Expression of tenascin and fibronectin in benign epithelial hyperplastic lesions and squamous carcinoma of the larynx.

BACKGROUND: Tenascin (T) and fibronectin (FN) are glycoprotein components of the extracellular matrix presumably involved in cancer progression. We analyzed their expression in epithelial hyperplastic lesions (EHL) and squamous carcinoma (SC) of the larynx. MATERIALS AND METHODS: Samples from resected larynges of 30 patients with SC, and laryngeal biopsies of 28 patients with EHL, SC or benign reactive conditions were included. Immunohistochemistry was performed with antibodies against T and FN. RESULTS: T and FN gradually increased with the grade of EHL and were markedly increased in the majority of SC. In SC, expression of T and FN correlated with the degree of desmoplasia but was inversely related to the density of lymphocytic stromal infiltration and the differentiation of SC. T and FN were also positive in benign reactive conditions. CONCLUSION: T and FN immunostaining provides useful information on epithelial-stromal interaction in laryngeal EHL and SC but should not be regarded as a reliable stromal marker of malignancy. Our results supported the postulated diversified nature of the tumor stroma.

Clinical applicability of the Ljubljana classification of epithelial hyperplastic laryngeal lesions.

The diagnosis, prognosis, and choice of treatment of various laryngeal lesions depends almost entirely on the interpretation of changes in the covering epithelium. These abnormalities, referred to as epithelial hyperplastic laryngeal lesions, have been graded according to the Ljubljana classification into simple, abnormal and atypical (risky epithelium) hyperplasia and carcinoma in situ. The aim of this study was to evaluate the clinical applicability and prognostic value of this classification and to determine the incidence of malignant transformation. A retrospective clinical-pathological analysis was performed in a series of 4167 patients with 4574 biopsies, treated from 1979 to 1994. Simple (benign prickle cell) hyperplasia was the predominant grade in nodules, polyps, Reinke's oedema, granulomas, and papillomas, accounting for 37.6-68.6% of cases. In chronic laryngitis, abnormal (benign basal cell) hyperplasia was predominant with 43.9% of cases. Atypical ('risky') hyperplasia was observed almost exclusively in patients with chronic laryngitis (16.1%) and papillomas (10.1%), and only exceptionally in patients with vocal cord nodules (0.9%) and Reinke's oedema (0.3%). The percentage of malignant transformation in atypical hyperplasia was 11.6% (13/112 patients in 2-12 years), while in simple and abnormal hyperplasia, it was 0.3% (8/2920 patients in 1.5-11 years). The data support the concept of the Ljubljana classification dividing epithelial hyperplastic laryngeal lesions into benign (simple and abnormal hyperplasia), potentially malignant (atypical hyperplasia) lesions and carcinoma in situ.

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.

Chromosomes 7,17 polysomies and overexpression of epidermal growth factor receptor and p53 protein in epithelial hyperplastic laryngeal lesions.

PURPOSE: To visualize directly a sequence of genetic changes underlying the entire spectrum of epithelial hyperplastic laryngeal lesions (EHLL) and laryngeal cancer by the use of non-isotopic in situ hybridization (ISH) for chromosomes 7 and 17 in correlation with overexpression of p53 protein and epidermal growth factor receptor (EGFR). The specific aim was to compare the results and prognostic significance between the two types of EHLL: isolated, mainly atypical hyperplasia or risky epithelium, and EHLL associated with squamous cell carcinoma (SCC). PATIENTS AND METHODS: 59 tissue specimens of EHLL obtained from 34 patients, graded according to the Ljubljana classification into simple (SH), abnormal (AbH) and atypical hyperplasia (AtH), and carcinoma in situ (CIS) were included in the study. Non-fluorescent ISH for chromosomes 7 and 17 was performed by biotinylated alpha-satellite DNA probes. Immunohistochemical staining for EGFR and p53 protein was analyzed on the same tissue samples. RESULTS: Polysomy for both chromosomes increased in correlation with progressive grades of EHLL. The most important finding was the statistically significant difference in chromosome copy numbers between the isolated AtH and AtH associated with SCC. Overexpression of EGFR and p53 protein was found in 61 (36/59) and 52% (31/59) of cases, respectively. The immunoreactivity for both markers increased with the grade of lesions, but the staining pattern was not so uniform in isolated EHLL. On the other hand, the immunoreactivity was expressed more constantly in EHLL adjacent to SCC. CONCLUSIONS: Numerical changes in chromosomes 7 and 17 might be associated with an upregulation of EGFR and p53 genes, and could contribute to critical events in laryngeal carcinogenesis. For daily practice, the cytogenetic and immunohistochemical analyses could be of assistance in distinguishing between low- and high-risk groups of AtH. However, the isolated forms of atypical hyperplasia need considerable further study by evaluating genetic changes with the described methods regarding their ultimate transformation to carcinoma.

