Telomerase reactivation is an early event in laryngeal carcinogenesis.

The exact role and timing of reactivation of telomerase, a key enzyme implicated in cellular immortalization and transformation in the multistep process of laryngeal carcinogenesis, is still unknown. We attempted to (1) determine that quantitative differences exist in the levels of telomerase catalytic subunit (hTERT) mRNA expression among different grades of laryngeal epithelial abnormalities classified according to the Ljubljana classification; (2) determine that telomerase reactivation is an important, most probably early event in laryngeal carcinogenesis; and (3) analyze whether the relative quantity of hTERT mRNA can be used as a molecular biomarker in the early detection of precancerous lesions. The relative quantity of hTERT mRNA, expressed as an hTERT index, was analyzed in 140 frozen laryngeal tissue specimens representing different morphological stages of laryngeal carcinogenesis by using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. The presence and relative quantity of hTERT mRNA in laryngeal epithelium increases progressively with the degree of epithelial abnormalities. hTERT mRNA was detectable in 1/15 normal laryngeal epithelia (7%, mean hTERT index 0.02), 3/15 simple hyperplasias (20%, mean hTERT index 0.09), 10/27 abnormal hyperplasias (37%, mean hTERT index 0.18), 9/12 atypical hyperplasias (75%, mean hTERT index 0.74), 8/9 intraepithelial carcinomas (89%, mean hTERT index 1.82), and 53/62 invasive laryngeal squamous cell carcinomas (85%, mean hTERT index 2.51). Statistical analysis revealed two groups of laryngeal epithelial changes with significant differences in the levels of hTERT mRNA expression (P <.0033): (1) normal and reactive hyperplastic laryngeal epithelium (simple and abnormal hyperplasia) and (2) atypical hyperplasia (precancerous lesion), intraepithelial and invasive laryngeal squamous cell carcinoma. The results of the present study suggest that telomerase reactivation is an early event in laryngeal carcinogenesis, detectable already at the stage of precancerous laryngeal epithelial changes. Nevertheless, other genetic abnormalities appear to be necessary for progression of these epithelial abnormalities toward invasive laryngeal squamous cell carcinoma.     

Immunohistological comparison of the World Health Organization (WHO) and Ljubljana classifications on the grading of preneoplastic lesions of the larynx

There is still controversy about the classification of laryngeal preneoplastic lesions. In this study, we compared the World Health Organization (WHO) and Ljubljana histological classifications with regard to laryngeal epithelial hyperplastic-dysplastic lesions in a single series to determine their relation to statistically significant criteria when the mechanisms of neoplastic progression are considered. Emphasis was put on p53 expression, proliferative activity, and angiogenesis. Fifty-four laryngeal biopsies with preneoplastic changes were re-evaluated and classified according to both classifications. The streptavidin-biotin method was used for immunohistochemical staining for Ki-67, p53, and CD34 antibodies. A positive correlation was obtained between the histological categories and Ki-67, p53, and CD34 expressions using both classifications (Spearman's Correlation test). There was a significant difference between the histological categories of both the WHO and the Ljubljana classifications, when the expression of the three markers was compared (Kruskal Wallis test, p = 0.000 for each). Further evaluation revealed a statistically significant difference between all categories of both classifications, excluding the p53 overexpression scores and vessel counts in mild and moderate dysplasia categories (Mann-Whitney U-test, p = 0.209, and 0.091 respectively), and the p53 overexpression scores in severe dysplasia and carcinoma in situ categories (Mann-Whitney U-test, p = 0.249) of the WHO classification. Similarly, no significant differences were found between severe dysplasia and carcinoma in situ, as well as between atypical hyperplasia and carcinoma in situ categories using both classifications for the Ki-67 expression (Mann-Whitney U-test, p = 0.806, and 0.111, respectively). Our results suggest that regarding the mechanisms of neoplastic progression such as p53 expression and angiogenesis, the histological categories of the Ljubljana classification seem to depend on additional evidence. Therefore, we support the use of the Ljubljana classification.     

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.     

