervix

We have used immunohistochemistry to test the hypothesis that components of the desmosome are disrupted during neoplastic progression of squamous epithelial cells in the uterine cervix. Sections of normal cervix and squamous intraepithelial lesions (SILs) were immunostained for desmosomal proteins and glycoproteins, and results were assessed using a semi-quantitative grading system. No difference between normal cervix and low-grade SIL (LSIL) was found. A significant reduction in expression of desmogleins was seen between high-grade SIL (HSIL) and LSIL (P<0.01) and normal cervix (P<0.001). Desmocollin expression was not reduced significantly, although scores showed significantly greater variation in HSIL compared with LSIL (P<0.05) and normal cervix (P<0.05). There was no significant difference in desmoplakin expression among the three groups. The results suggest that there may be sequential disruption of desmosomal function during neoplastic progression of cervical squamous intraepithelial cells, with downregulation of desmogleins during the progression from LSIL to HSIL and loss of desmocollin expression occurring in some cases of established HSIL.     

Correlation of the Papanicolaou smear and human papillomavirus type in women with biopsy-proven cervical squamous intraepithelial lesions.

The authors correlated Papanicolaou smear diagnoses with the presence of human papillomavirus (HPV) as determined by in situ hybridization in concurrent biopsy-proven cervical squamous intraepithelial lesions (SILs) in 132 women. Infection by HPV 6 or 11 was associated with a simultaneous normal Papanicolaou smear in 4 of 29 (14%) cases. This result was significantly greater (P less than 0.05) than that found in cases of infection by an oncogenic HPV type (types 16, 31, 33, 35, and others), in which the rate of a concurrent normal Papanicolaou smear was 5 of 88 (5%). Infection by one of these oncogenic types was associated with a Papanicolaou smear diagnostic of SIL in 55 of 88 (63%) cases, whereas infection by HPV 6 or 11 was associated with a Papanicolaou smear diagnostic of SIL significantly (P less than 0.05) less frequently (6 of 29, 18%). It is concluded that, for women with SILs, the likelihood of a Papanicolaou smear diagnostic of the lesion is greater for women with HPV types of known oncogenic potential.     

Increased secretion patterns of interleukin-10 and tumor necrosis factor-alpha in cervical squamous intraepithelial lesions

Cytokines are released in response to infection of the uterine cervix by high-risk HPV. By using enzyme-linked immunosorbent assay, we measured the levels of tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (INF-gamma), interleukin-6 (IL-6), and interleukin-10 (IL-10) in the cervical secretions of 120 cytologically normal or equivocal and 91 abnormal Japanese women. HPV infection of the cervical cells was typed by the LCR-E7 PCR method. The HPV DNA-negative samples were classified as either normal or inflamed, and the HPV DNA-positive samples were classified as HPV positive(+) n-ormal and as low- or high-grade squamous intraepithelial lesions (SILs). Compared with the normal cervices, all of the cytokines tested were elevated in inflamed, HPV+ normal, low-grade SILs (LSIL), and high-grade SILs (HSIL). The level of IL-10 was statistically higher in LSIL, and the level of TNF-alpha was higher in HSIL, relative to the cytokine levels in the inflamed and HPV+ normal samples (P <0.05; Mann-Whitney test). Multivariate analyses confirmed that increased levels of IL-10 were associated with LSIL (relative risk [RR]=3.9, 95% confidence interval [CI]=1.7-8.8) and that increased levels of TNF-alpha (RR=4.6, 95% CI=1.4-15) and age older than 40 years (RR=8.5, 95% CI=1.3-56) were associated with HSIL. The levels of INF-gamma and TNF-alpha (Th1-cytokines) correlated negatively with those of IL-6 and IL-10 (Th2-cytokines) in HPV+ normal and LSIL subjects, whereas no such correlation was observed for HSIL. The up-regulated secretion of IL-10 may inhibit immune responses against HPV infection in early cervical lesions, whereas up-regulated TNF-alpha and uncoordinated cytokine secretion (elevated both Th1 and Th2 cytokines) may reflect impaired or invalid responses in advanced stage lesions. The detection of IL-10 and TNF-alpha in cervical secretions may be a useful indicator of local immune responses and of the stage of the cervical lesions induced by HPV infection.     