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.

p53 protein overexpression in laryngeal squamous cell papillomas.

We retrospectively investigated p53 protein immunoreactivity in 103 laryngeal squamous cell papillomas (LP) previously revealed to be human papillomavirus type 6 or 11 positive by in situ hybridization and/or the polymerase chain reaction. 21 LP failed to show any detectable level of p53 protein reactivity. In 45 cases only occasional strongly positive cells were observed in almost the whole thickness of the epithelium. In 26 LP, p53 protein immunoreactivity was found to be almost exclusively restricted to the basal epithelial cells. Finally, in 11 cases, basal cell layer immunoreactivity was accompanied by aggregates of p53-positive cells in the lower two thirds of the epithelium. This staining pattern was found predominantly in LP with atypical hyperplasia. We think that the observed patterns of p53 immunoreactivity in a majority of cases are a result of immunohistochemical detection of the stabilized wild-type p53 protein rather than the mutated p53 protein.

Epithelial hyperplastic lesions--a challenging topic in laryngology.

Adequate diagnosis, treatment and prognosis of particular pathologic entities of the laryngeal mucosa depend entirely on the histological changes of the epithelium. The basis for the classification of epithelial hyperplastic lesions of the larynx (EHLL) is the progression of the histologic features of these lesions to cancer. Considering various criteria thought to be typical for the transformation of benign EHLL to carcinoma, they are most frequently classified into three different groups. However, difficulties begin with the lack of uniformity and inconsistency of terminology, and the fact that the histomorphological features and biological behavior are not always in accordance. It is emphasized that the surface keratosis in laryngeal pathology has no prognostic significance per se and that the term risky epithelium should replace the expression precancerosis. This term does not predict the evolution of the lesion in any way, and it does not exclude any possible future development; but it warns the laryngologist to exercise extreme caution and to follow the patient closely. Present knowledge of laryngeal cancer, its therapeutic options and consequences, force us to pay increasing attention to early detection and adequate treatment of EHLL.

Quantitative pathology of laryngeal epithelial hyperplastic lesions.

We studied 40 laryngeal biopsies samples in order to ascertain the reliability of light microscopical criteria for subdividing laryngeal epithelial hyperplastic lesions (EHL) and carcinoma in situ as well as to determine the relationship between proliferative activity of their epithelial cells and the histological grade. The biopsies were divided into four groups in accordance with the Kambic-Lenart classification: simple, abnormal and atypical hyperplasia and carcinoma in situ. 10 cases in each group were included. The morphometrical analysis was carried out by a semiautomatic image analysis system. The proliferative activity was determined by the high percentage of cell nuclear antigen (PCNA) and Ki-67 positive epithelial cells and with counting nucleolar organizer regions (Ag-NORs) per nucleus. Our results suggest that measuring the nuclear area of the basal cells. augmented with basaloid cells and carcinomatous cells, is the most useful morphometrical method of differentiating three types of laryngeal EHL and carcinoma in situ, while the proliferative activity progressively increased with the degree of epithelial hyperplasia. Morphometrical methods and proliferative activity should be regarded as useful in conjunction with the traditional histopathological methods allowing more of objective grading of EHL.

Human papillomaviruses: a study of their prevalence in the epithelial hyperplastic lesions of the larynx.

This study investigated the prevalence of human papillomavirus (HPV) infection in various laryngeal epithelial hyperplastic lesions using the Kambic classification from simple hyperplasia to invasive squamous cell carcinoma. For detection of HPV infection polymerase chain reaction (PCR) with 3 different HPV consensus primer sets and in situ hybridization were used. The presence of the HPV DNA was detected in only 2 of the 88 specimens tested: HPV type 6 was detected in 1 case of simple hyperplasia and HPV type 16 in 1 case of invasive squamous cell carcinoma. In conclusion, our study suggests that most laryngeal epithelial hyperplastic lesions are not associated with HPV infection and that other pathogenic mechanisms are more important in the etiology of these lesions.