Changes in retinoblastoma gene expression during cervical cancer progression

The role of tumour suppressor genes in the development of human cancers has been studied extensively. In viral carcinogenesis, the inactivation of suppressor proteins such as retinoblastoma (pRb) and p53, and cellular oncogenes overexpression, such as c-myc, has been the subject of a number of investigations. In uterine-cervix carcinomas, where high-risk human papillomavirus (HPV) plays an important role, pRb and p53 are inactivated by E7 and E6 viral oncoproteins, respectively. However, little is known about the in situ expression of some of these proteins in pre-malignant and malignant cervical tissues. On the other hand, it has also been demonstrated that c-myc is involved in cervical carcinogenesis, and that pRb participates in the control of c-myc gene expression. By using immunostaining techniques, we investigated pRb immunodetection pattern in normal tissues, squamous intraepithelial lesions (SILs) and invasive carcinomas from the uterine cervix. Our data show low pRb detection in both normal cervical tissue and invasive lesions, but a higher expression in SILs. C-Myc protein was observed in most of the cellular nuclei of the invasive lesions, while in SILs was low. These findings indicate a heterogeneous pRb immunostaining during the different stages of cervical carcinogenesis, and suggest that this staining pattern could be a common feature implicated in the pathogenesis of uterine-cervix carcinoma.     

E-cadherin, CD44 and CD44v6 in squamous intraepithelial lesions and invasive carcinomas of the uterine cervix: an immunohistochemical study.

OBJECTIVE: To evaluate the immunoexpression pattern of E-cadherin, CD44std and the variant isoform v6 in normal squamous epithelium, low and high squamous intraepithelial lesions (SILs) and invasive squamous cell carcinomas (ISCCs) of the uterine cervix. The purpose was to determine whether any distinctive change in antigenic expression could contribute to the recognition of the earliest commitment to neoplasia and/or the onset of the invasive phenotype. METHODS: Immunohistochemistry using the avidin-biotin indirect immunoperoxidase method was used to study the protein expression of epithelial cadherin (E-cadherin), cluster differentiation 44 (CD44), and the isoform v6 (CD44v6) in 124 human cervical samples (5 normal, 39 low-grade, 54 high-grade and 26 ISCCS) in formalin-fixed, paraffin-embedded tissue blocks. RESULTS: Membranous expression of E-cadherin, CD44 and CD44v6 was preserved in normal squamous epithelium and in low-grade squamous intraepithelial lesions. A significant association was observed with the histological grade of the SILs and the immunoreactivity (membranous versus cytoplasmic) pattern of E-cadherin (p < 0.001), CD44std (p = 0.027) and CD44v6 (p < 0.001). A loss of membranous staining and a progressive increase in cytoplasmic staining was observed from low to high grade SILs to ISCCs. CONCLUSIONS: Our study demonstrates that during the development of cervical lesions substantial qualitative (subcellular localization-membrane to cytoplasmic) and quantitative alterations (changes in expression) occur in the protein expression of E-cadherin, CD44, and CD44v6 in cervical cancer. The most striking observation was the decrease in membranous immunoreactivity and the progressive increase in cytoplasmic staining of E-cadherin, CD44 and CD44v6, relating to loss of differentiation as a consequence of neoplastic transformation.     

Telomerase catalytic subunit in laryngeal carcinogenesis--an immunohistochemical study.

We recently demonstrated that (1) telomerase catalytic subunit messenger RNA (mRNA) relative quantities increase progressively with the degree of laryngeal epithelial abnormalities and that (2) telomerase catalytic subunit gene re-expression represents an early event in laryngeal carcinogenesis. The aim of the study was to determine whether telomerase catalytic protein immunohistochemisty reflects telomerase catalytic subunit gene expression in different grades of laryngeal epithelial abnormalities and squamous cell carcinomas of the larynx. Telomerase catalytic protein was analysed immunohistochemically in 106 laryngeal epithelial tissue samples: 10 normal epithelia, 15 squamous cell hyperplasias, 14 basal/parabasal cell hyperplasias, 10 atypical hyperplasias, eight intraepithelial carcinomas and 49 squamous cell carcinomas. At least 200 nuclei of each lesion were quantified per slide and the number of positive signals per nucleus was expressed as a telomerase catalytic protein index. The mean telomerase catalytic protein index increased progressively with the degree of laryngeal epithelial abnormalities: from 0.17 in normal epithelia, 0.44 in squamous cell hyperplasia, 0.54 in basal/parabasal cell hyperplasia, 0.91 in atypical hyperplasia, 1.05 in intraepithelial carcinoma to 0.96 in squamous cell carcinomas. Statistical analysis revealed three different groups of laryngeal epithelial changes according to the number of telomerase catalytic protein signals per nucleus: (1) normal epithelium, (2) regenerative epithelium (squamous cell hyperplasia, basal/parabasal cell hyperplasia), and (3) atypical hyperplasia, intraepithelial carcinoma and squamous cell carcinoma (P<0.0033). Telomerase catalytic protein immunohistochemistry parallels well with telomerase catalytic subunit mRNA relative quantities in laryngeal carcinogenesis. In normal and regenerative laryngeal epithelia, telomerase catalytic protein is present in occasional basal/parabasal nuclei, becomes undetectable with maturation or differentiation of epithelial cells, and reflects the regenerative capacity of squamous epithelium. Nevertheless, several telomerase catalytic protein signals in the majority of nuclei in precancerous lesions, intraepithelial carcinomas and squamous cell carcinomas, are consistent with telomerase catalytic subunit gene re-expression, an early event in laryngeal carcinogenesis.     

