Current review on squamous intraepithelial lesions of the larynx

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1 Histopathology 2009, 54, DOI: /j x REVIEW Current review on squamous intraepithelial lesions of the larynx Nina Gale, Leslie Michaels, 1 Bostjan Luzar, Mario Poljak, 2 Nina Zidar, Janez Fischinger 3 & Antonio Cardesa 4 Institute of Pathology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia, 1 Department of Histopathology, University College London, London, UK, 2 Institute of Microbiology and Immunology, Faculty of Medicine, University of Ljubljana, 3 Department of Otorhinolaringology and Cervicofacial Surgery, University Clinical Centre, Ljubljana, Slovenia, and 4 Department of Anatomic Pathology, Hospital Clinic, University of Barcelona, Barcelona, Spain Gale N, Michaels L, Luzar B, Poljak M, Zidar N, Fischinger J & Cardesa A (2009) Histopathology 54, 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. Keywords: aetiology, follow-up, histopathological classifications, larynx, molecular biology, squamous intraepithelial lesions, treatment Abbreviations: CI, confidence interval; CIS, carcinoma in situ; EGFR, epidermal growth factor receptor; eif4e, eukaryotic initiation factor 4E; FHIT, fragile histidine triad; GERD, gastroesophageal reflux disease; HNSCC, head and neck squamous cell carcinoma; HPF, high-power field; HPV, human papillomavirus; htert, telomerase catalytic subunit; LIN, laryngeal intraepithelial neoplasia; LOH, loss of heterozygosity; Mcm, minichromosome maintenance proteins; MSI, microsatellite instability; OR, odds ratio; PCR, polymerase chain reaction; Rb, retinoblastoma; SCC, squamous cell carcinoma; SIL, squamous intraepithelial lesion; SIN, squamous intraepithelial neoplasia; TGF-b, transforming growth factor-beta; TSG, tumour-suppressor gene; WHO, World Health Organization Address for correspondence: Professor Nina Gale, MD, Institute of Pathology, Faculty of Medicine, University of Ljubljana, Korytkova 2, 1000 Ljubljana, Slovenia. nina.gale@mf.uni-lj.si Ó 2008 The Authors. Journal compilation Ó 2008 Blackwell Publishing Limited.

2 640 N Gale et al. Introduction It has been widely accepted that transition from a normal epithelium to squamous cell carcinoma (SCC) of the larynx is a lengthy, comprehensive and multistage process, causally related to progressive accumulation of genetic changes leading to the selection of a clonal population of transformed epithelial cells. 1 The whole spectrum of histological changes occurring in this process has been recently cumulatively designated squamous intraepithelial lesions (SILs), ranging from squamous hyperplasia to carcinoma in situ (CIS). 2,3 In their evolution, some cases of SILs are self-limiting and reversible, some persist, and some progress to SCC in spite of careful follow-up and treatment. 4,5 During recent decades, various aspects of laryngeal carcinogenesis have been intensively studied, including the aetiology, histological classification, treatment, frequency of malignant transformation and predictive factors. Many researchers have provided meticulous analyses of histological classifications of SILs in relation to their biological behaviour. 2 4,6 27 These analyses have been recently further supplemented by molecular genetic investigations trying to elucidate the pathogenic mechanisms of SCC development, and to improve the prognostic evaluation of SILs. 1,28 51 Particular interest in the spectrum of SILs has always been focused on lesions with the highest rate of malignant transformation. 3,8,23 25,52 54 These lesions are variously designated as potentially malignant, risky, precursor, preneoplastic or precancerous lesions. A fundamental, but still unsolved problem concerning preneoplastic lesions remains: when, and under what conditions, if ever, these changes progress to overt malignancy. 3,4,54 Terminology A precise and uniform terminology of SILs is essential for successful collaboration among pathologists, as well as for proper communication with clinicians. The terminology of SILs used in clinical and pathological reports has changed significantly over the last six decades. Common agreement has recently been achieved for terms that are used only for the clinical appearance and do not have any histopathological and prognostic implications. The most frequently applied clinical diagnoses are laryngeal leukoplakia, erythroplakia, and chronic laryngitis. 23,25,26,55 In contrast, keratosis remains a controversial term, since it is often wrongly applied interchangeably to macroscopic and microscopic features. However, keratosis is strictly a histological term denoting the appearance of a keratin layer on the surface of the squamous epithelium. 23 Unfortunately, inconsistent terminology still exists for the histological classification of SILs. The spectrum of epithelial changes has been variously described as keratosis, dysplasia, squamous intraepithelial neoplasia (SIN), laryngeal intraepithelial neoplasia (LIN), SILs, etc., to list only the most commonly used terms. Because of our inability to harmonize different views and establish a single classification of SILs, there are three classification schemes in the most recent edition of the World Health Organization (WHO) Classification of tumours, pathology of the head and neck tumours, as follows: (i) dysplasia system, (ii) SIN system, and (iii) Ljubljana classification. 