HPV typing of cervical squamous lesions by in situ HPV DNA hybridization: Influence of HPV type and therapy on the follow-up of low-grade squamous cervical disease

Papanicolaou (Pap)-stained cervical specimens from 160 squamous lesions were processed for the detection of human papillomavirus (HPV) DNA by an in situ hybridization (ISH) assay. Three biotinylated HPV DNA probes were employed, each containing HPV genotypes 6/11, HPV genotypes 16/18, or HPV genotypes 31/35/51. The HPV etiology of 86 lesions was ascertained (53.8%). In 74 out of 135 (58.8%) HPV-typed low-grade squamous intraepithelial lesions (SILs), HPV 6/11 was found in nine (6.6%), HPV 16/18 in 46 (34.2%), and HPV31/35/51 in 19 lesions (14.1%); in 11 out of 18 HPV-typed high-grade SILs (61.1%), seven lesions (38.9%) were typed for HPV 16/18 and four (22.2%) for HPV 31/35/51. Of seven invasive carcinomas, only one (14.3%) reacted with the HPV 16/18 DNA probe. A cohort of 124 low-grade SILs was followed cytologically for a year. The results of this study are discussed in light of HPV type association and therapy. Diagn Cytopathol 1994; 11:28–32. © 1994 Wiley-Liss, Inc.     

Expression of the 67 kD laminin receptor in human cervical preneoplastic and neoplastic squamous epithelial lesions: an immunohistochemical study

Interactions of cancer cells with laminin play a critical role during the progression of solid malignant tumours. Increased expression of the 67 kD laminin receptor (67LR), one of the several laminin binding proteins, is associated with the invasive and metastatic capacity of various types of cancer, including breast, colon, ovary, lung, and endometrial carcinoma. In this study, 67LR expression was analysed in a series of cervical biopsy specimens including 16 normal cervical tissues, 36 low-grade squamous intraepithelial lesions (SILs), 24 high-grade SILs, and 11 invasive carcinomas. Detection of the 67LR was performed using immunoperoxidase staining and the monoclonal antibody MLuC5 which specifically recognizes the 67LR. Immunostaining of the 67LR was correlated with human papillomavirus (HPV) type detected by in situ hybridization and with proliferative activity of the lesion determined by immunohistochemistry with the MIB-1 monoclonal antibody, specific for the Ki67 antigen. Increased expression of the 67LR was correlated with the histological severity of the lesions, with the strongest immunoreactivity being found in invasive carcinomas. Significant differences in 67LR expression were found between normal cervical epithelium and high-grade SILs (P<0·05, non-parametric Mann-Whitney test) or invasive carcinomas (P<0·001), as well as between low- or high-grade SILs and invasive carcinoma (P<0·01 and P<0·05, respectively). Ki67 antigen expression also increased with the severity of the lesions. There was a positive correlation for each type of lesion between expression of the 67LR and of the Ki67 antigen. No specific relationship was found between 67LR or Ki67 antigen immunostaining and HPV type detected in SILs, segregated into low-grade and high-grade lesions. These data add weight to the evidence that increased expression of the 67LR is a consistent, but not sufficient feature of the invasive and metastatic phenotype and suggest that high expression of the 67LR might be associated with both more proliferative and more aggressive cervical (pre)neoplastic lesions. © 1997 John Wiley & Sons, Ltd.     