Langerhans and other immunocompetent cells in vocal cord epithelial hyperplastic lesions of patients with chronic laryngitis.

The aim of the study was to evaluate the intraepithelial and stromal density of Langerhans cells and lymphoid infiltrate in different stages of carcinogenesis in vocal cord biopsies of 24 randomly selected patients with chronic laryngitis. The Langerhans and lymphoid cells were counted using immunolabelling with antibodies against CD1a, S100, CD3, CD20, and CD68 on paraffin-embedded sections of 24 archival laryngeal vocal cord mucosa biopsy specimens, 6 classified as simple, 7 as abnormal, and 11 as atypical epithelial hyperplasia. Results were statistically evaluated using the Kruskal-Wallis and Wilcoxon sign rank tests. The mean number of Langerhans cells and T lymphocytes per mm2 of cross-sectioned epithelium was found to increase from simple to atypical hyperplasia. There were statistically significant differences in Langerhans cell density between atypical hyperplasia and each of the other 2 grades, simple and abnormal hyperplasia, with p < 0.05. Our study suggests the involvement of immune mechanisms, particularly cell mediated, during laryngeal carcinogenesis and the possibility that the assessment of Langerhans cell density might be of prognostic significance.

Langerhans cells in human papillomaviruses types 6/11 associated laryngeal papillomas.

Some studies have shown a reduced density of Langerhans cells (LCs) within epithelium infected by human papillomaviruses (HPV) types 16/18. However, data on a correlation between HPV types 6/11 infection and LCs have been missing. To solve this problem, we analysed 24 biopsy specimens of laryngeal papillomas, selected randomly, 20 multiple and 4 solitary. The presence of HPV 6 and 11 was proven by polymerase chain reaction (PCR) using 2 different sets of primers in 23 biopsy specimens. Abnormalities of the covering stratified squamous epithelium were graded according to the Kambic-Gale classification. LCs were immunohistochemically labelled with 2 different antibodies, CD1a and S100. Quantitative analysis was performed to determine the density of LC per mm2 in different grades of epithelial abnormalities covering laryngeal papillomas. Although no statistically significant differences in the mean number of LCs per mm2 of the cross-sectioned epithelium covering laryngeal papillomas were observed comparing simple, abnormal and atypical hyperplasia groups, the mean number of LCs per mm2 in laryngeal papillomas associated with HPV types 6/11 infection substantially exceeded that of the vocal cord surface epithelium in patients with chronic laryngitis.

Epidermal growth factor receptor, c-erbB-2 and p53 overexpressions in epithelial hyperplastic lesions of the larynx.

An immunohistochemical analysis of overexpression of epidermal growth factor receptor (EGFR), c-erbB-2, and p53 proteins was performed on 43 biopsies of laryngeal epithelial hyperplastic lesions (EHLL), classified according to the Kambic-Lenart classification, and in 11 cases of laryngeal carcinoma (SCCL). The aim of the present study was to determine whether there is a correlation between the staining patterns of these proteins and different grades of EHLL, and to reveal their possible prognostic value. We compared the staining patterns of atypical hyperplasia adjacent to cancer with the same type of lesions which have not turned malignant. p53 and EGFR overexpressions were detected in 28/54 (52%) and 33/54 cases (61%), respectively, and tend to increase with the degree of epithelial changes. The intensity of staining in various grades of EHLL adjacent to cancer was more pronounced than the same type of lesions which have not progressed to cancer. c-erbB-2 was weakly positive in the majority of cases, and changed from predominantly membranous in simple hyperplasia to cytoplasmic staining in abnormal and atypical hyperplasias. There was no significant statistic correlation between the amount of positive cells for all proteins and the grade of epithelial abnormalities. We conclude that the overexpression of each biomarker itself adds little predictive value over routine histomorphology, and cannot be regarded as a reliable prognostic factor for EHLL. However, the histologic characteristics of atypical hyperplasia together with the immunostaining patterns of EGFR and p53 up to two-thirds or more of the epithelial thickness could be considered a reliable pattern which correlates with the progression to cancer.