2 These classifications differ conceptually and terminologically, and analogy between them can only be approximate. Epidemiology SILs are mainly limited to the adult population and affect men more often than women. This disparity is especially pronounced after 54 years of age. 20 The estimated incidence varies worldwide and depends upon the amount, manner and types of exposure to various carcinogens. Epidemiological studies of laryngeal SILs are scarce in comparison with similar studies of oral leukoplakia, necessitating the use of hospitalbased data or the results of epidemiological studies of smaller populations. 20,23,52 The incidence of clinically diagnosed laryngeal keratosis in the study of Bouquot et al. in a Rochester (MN, USA) population between 1934 and 1984 was shown to be 10.2% for men and 2.1% for women, whereas the mean annual incidence rate was 5.8%. 20 Another series of as yet unpublished data of 1268 patients, 1042 (82.2%) male and 226 (17.8%) female, clinically diagnosed as laryngeal leukopakia and chronic laryngitis within the period from 1979 to 2004, was studied at the University of Ljubljana, Institute of Pathology, Faculty of Medicine, Ljubljana, Slovenia. Clinical, surgical and histopathological data were considered. The male:female ratio was 4.6. The male patients ages ranged from 14 to 86 years (mean 51.5) and the female patients ages ranged from 19 to 89 years (mean 44.8). The incidence of patients, covering a region with approximately inhabitants or 40% of the population of Slovenia, varied for the benign group of SILs (squamous hyperplasia and basal parabasal cell hyperplasia) from 0.84 to inhabitants (mean value inhabitants, SD = 1.10). The incidence of patients for atypical hyperplasia ranged from 0.25

3 Laryngeal squamous intraepithelial lesions 641 to inhabitants (mean value inhabitants, SD = 0.49). Aetiology cigarette smoking and alcohol abuse Most reports agree that cigarette smoking and alcohol abuse, and especially a combination of these two detrimental factors, are major identifiable risk factors for laryngeal SCC, as well as for SILs. The role of smoking has been proven both clinically and experimentally. The risk of SIL development was found to be related to the age of the patient at the start of smoking, duration of smoking and the quality of tobacco Bosatra et al. analysed 97 dysplastic lesions of the head and neck region, including 47 cases of laryngeal dysplasia, and found a direct correlation between the degree of dysplasia, malignant transformation and amount of cigarette smoking and alcohol consumption. 67 Neuwirth-Riedl et al. also reached the same conclusions. 59 They found in a study of 63 patients with histologically proven laryngeal dysplasia that the percentage of smokers significantly exceeded that of the general population. 59 Bouquot et al. also established in a series of 108 patients with laryngeal keratosis that 84.3% were smokers, with an average usage of 36 cigarettes a day, and 35.2% were alcoholics or heavy drinkers. 20 Interesting data about aetiological factors in laryngeal carcinogenesis have also been published by Gallus et al. 68 This study, which included a uniquely large number of laryngeal cancers in women, provided definite evidence that cigarette smoking is a prominent risk factor for laryngeal cancer in women, since women accounted for 78% of cases in this population. Furthermore, alcohol and selected dietary aspects accounted for 30% of cases. 68 New data on a decline in laryngeal cancer risk after cessation of smoking were presented by Vaezi et al. 69 The authors found that increased risk might last for up to years after cessation of smoking, whereas previous data have reported a decline in laryngeal cancer risk after 15 years of such cessation in the general population. 69,71 gastroesophageal reflux Whereas tobacco has been established as the principal aetiological factor of SIL development for more than half a century, several authors have recently devoted much more attention to the potential role of gastroesophageal reflux disease (GERD). The association of GERD with laryngeal cancer was first suggested in 1983 by Olson, who identified laryngeal cancer in a series of patients with GERD. 72 Since then, several studies have reported a high prevalence of GERD in patients with laryngeal SILs and cancer. 69,72 79 It has been shown that demographic variability and the selection of inappropriate control groups have resulted in conflicting reports of an association between GERD and laryngeal cancer. 69 A matched case control study with controls from internal medicine clinics, since this group closely resembles the general population, continued to support an association between GERD and laryngeal cancer. 69 Lewin et al. in 2003 published the first study of laryngeal dysplasia and early cancer in relation to GERD. 77 The authors additionally found a high incidence of GERD in patients with early carcinogenic changes in the larynx. 77 Similar data from Cianci et al. also showed that of 93 patients with gastric resection, seven (8%) had current or previous laryngeal malignancies or current precancerous lesions. In the control group, in contrast, only one patient showed mild dysplasia of the vocal cord. 80 However, most authors have noted the need for further investigations with larger sample sizes and matched controls to objectivize the role of GERD in the development of laryngeal cancer. 