p53 Immunoreactivity in inflammatory and neoplastic diseases of the uterine cervix

Immunoreactivity for the tumour suppressor gene product p53 is commonly found in many different human malignancies and few premalignant lesions. Data on cervical neoplasms, however, are still lacking. We retrospectively investigated p53 immunoreactivity in 92 lesions of the uterine cervix, including 44 cases of chronic cervicitis, 29 squamous intraepithelial lesions (SILs), and 19 invasive carcinomas. p53 immunoreactivity confined to the basal cell layer, was detected in 74 per cent of cases showing chronic cervicitis and in all cases with low-grade SILs. Conversely, suprabasal and/or diffuse p53 immunoreactivity was exclusively demonstrated in 25 per cent of high-grade SILs and in 74 per cent of invasive carcinomas. The results of this investigation document a high prevalence of p53-immunoreactive malignant tumours of the uterine cervix. In high-grade SILs, p53-immunoreactive cells paralleled the height of involvement by dysplastic changes within the squamous epithelium. A prolonged half-life of the protein is the most likely explanation for the occurrence of p53 immunoreactivity in neoplastic cells. The unexpected finding of p53-immunoreactive cells in inflammatory lesions, though possibly related to an increased proliferation rate of the basal cell compartment, requires further study and underlines the need for a careful approach to p53 immunocytochemistry.     

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.     

Criteria for grading in the Ljubljana classification of epithelial hyperplastic laryngeal lesions. A study by members of the Working Group on Epithelial Hyperplastic Laryngeal Lesions of the European Society of Pathology

AIMS: To validate histological criteria for the grading of epithelial hyperplastic laryngeal lesions (EHHL) (dysplastic laryngeal lesions), we used a system that had been devised and tested in Ljubljana, Slovenia over many years and was felt to be more appropriate to laryngeal pathology than is the commonly-used model of intraepithelial neoplasia in the cervix. METHODS AND RESULTS: Vocal cord biopsies of 45 patients with a broad spectrum of EHLL were reviewed. Detailed histological criteria were formulated for each of the four grades of EHLL in the Ljubljana classification, comprising simple hyperplasia (benign spinous layer augmentation), abnormal hyperplasia (benign basal and parabasal layer augmentation), atypical hyperplasia (risky for malignancy) and carcinoma in situ (actually malignant, but without invasion). CONCLUSIONS: Using these criteria a high degree of concordance of histological diagnoses of grading levels for the Ljubljana classification was achieved between the pathologists of the Working Group. The system was found to be more precise for routine diagnostic work than the others in vogue. The different grades of the Ljubljana classification correspond to significantly different levels yielded in each grade by the semiobjective methods of quantitative morphometry and immunohistochemistry.     

Molecular markers of early cervical neoplasia

Pure morphological distinction of high-grade squamous intraepithelial lesions (HSILs) from their mimics can be challenging. Diagnosis can be difficult with nonconventional HSILs associated with a metaplastic phenotype, squamous intraepithelial lesions (SILs) that defy precise classification such as “eosinophilic dysplasias”, and those that overlap with columnar neoplasms, including stratified variants of adenocarcinoma in situ (“SMILE”). Gene expression and protein profiling have identified biomarkers with the potential to decrease diagnostic variability and increase specificity of histological and cytological analysis. Among the ones clinically useful for HSIL detection are p16^INK4A and MIB-1 which complement each other, differentiating SIL from normal/atrophic (MIB-1 low) or reactive/immature metaplastic (p16^INK4A scattered) epithelium. Additional markers, including ProEx™ C, have been proposed but their added value is yet to be established. In the final analysis, biomarkers are most helpful for distinguishing benign immature or atrophic proliferations from HSIL. The distinction of LSIL from HSIL must be made on the haematoxylin and eosin-stained section and should be made with care, given the potential consequences of a diagnosis of CIN2 or CIN3.     

