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.     

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.     

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.     

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.     

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.     

Expression of CD34, alpha-smooth muscle actin, and transforming growth factor beta1 in squamous intraepithelial lesions and squamous cell carcinoma of the larynx and hypopharynx

AIM OF THE STUDY: There is increasing evidence that stromal reaction in cancer has an important diagnostic and prognostic significance. Recent studies have shown that CD34-positive stromal cells and myofibroblasts may play an important role in host response to invasive cancer. The aim of our study was to analyze the expression of CD34, alpha-smooth muscle actin (SMA), and transforming growth factor beta1 (TGFbeta1) in squamous intraepithelial lesions (SILs) and squamous cell carcinoma (SCC) of the larynx and hypopharynx, to establish their significance, and to elucidate the mechanism of myofibroblast formation. METHODS: Immunohistochemistry was performed on samples of 42 resected larynges and 12 laryngeal biopsies of SILs and SCC using antibodies against SMA, CD34, CD31, TGFbeta1, and TGFbeta1 receptors. The expression of TGFbeta1 mRNA was detected with RNA in situ hybridization using specific oligonucleotides for TGFbeta1. RESULTS: The stroma in normal laryngeal mucosa and SILs contained scattered CD34-positive cells, but there were no SMA-positive myofibroblasts. In contrast, the stroma of SCC contained SMA-positive myofibroblasts, but there were no CD34-positive stromal cells. This pattern of stromal reaction was also observed in the peritumoral zone. In adjacent normal tissue, there were CD34-positive stromal cells and no myofibroblasts. We found more intense TGFbeta1 expression in carcinoma cells than in the normal laryngeal epithelium and positive staining for both TGFbeta1 receptors on stromal cells of the normal mucosa. In SCC, many myofibroblasts expressed TGFbeta1 and both receptors for TGFbeta1. Expression of TGFbeta1 mRNA was similar to expression of TGFbeta1 protein. CONCLUSION: Our study shows that disappearance of CD34-positive stromal cells and appearance of SMA-positive stromal myofibroblasts are associated with transformation of laryngeal SILs to SCC. This pattern of stromal reaction was found not only in the tumor but also in the peritumoral zone, defined as a band of host tissue between the invasive tumor front and adjacent normal tissue. Our findings also support the suggestion that overproduced TGFbeta1 in carcinoma cells mediates one of the mechanisms of transformation of stromal cells to myofibroblasts in laryngeal carcinogenesis.     

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.     

Immunoreactive alpha interferon in cervical flat koilocytic lesions and intraepithelial neoplasia.

Immunocytochemical staining for alpha-interferon was carried out on cervical biopsy specimens showing non-condylomatous koilocytic atypia (n = 12) and cervical intraepithelial neoplasia (n = 18), both of which are associated with human papilloma virus (HPV) infection. Normal cervical tissue obtained from hysterectomy specimens was also assessed. Koilocytes were not immunoreactive for alpha interferon and keratinocyte staining was observed in only four cases of intraepithelial neoplasia. HPV infection alone does not therefore seem to induce the production of alpha interferon in cervical squamous epithelium. There was variable but, in some cases, prominent staining of cells in the stromal inflammatory infiltrate as well as intraepithelial cells which had morphological and immunocytochemical characteristics of Langerhans' cells. Alpha interferon immunoreactivity in Langerhans' cells is in keeping with derivation from the mononuclear phagocyte system and may be important in the host response to HPV infection.     

Cytologic correlates of papillary immature metaplasia (immature condyloma) of the cervix

Papillary immature metaplasia (PIM) of the cervix (immature condyloma) is a variant of low-grade squamous intraepithelial lesions (LSIL). It is frequently associated with human papillomavirus (HPV) types 6 and 11. The purpose of this study was to characterize the cytologic changes associated with this lesion. We analyzed 10 cases of PIM from our files and reviewed the Papanicolaou smears taken proximate to the time of the biopsy. Four cases had either reactive epithelial changes (2 cases) or cytologic findings typical of low-grade SIL, with koilocytosis (2 cases). Six cases displayed a spectrum of metaplastic cells with varying maturation that ranged from atypical reactive cells to atypical immature metaplastic cells. Binucleation was common. Some cells exhibited features characteristic of SIL, although the degree of nuclear atypia generally was less than that associated with high-grade SIL. Papanicolaou smears from all cases were interpreted as atypical (ASCUS) metaplasia or low-grade SIL. Follow-up biopsy in one case revealed a PIM in association with a high-grade SIL, the latter undiagnosed by smear alone. PIM is a distinct histologic entity that can present with a spectrum of cytologic findings. Its recognition histologically can resolve some cytologic/histologic discrepancies. Confusion with an immature HSIL or atypical immature metaplasia can occur in some instances and the diagnosis of PIM by cytology alone is not recommended, unless the diagnosis is qualified. Diagn. Cytopathol. 1998;18:416–421. © 1998 Wiley-Liss, Inc.     