Changing Roles of Cadherins and Catenins during Progression of Squamous Intraepithelial Lesions in the Uterine Cervix

Uterine cervix represents a convenient model for the study of the gradual transformation of normal squamous epithelium via low- to high-grade squamous intraepithelial lesions (SILs). Because SIL, on the basis of the cytokeratins expressed, are thought to originate from the reserve cells, we analyzed whether SILs also show a reserve cell phenotype with respect to intercellular interactions. The changes in expression and subcellular localization of the components of the adherens junction and desmosomal complexes were investigated in normal, metaplastic, and premalignant cervical epithelium, as well as in cell cultures derived from these tissues. The results suggest that 1) during progression of SILs, E-cadherin is suppressed, with its role in cell-cell connections diminishing; 2) P-cadherin, in contrast, becomes the predominant cadherin in high-grade SILs; 3) the level of cellular alpha-catenin is dramatically decreased in high-grade SILs; 4) the level of beta-catenin is decreased during progression of SILs, with plakoglobin suggestively becoming the predominant catenin mediating connection of cadherins to the cytoskeleton; 5) the assembly of desmosomes is affected during progression of SILs and is accompanied by a dramatically decreased expression for desmogleins and desmoplakins (I, II); and 6) expression of differentiation markers (involucrin, CK13) in high-grade SILs seems to be controlled by P-cadherin as opposed to E-cadherin in the normal tissue counterpart. We conclude that during development of cervical lesions substantial (both quantitative and qualitative) changes occur in cell-cell junctions, making the interactions of cells in lesions dissimilar from those of reserve cells, basal cells, or cells of immature squamous metaplasia, despite existing morphological similarity between all of these cell types and cells of high-grade lesions.     

Extensively keratinized squamous intraepithelial lesions of the cervix are difficult to grade

We tested the hypothesis that extensively keratinized squamous intraepithelial lesions (SILs) are difficult to grade precisely by identifying 100 Papanicolaou smears with a keratinizing SIL that had been originally judged difficult to grade. Of these, 65 were confirmed as low-grade SIL (LSIL) or high-grade SIL (HSIL) on subsequent biopsy. The 65 smears were reviewed independently by 3 cytopathologists who graded each case as LSIL or HSIL (by Bethesda System criteria). The accuracy of the grade was determined by the subsequent biopsy results; accuracy was compared with that of a historic control group of SILs with biopsy follow-up. In the study group, biopsies showed LSIL in 41 cases and HSIL in 24. The mean accuracy for a smear diagnosis of LSIL was 60% for the study group and 92% for the control group. For a smear diagnosis of HSIL, the accuracy was 60% for the study group and 95% for the control group. The overall kappa value for the study group confirmed poor interobserver agreement. Some keratinizing SILs are difficult if not impossible to grade precisely using standard criteria. For such lesions, the diagnosis "SIL, grade cannot be determined due to extensive keratinization" is justified.     

Increased angiogenesis in the uterine cervix associated with human papillomavirus infection

We studied the relationship between angiogenesis (using the CD34 antibody), the presence of human papilloma virus (HPV) infection, HPV E6 protein expression and the accumulation of p53 protein at various phases of tumour progression in the uterine cervix. Expression of CD34, p53 and HPV E6 protein was evaluated by immunocytochemistry. Presence of the mutant p53 was detected using a mutant specific ELISA, and the type of HPV was determined by the Polymerase Chain Reaction. A total of 230 cervical tissue samples were analyzed and included 40 cases of apparently normal cervical epithelium, 37 low grade squamous intraepithelial lesions (SILs), 43 high grade SILs, 36 well-differentiated squamous cell carcinomas (DSCC), 31 moderately differentiated (MDSCC) and 43 poorly differentiated carcinomas (PDSCC). There was an excellent correlation between the extent of angiogenesis and histological abnormality (r = 0.912, p = 0.000004). The least extent of angiogenesis was seen in normal cervical tissue and low grade SILs where the mean (low power) intra lesional vascular density (ILVD) was 12 +/- 1.13 and 25.66 +/- 5.20, respectively. In high grade squamous intraepithelial lesions (SILs), the mean ILVD value was 80.84 +/- 25.57. In well-differentiated squamous cell carcinomas (WDSCC's) the mean value was 144.22 +/- 28.67 while in moderately differentiated squamous cell carcinomas (MDSCC's) the mean value was 166.29 +/- 34.95 and in poorly differentiated tumours (PDSCC's) 192.42 +/- 27.98. The extent of angiogenesis also correlated to presence of HPV (r = 0.505, p = 0.00001). Increased CD34 expression was associated with the presence of HPV types 16 and 18. A similar correlation was also evident in HPV, 16/18 infected cases expressing the E6 protein (r = 0.612, p = 0.000001). CD34 expression also correlated well with p53 accumulation (r = 0.859, p = 0.000002). Presence of HPV infection significantly correlated with the extent of histological abnormality (r = 0.467, p = 0.00001). Expression of E6 also showed this significant correlation (r = 0.644, p = 0.00002). Accumulation of p53 was significantly more elevated in HPV 16-infected lesions (r = 0.518, p = 0.00001) and E6-expressing cells (r = 0.650, p = 0.000004). Only 12 of the 230 cases analyzed showed presence of the mutant p53 protein. Angiogenesis appears to increase with histological abnormality in the uterine cervix. Angiogenesis also appears to be influenced by high risk HPV infection, the expression of the E6 transforming protein and the p53 tumour suppressor protein.     