47,69,77 human papillomavirus infection Human papillomaviruses (HPV) are aetiologically involved in virtually all SCCs of the uterine cervix, since persistent infection with high-risk genotypes (most often HPV-16 and HPV-18) is a necessary, although not sufficient factor for the development of this SCC. 81 Early studies have indicated that HPV may also play a similar aetiological role in laryngeal SCC. This association was first suggested by detecting typical HPV cytopathic effects in laryngeal carcinoma, 82 subsequently confirmed by immunohistochemistry 83 and studied by using various DNA hybridization and amplification techniques, mainly polymerase chain reaction (PCR). 84,85 However, in contrast to anogenital carcinomas, the prevalence of HPV in laryngeal carcinoma has varied considerably, e.g. between 0% and 54.1%, depending on the population studied, type of clinical specimen examined and HPV detection method used. 84,86 89 The overall published prevalence of HPV infection on approximately 1800 laryngeal cancers tested until December 2007 is around 24%. 84,85,90 HPV+ laryngeal carcinomas harbour mainly high-risk HPV genotypes, with HPV-16 being the most frequent genotype. 84,85 In some laryngeal cancers, HPV DNA was found integrated into the host genome. 89,91 A recent systematic review and meta-analysis has shown a weak association

4 642 N Gale et al. between HPV-16 and laryngeal cancer [odds ratio (OR) 2.0, 95% confidence interval (CI) 1.0, 4.2]. 85 In contrast, the association between HPV-16 and tonsillar cancer was within a magnitude which is consistent with an infectious aetiology (OR 15.1, 95% CI 6.8, 33.7). 85 The correlation between HPV and increased p53 accumulation in laryngeal carcinomas also has produced conflicting results. 87,92,93 In the same fashion as laryngeal cancer, the prevalence of HPV infection in laryngeal SILs has varied widely, between 0% and 56%, with an overall prevalence of HPV infection of 12.4% HPV+ SILs harbour mainly high-risk HPV genotypes, with HPV- 16 being the most frequent. In addition to laryngeal carcinoma and SILs, HPV DNA has also been detected in a substantial proportion, 12 25%, of individuals with clinically and histologically normal laryngeal mucosa. 102,103 These results suggest that occasional findings of HPV DNA in laryngeal carcinomas and SILs may be the result not of viral infection, but rather of an incidental HPV colonization of laryngeal mucosa. 104 In conclusion, at present the evidence linking HPV to laryngeal carcinoma and SILs is considered to be incomplete. 84,85,90 Whether, in the same fashion as oropharyngeal cancer, subsets of laryngeal carcinomas exist, i.e. HPV-related and HPV-non-related, remains to be seen. A large-scale multicentre case control study with adequate statistical power is needed to elucidate fully the association between HPV and laryngeal carcinoma and to examine the potential relationship between HPV and other risk factors. 104 otherriskfactors The majority of patients with laryngeal cancer are industrial workers who are exposed to a variety of hazardous working materials, such as polycyclic aromatic hydrocarbons, cement dust, asbestos or varnish lacquer, etc. 56,66 In addition to these possible causal agents, nutritional problems, lack of vitamins A, C and E, voice abuse, obstructions in the upper respiratory tract, chronic infections and hormonal disturbances may trigger pathways not yet entirely elucidated in the pathogenesis of laryngeal carcinoma. 23,64 66, Macroscopic appearance and clinical features Chronic laryngitis, leukoplakia or, occasionally, erythroplakia, appear mainly along the true vocal cords and, rarely, in other parts of the larynx, such as the epiglottis. Lesions can be either sharply circumscribed and grow exophytically, or be predominantly flat and diffuse, related in part to the amount of keratin layer. Their surface is rough, may be muddy brown to red (erythroplakia), perhaps with increasingly visible vascularity, or coated with diffuse or dispersed circumscribed whitish plaques. A circumscribed whitish thickening of the mucosa may be observed, covered by irregularly exophytic warty plaques. A speckled appearance of lesions can also be present, caused by an unequal thickness of the keratin layer. 2,23,109 Some leukoplakic lesions are ulcerated (6.5%) or combined with erythroplakia (15%). 52 In general, leukoplakic lesions are thought to have a low risk of malignant transformation, mixed white and red lesions, or speckled leukoplakia, an intermediate risk, and pure erythroplakia (red lesions) the highest risk of cancer development. 55 However, none of these laryngoscopic features can be used as an indicator of the overlying changes of the epithelium, and histological analysis of these lesions is mandatory to determine their biological potential. Symptoms depend on the location and severity of the disease and usually last a few months before clinical notice. Patients may complain of fluctuating hoarseness, throat irritation, sore throat, and or chronic cough. 2,3,23,110 Histopathological features of SILs Traditional light microscopic examination, in spite of a certain subjectivity in interpretation, remains the most reliable method for determining an accurate diagnosis of SILs. Since 1923, when Jackson first defined chronic laryngitis and keratosis as precancerous lesions, 111 numerous studies and classifications have attempted to correlate phenotypic and genetic changes with the biological behaviour of the lesions. 4,22 25,112 Regrettably, neither generally accepted criteria nor unified terminology have to date been provided for a histological grading system of laryngeal SILs. Evidence of the inability of pathologists to set up a single, unified classification of SILs was manifest in the WHO Classification of head and neck tumours, published in 2005, where the dysplasia system is presented as the 2005 WHO classification simultaneously with the classification of SIN and the Ljubljana classification. 2 The majority of current classifications, such as the traditional dysplasia system, 2,11,22,113 keratosis without (KWA) and with atypia CIS, 9,114 SIN, 5,18 and LIN, 115,116 follow criteria similar to those commonly used for epithelial lesions of the uterine cervix. However, the different aetiology of laryngeal lesions and their particular clinical and histological features