Correlation of histomorphological criteria used in different classifications of epithelial hyperplastic lesions of the larynx.

Different classifications of epithelial hyperplastic lesions of the larynx were proposed, but none of them has been generally accepted. The basic distinction among these gradings is evaluation of carcinoma in situ as a precancerosis or a distinct and separate entity. In the present study, atypical hyperplasia and carcinoma in situ are evaluated according to the proposed histomorphological criteria of the Kambic-Lenart classification. In an attempt to separate more objectively the histomorphological differences between these 2 lesions, in addition to traditional light microscopical examination, we also performed semiquantitative analysis with statistical evaluation using the Wilcoxon signed rank test. These results revealed a significant morphological and statistical difference comparing atypical hyperplasia and carcinoma in situ on the basis of the following criteria: abnormal mitotic figures (p = 0.005), mitotic activity (p = 0.014), nuclear pleomorphism (p = 0.006), cellular atypia (p = 0.005), dyskeratosis (p = 0.008), and stromal infiltration (p = 0.015). These results confirm our view that atypical hyperplasia and carcinoma in situ are 2 consecutive but different entities in the process of carcinonogenesis.

Human papillomavirus infection and expression of p53 and c-erbB-2 protein in laryngeal papillomas.

Laryngeal papilloma (LP) is the most frequent benign laryngeal epithelial tumor caused by human papillomaviruses (HPV) types 6 and 11. In the present study, we were interested in whether we can find any prognostic markers which might reflect the biological behavior of the covering epithelium in LP. We focused our attention on the determination of HPV infection, the detection of p53 protein, and c-erbB-2 protein in 24 biopsy specimens of LP. We confirmed the HPV 6 and 11 etiology in 23 of 24 LP. In these lesions the overexpression of p53 protein increased with the grade of epithelial abnormalities. The distribution of positive cells changed from scattered and focal, in simple and abnormal hyperplasia, to diffuse in atypical hyperplasia. It has been shown that in the presence of HPV types 6 and 11 found in LP, p53 can still preserve its tumor suppressor activity. Infection with HPV types 6 and 11 might therefore account for the significantly lower rate of malignant transformation in LP. Two staining patterns for c-erbB-2 protein were observed in the hyperplastic epithelium covering LP: membranous and cytoplasmic. With the increasing grade of epithelial abnormalities, cytoplasmic staining became predominant, and c-erbB-2 positivity sometimes occupied the whole epithelial thickness. This may represent either an alteration in the processing stability of the c-erbB-2 mRNA, gene amplification, or even an artefact.

Overexpression of p53 protein in benign and malignant laryngeal epithelial lesions.

This study investigated the immunohistochemical expression of p53 protein in various laryngeal lesions using Kambic's classification from simple hyperplasia to invasive squamous cell carcinoma (SCC), and analyzed the relationship between p53 protein overexpression and the severity of epithelial abnormalities. p53 overexpression was observed in 10/19 (53%), in 9/16 (56%), and in 9/13 (69%) cases of simple, abnormal, and atypical hyperplasia respectively, and also in 8/10 (80%) cases of SCC. The proportion of immunoreactive cells and staining intensity increased with the progression of the epithelial hyperplastic lesions. Our study confirms the association of p53 protein overexpression with laryngeal epithelial hyperplastic lesions which have the potential to transform into malignancy, but considering the follow-up of the patients, p53 expression cannot be considered a reliable prognostic factor for any group of laryngeal epithelial hyperplastic lesions regardless of the severity of the lesions.

Expression of Ki-67 antigen and proliferative cell nuclear antigen in benign and malignant epithelial lesions of the larynx.