Genetic predisposition and parameters of malignant progression in K14-HPV16 transgenic mice.

Reproducible multi-stage progression to invasive squamous carcinoma of the epidermis has been achieved in transgenic mice expressing the HPV16 early-region genes, including the E6/E7 oncogenes, under the control of the human keratin-14 promoter/enhancer. Although 100% of K14-HPV16 transgenic animals develop hyperplastic and/or dysplastic lesions in several inbred backgrounds, including C57BL/6, BALB/c, and SSIN/SENCAR, only mice backcrossed into the FVB/n background progress to malignant squamous cell carcinomas of two pathological grades, well differentiated and moderate/poorly differentiated (WDSC or MPDSC, respectively), each displaying characteristic patterns of malignant behavior. WDSCs typically arise within the epidermis of the ear and invade deeply into the underlying dermis but fail to metastasize, whereas MPDSCs develop on the chest and truncal skin and invariably metastasize to regional lymph nodes. The transition to the malignant state, in 21% of FVB/n transgenic mice, is characterized by alteration of the repertoire of keratin intermediate filament proteins expressed within neoplastic epidermis, such that WDSCs maintain expression of keratins common to terminally differentiating stratified keratinocytes (K10), whereas MPDSCs are distinguished from WDSCs by activation of embryonic and mucosal keratins (K13, K8, and K19). Precursor hyperplastic and dysplastic lesions are characterized by a progressively increased proliferative index, striking morphological alterations in keratinocyte cell-cell and cell-matrix interactions, and extensive remodeling of the underlying dermal stroma. Remarkably, this extensive stromal remodeling, which may facilitate both angiogenesis and eventual tumor cell invasion, develops early at the dysplastic stage in all animals well before malignant conversion.     

Expression of the p16(INK4a) gene product in premalignant and malignant epithelial lesions of the gallbladder

The sequence of molecular changes leading to neoplastic transformation in the gallbladder remains elusive. The aim of this study is to characterize the spectrum of nuclear p16 protein product immunohistochemical expression in tissue microarray cores taken from resected gallbladders, comprising histologically normal gallbladder epithelia (n = 29), dyplastic epithelia (n = 19), reactive atypia (n = 7), and gallbladder adenocarcinoma (n = 23). Nuclear staining for p16 was evaluated for intensity (range, 0-3) and distribution (range, 0-3), and a summary staining score (range, 0-6) was obtained. Pairwise comparisons were significant for increased p16 protein expression in dysplastic epithelium (6/19, 31.6%) and invasive carcinoma (9/23, 39.1%) when compared to normal epithelium (0/29, 0%) (P = .003 and P = .0006, respectively). A quantitative increase of nuclear expression of p16 in reactive atypia (1/7, 14%) when compared to normal was also statistically significant (P = .049). Our data demonstrate that nuclear p16 expression is absent in normal gallbladder epithelium and is a frequent event in high-grade dysplasia of the gallbladder and gallbladder adenocarcinoma.