5 Laryngeal squamous intraepithelial lesions 643 require a grading system more appropriate to this region. 24 As early as 1963, Kleinsasser suggested one of the first laryngeal classification systems with three subgroups: grade I (simple squamous epithelial hyperplasia) is characterized by thickening of the epithelium with normal maturation; grade II (epithelial hyperplasia with occasional atypia) represents slight localized cellular atypias and disturbance of differentiation only to a certain degree, so that the lesions cannot be classified as premalignant; grade III (CIS, precancerous epithelium) includes high-grade dysplasia and CIS. 109 This classification has recently been modified using different terminology, such as three grades of intraepithelial neoplasia. 117 In 1971, the Ljubljana classification was devised by Kambič and Lenart. 118 The authors clinically and histologically adapted the classification to the specific demands of the larynx. Criteria for histological grading were additionally formulated by a Working Group on Head and Neck Pathology of the European Society of Pathology in ,24,25 The Ljubljana system nominally recognizes four grades: simple and basal parabasal cell hyperplasia includes mainly benign categories with a minimum risk of malignant alteration, atypical hyperplasia (risky epithelium) is potentially, and CIS actually a malignant lesion. One can object that grading SILs, in spite of the clear histological criteria, is an attempt to impose arbitrary distinct categories of a continually progressing process without naturally and sharply defined borders. 119,120 However, this continuous process, which is of long duration, may eventually stop, regress or progress, depending above all on the influence of various detrimental factors causing genetic and, consequently, phenotypic epithelial changes. When a laryngeal biopsy is performed with a representative tissue sample, the established histological changes still serve at present as the main guidance for clinicians on how to treat the patient, as well as being the most reliable prognostic factor of the biological behaviour of the disease. the dysplasia system compared with the ljubljana classification and the sin classification Although histological assessment of the dysplasia system and the Ljubljana classification are based on the same architectural and cytological changes (irregular epithelia stratification, loss of polarity of basal cells, increased number of mitoses with atypical mitotic figures, increased number of dyskeratotic cells, cellular and nuclear atypias, increased nuclear cytoplasmic ratio, increased number and size of nucleoli and hyperchromatism), there is no simple relationship or overlapping between the two grading systems. 2 Dysplasia is defined as architectural disturbance, accompanied by cytological atypia. Furthermore, in the dysplasia system, there is difficulty in recognizing its earliest manifestations, and no single combination of the above-listed histological features allows consistent distinction between hyperplasia and the earliest stages of dysplasia, as well as in attempting rigidly to divide the spectrum of dysplasia into mild, moderate and severe categories. CIS remains separated from severe dysplasia. 2 The lack of defined criteria for grading laryngeal dysplasia is an important source of inconsistent and poorly reproducible results. 119 This disturbing problem also appears in other parts of the body in which the dysplasia classification is used. 120 In addition, some advocates of the laryngeal dysplasia system have proposed that a lack of definition of moderate dysplasia precludes confidence in histopathological reports of this grade and suggest utilizing a two-grade system, such as low- and high-grade dysplasia. 113 In the Ljubljana classification, critical interobserver agreement has been reached for defining each particular grade of SIL. 24,25 General guidelines concerning the important histological features of the Ljubljana classification are the following. Benign lesions squamous hyperplasia Hyperplastic epithelium. Increased prickle layer. Basal-parabasal layer unchanged. Normal maturation. No cellular atypia. Infrequent regular mitoses in basal layer (Figure 1). basal-parabasal hyperplasia Augmentation of basal and parabasal cells lower part of epithelium, occasionally slightly more. Unchanged prickle cells in the upper part. Stratification smooth transition of basal cells with perpendicular orientation to basement membrane to prickle cells oriented horizontally to the basement membrane. Parabasal cells slightly increased cytoplasm compared with basal cells, no intercellular bridges. Parabasal cells slightly enlarged nuclei, uniformly distributed chromatin. Rare regular mitoses in or near basal layer. Less than 5% of dyskeratotic cells (Figure 2).