Cervical cancer precursors: cytohistologic correlation with the results of HPV testing

Persistence of high-risk types of human papillomavirus (HPV) is responsible for the development, maintenance and progression of squamous intraepithelial lesions (SILs). Cytohistologic correlation with the results of the HPV testing in 52 patients over a 3year period is presented. Two patients out of the 52 patients presented in this histologic follow up bore the diagnosis high-grade squamous intraepithelial lesion (HSIL) with the former cytology ASC-H. Low grade squamous intraepithelial lesions (LSIL) were found in eight patients, half of them diagnosed identically on cytology. Another four cases were formerly diagnosed cytologically as ASC-US. All women with the histologically confirmed dysplastic changes were HR HPV DNA positive. Our results indicate that significant histologic lesions may be discovered in patients exhibiting the high-risk HPV DNA positivity in the category of ASC-US (Atypical Squamous Cells of Undetermined Significance) and especially ASC-H (ASC cannot exclude high-grade squamous intraepithelial lesion). A combined screening test thus offers the possibility of greater protection and /or longer screening intervals.     

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.     

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.     

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.     

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.     

Noncorrelating Pap tests and cervical biopsies: histological predictors of subsequent correlation

Lack of correlation between dysplastic cervicovaginal Papanicolaou (Pap) tests and subsequent cervical biopsies raises the concern that a significant squamous intraepithelial lesion (SIL) may go unconfirmed. Additional tissue sections of cervical biopsies may detect SILs after noncorrelation on initial sections. Complete step sectioning of paraffin blocks was undertaken on 111 noncorrelating biopsy specimens from 95 patients and selected slides were reviewed for the presence of SIL. The initial negative biopsy slides were evaluated for four histological features: chronic cervicitis, acute cervicitis, mucosal erosion, and squamous atypia. Twenty-seven biopsies (24.3%) demonstrated the presence of a SIL in deeper levels. The presence of squamous atypia was significantly associated with the presence of dysplasia deeper in the block (P < 0.002). Acute and chronic cervicitis was seen roughly equally. Additional tissue levels are a productive way of confirming SILs, and squamous atypia allows a refined selection of negative cervical biopsies most likely to reveal an SIL on review of deeper levels.     

Human telomerase catalytic subunit gene re-expression is an early event in oral carcinogenesis

AIMS: Detection of telomerase catalytic subunit (hTERT) mRNA has been used as a surrogate marker for estimation of telomerase activity. The exact role and timing of telomerase re-activation, a key enzyme implicated in cellular immortalization and transformation, in the multistep process of oral carcinogenesis is still unknown. The aim was to test the hypothesis that (i) quantitative rather than qualitative differences exist in the level of hTERT mRNA expression between normal oral mucosa, different grades of oral epithelial abnormalities and squamous cell carcinomas of the oral cavity, and that (ii) hTERT gene re-expression is an important, probably early event in oral carcinogenesis. METHODS AND RESULTS: The relative quantity of hTERT mRNA was analysed in 45 frozen oral epithelia representing different morphological stages of oral carcinogenesis classified according to the Ljubljana classification and in 37 oral squamous cell carcinomas, using a commercially available LightCycler Telo TAGGG hTERT Quantification kit. hTERT mRNA was not detected in normal or reactive hyperplastic oral epithelia, but was present in 43% of atypical hyperplasias (premalignant lesions), 60% of intraepithelial carcinomas and 68% of oral squamous cell carcinomas. Statistical analysis revealed two groups of oral epithelial changes, with significant differences in the levels of hTERT mRNA expression: 1, normal and reactive hyperplastic oral epithelium, and 2, atypical hyperplasia, intraepithelial carcinomas and squamous cell carcinomas. CONCLUSION: These data suggest that hTERT gene re-expression represents an early event in the multistep process of oral carcinogenesis, already detectable at the stage of precancerous oral epithelial changes. Nevertheless, other genetic aberrations appear to be necessary for progression of oral epithelial abnormalities towards invasive squamous cell carcinoma.     