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.     

Interobserver variability in human papillomavirus test results in cervicovaginal cytologic specimens interpreted as atypical squamous cells

We studied interobserver variability in the proportions of human papillomavirus (HPV)-positive results for atypical squamous cells of undetermined significance (ASCUS) and atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) diagnoses among 5 pathologists from the me laboratory during a 2-year period. These proportions were compared with individual pathologist's ASCUS/squamous intraepithelial lesion (SIL) ratios. Of 1,299 ASCUS diagnoses, 32.3% had HPV testing; 49.4% were HPV+. Positive findings by individual pathologists ranged from 38% to 67% (P = .057). There was a difference in the proportions of high risk HPV results for individual pathologists (P < .001). For the pathologist who diagnosed 38% (23/61) of samples as HPV+, the ASCUS/SIL was 0.58; the pathologist who diagnosed 67% (28/42) as HPV+ had a ratio of 1.02. Of the ASC-H diagnoses, 32.9% were tested for HPV; 63% (46/73) were positive. Although the HPV+ proportion by pathologist ranged from 54% to 83%, no significant differences were identified. Within the me laboratory, interobserver variability exists in the proportions of HPV positivity for ASCUS and ASC-H interpretations.     

Human papillomavirus types and recurrent cervical warts in immunocompromised women.

Little is known about the host's immunological response to human papillomavirus (HPV) infection, which is an important co-factor in genital tract cancers. This study analyzed the HPV type in pre- and posttreatment tissues in immunocompromised women with cervical squamous intraepithelial lesions (SILs) that recurred after ablative therapy. The HPV type was the same in the pre- and posttreatment lesion in nine of 12 recurrent SILs in women immunocompromised because of AIDS or long-term immunosuppressive therapy. In an extension of a previous study, it was noted that a different HPV type was present in the posttreatment SIL when compared to the pretreatment lesion in 13 of 14 cases in immunocompetent women. These observations support the hypothesis that a type-specific resistance may develop after ablative treatment, preventing re-infection by the same HPV type in immunocompetent women.     

Current review on squamous intraepithelial lesions of the larynx

Squamous intraepithelial lesions (SILs) of the larynx, clinically usually defined as leukoplakia and chronic laryngitis, have remained the main controversial topic in laryngeal pathology for decades as regards classification, histological diagnosis and treatment. SILs are caused by smoking and alcohol abuse. There is also mounting evidence that gastroesophageal reflux is a potential aetiological factor. Human papillomavirus infection seems to play little if any role in laryngeal carcinogenesis.
Histological classification of SILs is the central disputed aspect of these lesions. There are as yet no generally accepted criteria for histological grading of laryngeal SILs. Three currently used classifications of SILs are reviewed here: the dysplasia system, the Ljubljana classification and the binary system of squamous intraepithelial neoplasia. One of the most important issues of SILs is the risk of malignant transformation. Data in the literature are controversial because of inconsistent use of morphological criteria in different classifications. It is often difficult for clinicians to agree on the most appropriate therapeutic option for a particular grade of SIL that has been diagnosed. Transition from normal epithelium to SILs and squamous cell carcinoma is related to progressive accumulation of genetic changes leading to a clonal population of transformed epithelial cells. Despite extensive research into these genetic changes in laryngeal carcinogenesis, reliable genetic markers with diagnostic and prognostic value are still lacking.     

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.     

Squamous intraepithelial lesions in cervical smears of human immunodeficiency virus-seropositive adolescents