6 644 N Gale et al. Figure 1. Squamous cell hyperplasia. Hyperplastic epithelium with thickened prickle cell layer, the basal layer is unchanged. Potentially malignant atypical hyperplasia Stratification still evident. Increased number of altered epithelial cells with frequent orientation perpendicular to basement membrane. Nuclei with mild to moderate atypia (enlargement, irregular contours, marked variations in intensity of immunoreactivity with frequent hyperchromatism, nucleoli increased in number and size. Nuclear cytoplasmic ratio increased. Altered epithelial cells in lower half of epithelium up to entire epithelial thickness. Increased mitoses mainly in lower two-thirds of epithelium. Dyskeratotic and apoptotic cells frequent within entire epithelium. Two subdivisions: basal cell type with no intercellular prickles, no cytoplasmic eosinophilia, cells oriented perpendicularly to basement membrane; spinous cell type with intercellular prickles and increased cytoplasmic eosinophilia (Figure 3). Figure 2. Basal and parabasal cell hyperplasia. The epithelium is slightly thickened. Augmented cells of the basal and parabasal cell layer extend up to the midportion of the epithelium; the upper part of the epithelium is unchanged. carcinoma in situ Loss of stratification or maturation of epithelium, surface one to few layers compressed horizontally stratified and keratinized cells. Epithelial cells advanced cellular and nuclear atypias. Mitoses, also atypical, increased in whole epithelium, often five and more per high-power field (HPF). Dyskeratotic and apoptotic cells frequent within entire epithelium. Two subdivisions: basal cell type with no intercellular prickles, no cytoplasmic eosinophilia; spinous cell type with intercellular prickles and increased cytoplasmic eosinophilia (Figure 4). The general principles of the presented classification, which hold for all of its grades, are: the epithelium is generally thickened, although in a minority of cases the epithelium may show areas of diminished thickness; the basement membrane is generally preserved in all grades, with no definite evidence of even minimal invasion. The presence of a surface keratin layer, which

7 Laryngeal squamous intraepithelial lesions 645 Figure 3. Atypical hyperplasia risky epithelium. The whole thickness of the hyperplastic epithelium is occupied by the basaloid, moderately polymorphic epithelial cells with hyperchromatic nuclei and increased mitotic activity. Figure 4. Carcinoma in situ shows almost full thickened architectural disorder, pronounced cellular atypias, increased mitotic activity with abnormal mitoses. A focus of spinous differentiation is present. is often present in all grades of SIL, is of no importance in this classification. For practical purposes, the Ljubljana classification has been created solely on pathohistological criteria of traditional light microscopy. In fact, none of the supplementary techniques for assessing SILs that have been utilized has yet been shown to be superior to this basic method. The main features by which the Ljubljana grading system differs from other classifications are: the distinction between mainly benign (squamous hyperplasia and basal-parabasal hyperplasia) and potentially malignant lesions (atypical hyperplasia) and positive separation of CIS from atypical hyperplasia. The two entities differ in morphology and progression to invasive cancer. 3,23 25 This grading system has been used for more than a quarter of a century and has been found to be more precise for daily diagnostic work than other grading systems. 3,24,25,41,110, However, in spite of the well-defined morphological criteria of the Ljubljana classification, especially in cases of atypical hyperplasia, the likelihood of eventual malignant alteration remains enigmatic. Frequent recurrences of atypical hyperplasia (risky epithelium) and male patients who are not determined to change their life style, are still the most threatening features for the development of invasive SCC. Disagreement between the dysplasia system and the Ljubljana classification starts with the definition in the former that each grade of the whole series of dysplasia is considered to be a precursor or potentially malignant lesion, including mild dysplasia, which histologically very much resembles basal and parabasal cell hyperplasia, as the initial grade within a spectrum of SILs. On the other hand, basal and parabasal cell hyperplasia in the Ljubljana classification is considered to be a benign lesion with minimum risk of malignant transformation. Histologically, however, there are some similarities between the two entities. Atypical hyperplasia of the Ljubljana classification is similar to moderate dysplasia, but partially also includes severe dysplasia. The analogy is therefore only approximate. CIS is equivalent to CIS of the WHO 2005 classification. However, some cases of severe dysplasia would fall into the category of CIS of the Ljubljana classification, and the analogy is again only approximate. 3 The binary system of SIN is designed by specific criteria for precisely gauging grades of SIN into hyperplasia keratosis, SIN I (low grade) and SIN II