In an attempt to analyse the proliferative activity in benign and malignant laryngeal epithelial lesions, and to determine the relationship to their histologic grade, we studied the expression of proliferative cell nuclear antigen (PCNA) and Ki-67 antigen on 20 squamous carcinomas, and on 30 biopsies of epithelial hyperplasia categorized according to the Kambic-Lenart classification into simple, abnormal, and atypical hyperplasias. In simple hyperplasia, both antibodies stained the nuclei of the occasional cells in the basal layer. In abnormal hyperplasia (mild dyplasia), positive cells occupied up to a third, and in atypical hyperplasia (moderate and severe dysplasia) they occupied from two-thirds to the entire epithelial thickness. In squamous carcinoma, we have found a statistically significant correlation between its grade and the percentage of Ki-67-(p < 0.01) and PCNA-(p < 0.00001) positive cells. Our results suggest that the proliferative fraction progressively increases with the degree of epithelial hyperplasia and the grade of carcinoma. We conclude that the patterns of immunoreactivity to PCNA and Ki-67 antigen correspond to the histologic grade of both benign and malignant epithelial lesions of the larynx. This method should be regarded as a useful adjunct to traditional histological techniques allowing more objective grading of benign and malignant epithelial lesions.

Local immune response in hyperplastic lesions of the larynx.

A retrospective morphologic and immunohistochemical study of 25 benign and 5 malignant laryngeal hyperplastic lesions was performed concerning a local immune response which might be characteristic and of prognostic value for each particular group of these alterations, using Kambic's classification, especially for precancerous and cancerous lesions. On paraffin and frozen sections, 7 monoclonal antibodies against various leukocytic antigens were used. CD43 and CD45RO T lymphocytes were the predominant cells in the infiltrates, and their frequency increased according to the degree of hyperplastic lesions. Next in frequency were CD4 cells, and a predominance of CD4 over CD8 cells was an obvious finding. The infiltration of CD68-, CD57-, and CD20-positive cells was generally weak. The intensity and composition of the local reaction in all cases of atypical hyperplasias was nearly identical, regardless of their subsequent behaviour. No apparent cytotoxic effects on the epithelial cells, either in precancerous or in cancerous lesions, were observed. Thus, the immunocompetent cells in the epithelial and stromal tissue are most likely not an effective defense in preventing hyperplastic lesions from becoming malignant. It seems that laryngeal hyperplastic lesions do not provoke an essential defense immune response, but the present local inflammatory reaction might be a constituent part of etiologically different inflammations which may lead to unfavorable lesions.

Laryngeal papillomatosis: molecular, histopathological, and clinical evaluation.

Molecular, histopathological, and clinical studies were carried out on a series of 79 laryngeal papillomas (LP) from 36 patients in order to investigate the hypothesis that juvenile and adult LP may represent a biological entity causally related to Human papilloma virus (HPV) infection. Using in situ hybridization with biotin-labelled probes and polymerase chain reaction, we detected human papilloma virus (HPV) 6/11 in 28 of 29 juvenile LP, in 26 of 30 adult multiple, and in 17 of 20 adult solitary LP. None of LP was found to harbour HPV types 16, 18, 31, 33, and 51. There were no clear-cut histological differences between juvenile and adult LP, the presence of koilocytosis was equally observed in both, and there was no prevalent type of epithelial hyperplasia in either form, except that all three cases of atypical hyperplasias (precancerous lesions) were found among adult patients. During a 14 year follow-up, no carcinomatous transformation of LP was observed. All juvenile LP in our study had frequent recurrences of the disease, however, numerous surgical procedures were also required in 16 of 27 adult patients. Our study supports Lindeberg's hypothesis of a similar pathogenesis for all forms of LP caused by the HPV types 6/11.

Laryngeal hyperplastic lesions, follow-up study and application of lectins and anticytokeratins for their evaluation.

A retrospective study of 878 biopsy specimens from 692 patients with laryngeal hyperplastic aberrations was performed according to the Kambic-Lenart classification. Special attention was focused on 88 patients with persistent or recurring disease. In these carcinoma developed in 17 (2.4%) patients, 12 (1.7%) of whom had had atypical hyperplasia. We therefore propose that the term precancerosis, which so definitely implies cancer, should be replaced with the expression risky epithelium where nothing is determined in advance, but a careful follow-up of the patients is imperative. In particular cases of laryngeal hyperplastic lesions, mainly in abnormal and in atypical hyperplasias when the tissue specimens are cut tangentially, the exact identification and position of individual epithelial cells is essential. In such cases histochemical and immunohistochemical methods yield more precise evaluation. Lectins and cytokeratins provide good markers of epithelial maturation. These results contribute to a more useful evaluation of laryngeal hyperplastic lesions, crucial for the choice of adequate therapy.