Various authors have reported a high rate of human papillomavirus (HPV) infection and HPV-related neoplasias in human immunodeficiency virus (HIV)-seropositive women. On the other hand, young women are most susceptible to cervical infection because of immaturity of the cervix, as it appears that HPV has more access to the basal cells of the differentiating epithelium. The purpose of the present work was to study cervical smears of 82 adolescent HIV-seropositive women (13–21 years of age) to search for cytological evidence of cervical intraepithelial neoplasias. Twenty-one cases showed characteristic features of HPV infection and squamous intraepithelial lesions (SIL; 25.6%). Sixteen cases aged from 17 to 21 years (mean age 19.5 years) had low-grade SIL (LSIL; 19.5%) and five cases aged from 18 to 21 years (mean age 20.2 years) had high-grade SIL (HSIL; 6.1%). There was no significant difference between the mean age of patients with LSIL and HSIL. Two cases had atypical squamous cells of undetermined significance (ASCUS). In the present work it was found that HIV-seropositive adolescents have a high risk for preneoplastic lesions of the cervix (25.6%) as well as a high incidence of more aggressive lesions (6.1% of HSIL) when compared to the general population of adolescents. As it can be assumed that, if the age of acquisition of the infection in both groups (in the general population and HIV-seropositive women) is the same, it is probable that HIV infection in adolescents not only increases the frequency of HPV infections but also facilitates the evolution to more aggressive preneoplastic lesions of the cervix due to HPV. Diagn. Cytopathol. 1998;18:91–92. © 1998 Wiley-Liss, Inc.     

The value of duplicate slides on atypical squamous cells, cannot exclude high-grade intraepithelial lesion

Atypical squamous cells, cannot exclude high-grade squamous intraepithelial lesion (ASC-H) category was added to the 2001 Bethesda System. ASC-H accounts for a small percentage (0.2-0.6%) of abnormal Pap smears and includes heterogenous group of lesions. There are more high-grade cervical lesions (30-50%) in ASC-H than ASC-US (10-15%). An accurate Pap diagnosis is crucial for appropriate patient follow-up and treatment. A total of 43 consecutive ASC-H cases were collected from October 2007 to March 2008, and all duplicate and the original slides were reviewed blindly at the end of the study. On review of the duplicate Pap slides, 18 cases had diagnostic SIL cells (15 HSIL, 2 LSIL with ASC-H, and 1 LSIL). The duplicate slides could have potentially changed 18 (41.9%) ASC-H diagnoses to a more definitive SIL diagnosis. On review of the original Pap slides, 8 of these 18 cases also had HSIL cells. Twenty-one follow-up cervical biopsies (21/43, 48.8%) showed 12 CIN 2/3, 4 CIN 1, 1 VAIN 1, 2 cervical polyps, 1 negative for dysplasia, and 1 insufficient for diagnosis. The CIN 2/3 rate was 57.1% (12/21) based on the original ASC-H Pap diagnosis. The CIN 2/3 rates were 80% (8/10) with SIL cells on duplicate slides and 36.4% (4/11) without SIL cases on duplicate slides. Our study suggested that duplicate slides were very useful for further classification of ASC-H, but other ancillary tests might be necessary for some cases. We propose a systematic approach using combined duplicate slides and reflex HPV testing to further classify ASC-H.     

Immunohistochemical assessment of p16, COX-2 and EGFR in HPV-positive cervical squamous intraepithelial lesions

The protein capsid L1 of the human papilloma virus (HPV) - a key factor in the cervical carcinogenesis - is considered, together with p16, EGFR and COX-2, a characteristic marker for the evaluation of the malignancy progression and prognostic, in terms of tumoral aggressiveness. The purpose of the present study was to make a comparative assessment between the immunohistochemical pattern of p16, EGFR and COX-2 and immunochemical expression of L1 HPV capsid protein, in low grade and high-grade cervical squamous intraepithelial lesions, in order to determine the relationship of these tumoral markers with the infection status of HPV, and their practical applicability in patients diagnosis and follow-up. The study group included 50 women with cytological and histopathological confirmed LSIL (low grade SIL) and HSIL (high-grade SIL). The immunoexpression of L1 HPV protein was assessed on conventional cervico-vaginal smears and EGFR, COX-2 and p16 were immunohistochemically evaluated on the corresponding cervical biopsies. From all cervical smears, the HPV L1 capsid protein was expressed in 52% of LSIL and 23% of HSIL. From all cervical biopsies, p16 was positive in 64% of LSIL, 82% of CIN2 and 100% of CIN3, EGFR was overexpressed in 67% of HSIL (56% CIN2 and 43% CIN3) and 32% LSIL. For COX-2, the Allred score was higher in HSIL when compared to LSIL. Our data revealed 33 cases belonging to both LSIL and HSIL categories with the same Allred score. Immunochemical detection of L1 capsid protein, on cervico-vaginal smears, indicates an immune status induced by the HPV infection and may offer prognosis information, mainly in LSIL lesions. The assessment of p16, EGFR, and COX-2 allows to an integrative approach for the progression of squamous intraepithelial lesion, associated or not with the HPV infection.