The Ljubljana classification: a practical strategy for the diagnosis of laryngeal precancerous lesions

There is no internationally accepted classification of epithelial hyperplastic laryngeal lesions (EHLL). The majority of current classifications follow criteria similar to those commonly used for cervical epithelial lesions. However, the different etiology of laryngeal cancer and its particular clinical and histologic features necessitate a grading system more appropriate to this region. The Ljubljana classification of EHLL was devised in 1971 to cater to this requirement. Detailed criteria for histologic grading in this classification were formulated by a working group on EHLL of the European Society of Pathology in 1999. The system recognizes four grades: simple and abnormal hyperplasia are benign categories; atypical hyperplasia ("risky" epithelium) is potentially malignant, and carcinoma in situ actually malignant. The main features by which the proposed grading system differs from other classifications are: 1. the distinction between benign and potentially malignant lesions; 2. the positive separation of carcinoma in situ from atypical hyperplasia; 3. the lack of prognostic significance for any surface keratin layer. The eventual outcome of EHLL patients so graded justifies the proposal for separating the lesions into a benign group, showing malignant transformation in only 0.9% of cases, from a potentially malignant group showing malignant transformation in 11% of cases. For diagnostically difficult cases, supplementary techniques such as those using morphometry, immunohistochemical and molecular biology are advised to improve the accuracy of diagnosis and predictions of their biological behavior.     

Review of precancerous vulvar lesions

Classification of squamous vulvar precancerous lesions is based on the concept of vulvar intraepithelial neoplasia (VIN) and incorporates a three grade evaluation of the intensity of dysplastic changes (VIN I, II and III). On the basis of histological features, VIN has been subdivided into the usual VIN (u-VIN) and differentiated VIN (d-VIN), which represent the two basic pathways of the pathogenesis of vulvar squamous cell carcinoma. Although u-VIN is etiologically associated with the human papillomavirus (HPV) infection and histologically corresponds to cervical intraepithelial neoplasia, d-VIN represents the HPV-negative sequence of vulvar carcinogenesis, which is linked to lichen sclerosus (LS) and lichen simplex chronicus (LSC). u-VIN preferentially occurs in relatively young women with a history of cervical, vaginal or vulvar premalignant lesions. On the other hand, d-VIN usually affects postmenopausal women without anamnestic data of other dysplastic lesions of the lower female genital tract. d-VIN is characterized by a higher tendency of stromal invasion than u-VIN and its malignant potential is analogous to carcinoma in situ (VIN III). The histological appearance of d-VIN is subtle with basal atypia and a well-preserved differentiation of the superficial parts of the squamous epithelium, therefore it is frequently misdiagnosed for u-VIN I, LS or LSC in vulvar biopsies. Primarily because of the low diagnostic reproducibility of the u-VIN I category and the doubts about its precancerous potential as well as due to the questionable differentiation between u-VIN II and III, a revised VIN classification was proposed in 2004. The grading of vulvar precancerous lesions was abandoned, the u-VIN I category was discontinued and u-VIN II and III were merged. In the revised terminology, the term u-VIN represents HPV-associated high grade precancerous vulvar lesions (formerly u-VIN II and III) and d-VIN encompasses HPV-negative high grade dysplasias. Keywords: vulvar intraepithelial neoplasia - VIN of the usual type - VIN of the differentiated type - lichen sclerosus - lichen simplex chronicus - HPV.     

Differentiated dysplasia is a frequent precursor or associated lesion in invasive squamous cell carcinoma of the oral cavity and pharynx

The source of precursor lesions of squamous cell carcinoma (SCC) of the oral cavity and pharynx, their classification, and grading are controversial. In contrast, vulvar and penile cancer precursor lesions are known to be related to human papillomavirus or chronic inflammation and can be described using the vulvar intraepithelial neoplasia (VIN) classification system (VIN 1–3) or as differentiated vulvar intraepithelial neoplasia (dVIN), respectively. Oral and pharyngeal SCC precursor lesions are more etiologically diverse, and the spectrum of lesions may thus be wider. No international consensus exists regarding the histological types of precursor lesions or the significance of individual types. We therefore reviewed resection specimens and preceding biopsies of 155 patients with SCC of the oral cavity and pharynx (excluding tonsils) and identified five basic patterns of SCC-associated or precursor lesions: (1) pleomorphic (22/155), (2) basaloid (5/155), (3) differentiated (63/155), (4) mixed (42/155), and (5) verrucous (12/155). Keratinization was a common but variable feature in differentiated, mixed, and verrucous dysplasia. In 11/155 patients, no precursor lesion could be identified. Progression of isolated differentiated dysplasia (ranging from months to years) was documented in 13/155 (8 %) of patients. Our data suggest that full-thickness epithelial dysplasia of pleomorphic or basaloid type is present in <20 % of oral and pharyngeal SCC, and differentiated dysplasia is a frequent precursor or associated in situ lesion. Failure to recognize differentiated dysplasia results in the underdiagnosis of many patients at risk for invasive carcinoma. These results indicate a need to refine criteria to distinguish differentiated dysplasia from morphologically related lichenoid lesions.