8 646 N Gale et al. (high grade). 124,125 Hyperplasia keratosis is characterized by a thickened hyperplastic epithelium, rare mitoses, is confined to the suprabasal layer, with normal maturation, frequent surface keratinization and no nuclear pleomorphism. SIN I (low grade) can be identified by epithelial hyperplasia, increased mitoses, one to two per HPF, three or more layers of basallike cells and minor nuclear pleomorphism. In SIN II, one can observe epithelial hyperplasia, frequent mitoses in all layers, including abnormal forms, marked abnormalities of epithelial maturation with basal-like cells constituting the inner and middle onethird, premature keratinization, including the presence of pearls, prominent nuclear pleomorphism and increased chromatin staining. All three grades can also appear in atrophic forms. Compared with the Ljubljana classification, the histological criteria of hyperplasia and SIN I could approximately correspond to the histological criteria of squamous hyperplasia and basal and parabasal cell hyperplasia, being considered benign lesions with minimum risk of malignant transformation. In our opinion, therefore, the term neoplasia does not suit lesions with a small risk of malignant alteration. The histological features of SIN II correlate partially with atypical hyperplasia (risky epithelium) and partially with CIS of the Ljubljana classification. Cases of CIS that fulfil all the required histological criteria according to the Ljubljana classification are very uncommon. 23 In these rare established cases, the patient would benefit from receiving more radical treatment than with the grade of atypical hyperplasia. Moreover, in such a case, the search for possible foci of invasive carcinoma should be intensified. From the therapeutic point of view, it seems more reasonable to separate the grade of atypical hyperplasia from CIS. 22,25,126 Comparing all three discussed classifications, it is unlikely that they will come together in the very near future. On the other hand, future discoveries in the field of molecular biology could be the basis for a single, unified classification of laryngeal SILs. Molecular aspects of carcinogenesis The genetic changes and the sequence of genetic events underlying the progression of normal mucosa to invasive SCC in the head and neck area (HNSCC), and larynx in particular, are still not entirely recognized. Between six and 10 independent genetic events within a single cell have been estimated to be necessary for SCC development in the head and neck region. 127 They are believed to be morphologically expressed as different grades of epithelial abnormality. The latency period between carcinogen exposure and appearance of malignancy may last up to 25 years. 1 The process of tumorigenesis of solid tumours, including HNSCC, involves both activation of protooncogene products that stimulate growth, and inactivation of tumour-suppressor genes (TSGs), the products of which normally inhibit cell proliferation. 33,36 The identification and characterization of the comprehensive spectrum of genetic aberrations in SCC development may not only elucidate the process of carcinogenesis, but also provide promising diagnostic tools for early detection, prevention and assessment of cancer risk from precursor lesions. genetic progression model Califano and co-workers have made two studies of cytogenetic alterations in head and neck carcinogenesis, which showed an increasing number of chromosomal alterations with the progression of SILs, ranging from hyperplasia to CIS and invasive SCC. 1,33,36 The areas of allelic loss, and less frequently allelic gain, are decisive elements in the progression model involving HNSCC. The results of Califano s studies have revealed that the spectrum of chromosomal loss progressively increases at each histopathological step of SILs from benign hyperplasia to CIS and invasive SCC. The earliest alterations appear on chromosomes 9p21, where the p16 gene resides, at 3p with at least three putative tumoursupressor loci, and at 17p13 where the p53 gene is located. 1,33,36 Loss of chromosome region 9p21-22 appears to be the most common of all genetic changes in HNSCC, with a frequency of 70%. 128 Additional studies of microsatellite DNA allelic imbalance in laryngeal carcinogenesis have confirmed that dysplasia correlates with loss of heterozygosity (LOH) at 3p21, 5q21, 9p21 and 17q Yoo et al. have suggested that 9p21 is the earliest event, already appearing in squamous metaplasia, as well as in invasive and metastatic SCC. LOH at 17p13, 3p35 and 3p14 was observed as an intermediate event, occurring from dysplasia to metastatic SCC. 42 Microsatellite instability (MSI), a novel marker of genetic instability, was also applied in a study to assess the risk of malignant progression in laryngeal preinvasive lesions. The authors concluded that MSI is more common in preneoplastic laryngeal lesions that have progressed to invasive SCC. They suggest that MSI assessment may be useful in determining the risk of malignant alteration in patients for whom chemopreventive and multiple endoscopic protocols can be attempted. 129

9 Laryngeal squamous intraepithelial lesions 647 key tumour-suppressor genes in laryngeal carcinogenesis Gene p16 can be inactivated by a variety of mechanisms, such as mutation, homozygous deletion and promoter hypermethylation. 130,131 The p16 gene functions as an inhibitor of cyclin-dependent kinase 4 and 6, with subsequent abrogation of retinoblastoma (Rb) phosphorylation and G 1 cell cycle arrest. 132,133 Loss of chromosome region 9p21-22 occurs prior to the development of histological atypia, already at the level of hyperplastic mucosa, and is regarded as an early event in the development of HNSCC. 134 Another important region identified by allelic loss is chromosome 17p, the site of the p53 gene. It is involved in several key cell functions such as gene transcription, DNA synthesis and repair, cell-cycle coordination and apoptosis. Mutation inactivation of the p53 gene has been detected in approximately 50%, but may be present in as high as 80% of HNSCCs. 135 It remains unclear whether p53 gene inactivation is an early or late event of laryngeal carcinogenesis. According to Boyle and co-workers, it occurs in the transition from the preinvasive to invasive form. 136 Some others argue the opposite, presenting alterations of p53 among early steps of neoplastic transformation. 35 Furthermore, gene p53 mutation has been hypothesized to be the earliest event in the development of a genetically altered field in oral mucosa, identifying an area of clonally related cells with malignant potential. 137 Recently, Poeta et al. evaluated TP53 mutations and survival in a series 420 patients with HNSCC. 138 Mutations were classified into two groups, disruptive and non-disruptive, according to the degree of disturbance of protein structure predicted from the crystal structure of the p53 DNA complexes. They found that disruptive TP53 mutations in tumour DNA are associated with reduced survival after surgical treatment of the HNSCC. 138 Although LOH frequently appears in head and neck carcinogenesis at chromosome 3p, the genes at this region have not been well defined. 1 The fragile histidine triad (FHIT) gene has been identified on chromosome 3p14 as one candidate for TSG, altered by deletions in human tumours. The expression of FHIT protein has recently been studied in laryngeal SCC and premalignant lesions. 46 Loss of FHIT protein was observed in 42% of SCCs and 23% of premalignant lesions. There was no significant difference among the three grades of dysplasia and FHIT expression. The results of this study indicate that FHIT alterations may play an important role in early events of carcinogenesis. 46 key oncogenes in malignant alteration of sils Chromosome region 11q13 has been identified as the site of several putative oncogenes, such as Bcl-1, int-2, hst-1, EMS-1 and cyclin D1 PRAD Amplification of 11q13 is detected in approximately one-third of HNSCCs, but only cyclin D1 has shown consistent overexpression amplification. 32,139 The function of proto-oncogene cyclin D1 is to activate Rb via phosphorylation, thus facilitating progression from the G 1 phase to the S phase. 133 The normal function of the p16 gene is to decrease the phosphorylation of Rb by inhibiting cyclin D1. Despite these related, but opposite functions, the loss of p16 gene and gain of cyclin D1 are independent mechanisms leading to cell cycle dysregulation. 1 Overexpression amplification of cyclin D1 has been associated with the advanced stage of the disease and could indicate more aggressive behaviour of such HNSCCs. 140 key protein-based alterations in laryngeal carcinogenesis Protein overexpression can appear as a consequence of gene amplification, increased DNA transcription and translation. Several gene products can influence cancer progression in this manner. 1 Epidermal growth factor receptor (EGFR), located on chromosome 7p12, codes for transmembrane growthregulating receptor glycoprotein, which influences cell division, migration, adhesion, differentiation and apoptosis through a tyrosine kinase pathway. 141 The EGRF gene was found to be amplified in 25%, and its mrna was overexpressed in 43% of laryngeal SCCs. Half of the expressed cases occurred in the absence of detectable gene amplification. Both alterations appeared in advanced laryngeal SCC. 142 Furthermore, overexpression of EGFR protein is an early event in carcinogenesis, rising with increasing degree of epithelial abnormalities, mainly in the progression of SILs to SCC. 143,144 Eucaryotic initiation factor 4E (eif4e) is a 24-kDa protein, which binds to mrna as the initial ratelimiting step in protein synthesis. Amplification and overexpression of the eif4e gene, located at chromosome 4q21, has been associated with malignant transformation in breast cancer and HNSCC. 145 The proto-oncogene eif4e was found to be elevated in 100% of HNSCCs and is of prognostic value in predicting recurrence. 146 It has been suggested that eif4e gene amplification and overexpression may precede morphological changes. Several studies have shown that surgical margins that appear tumour free

10 648 N Gale et al. microscopically may have elevated eif4e protein and that this thus predicts early recurrence In 2001, Nathan et al. reported an immunohistochemical analysis of expression of cyclooxygenase 2 and eif4e in dysplasias and SCC in the head and neck area. They found a significant correlation in the expression of both markers for all groups of dysplasias and, furthermore, established that eif4e appears to fulfil the criteria of being a biomarker in epithelial carcinogenesis, since its expression in normal tissue is different from that in high-risk tissue, and is of a quantity or pattern that can be correlated with the stage of carcinogenesis. 151 Immunohistochemical study of the expression of eif4e in laryngeal SILs has also been performed by the Ljubljana group (unpublished data). The study focused particularly on the hypothesis that increased eif4e expression in atypical hyperplasias predicts malignant alterations. Two groups of atypical hyperplasia were included: seven cases of atypical hyperplasia with subsequent malignant alteration and five cases of atypical hyperplasia without progress to SCC. Five of seven cases of atypical hyperplasia with malignant progression showed diffuse intracytoplasmic positivity for eif4e protein in the whole epithelial thickness, but the keratin layer remained immunonegative (Figure 5). Two cases showed only weak, unspecific reactivity. Overexpression of the eif4e protein also was found in six of seven SCCs. In the group of atypical hyperplasia without progression to SCC, one case was immunopositive, one negative and three cases showed weak, unspecific positivity for eif4e protein These findings, although obtained in a small group of patients, do not support the use eif4e protein as a reliable marker for malignant alteration. field cancerization In early 1953, Slaughter et al. proposed the clinical concept of field cancerization to explain the development of multiple cancers and precursor lesions in the head and neck area, particularly in the oral cavity. 152 Their concept is based on long-term carcinogenic exposure, which causes the independent transformation of multiple epithelial cells at separate sites. Polyclonal tumours may independently arise from these spots. The so called histologically-based field cancerization model has been gradually succeeded by a new one established on the basis of molecular changes of the affected mucosa. The hypothesis, proposed by Bedi et al. and Califano et al., advocates a micrometastatic spread or a monoclonal theory, suggesting that a precancerous field of mucosa may derive from an early genetic event that has undergone clonal expansion and Figure 5. Atypical hyperplasia with subsequent progression to invasive cancer. eif4e immunoreactivity in the full thickness of atypical hyperplasia, the keratin layer remains immunonegative. lateral migration or expansion. 1,33,36,153 Subsequent genetic alterations produce genetic divergence and various phenotypic alterations, resulting in a variety of histopathologically diverse regions in the local anatomical area and in the selection of various subclones. The theory, therefore, proposes a clonal origin of premalignant cells with successive lateral migration, and possible multiple primary tumours would not be monoclonal, but clonally related. 1,154 telomerase reactivation in malignant alteration of sils The telomerase enzyme is a specialized multisubunit complex, with telomerase catalytic subunit (htert) functioning as a reverse transcriptase that can synthesize the telomeric ends at each cell division. 155 Telomerase has been found to be re-activated in 90% of malignant neoplasms, including laryngeal SCC Recent studies have confirmed a close relationship between htert mrna expression and telomerase activity, suggesting that quantification of htert gene expression can be used as an alternative to measure-

11 Laryngeal squamous intraepithelial lesions 649 ment of telomerase activity. 158,159 The presence and relative quantity of htert mrna was recently studied, expressed as an htert index, in 140 frozen laryngeal tissue specimens representing different morphological stages of laryngeal carcinogenesis. 160 The authors demonstrated that the presence and relative quantity of htert mrna in the laryngeal epithelium increases progressively with the degree of epithelial abnormality. Specifically, htert mrna was detectable in 7% of normal laryngeal epithelia, 20% of squamous hyperplasias, 37% of basa-parabasal hyperplasias, 75% of atypical hyperplasias, 89% of CIS and 85% of invasive laryngeal SCC. Statistical analysis revealed two groups of laryngeal epithelial changes, with significant differences in the levels of htert mrna expression (P < ): (i) normal and reactive hyperplastic laryngeal epithelium (squamous and basal-parabasal hyperplasia), and (ii) atypical hyperplasia (potentially malignant lesion), CIS and invasive laryngeal SCC. These results suggest that telomerase re-activation is an early event in laryngeal carcinogenesis, already detectable at the stage of precancerous laryngeal epithelial changes. Nevertheless, other genetic abnormalities appear to be necessary for the progression of these epithelial abnormalities towards invasive laryngeal SCC. 160 other markers of malignant alterations of laryngeal sils Several studies of laryngeal SILs generally agree that the severity of epithelial abnormality reflects the degree of risk of SCC development. 44 No marker or group of markers has so far been identified as a reliable predictor of malignant progression of SILs. It is therefore understandable that numerous studies have been devoted to the progression of laryngeal SILs to invasive SCC. The role of cell-cycle proteins such as p16, p21, p27, p53, cyclin D1 and E have been extensively studied over the last two decades. 29,44,50,95,144, However, none of these markers has been found to have reliable predictive value. In addition, detection of proliferative activity, mainly as immunohistochemical labelling for proliferating cell nuclear and Ki67 antigens, can be used only as adjuncts to light microscopy for more objective and reliable histological grading of SILs. 168, Recent study of the transforming growth factor-beta (TGF-bRII) has indicated that its down-regulation is an early event in laryngeal carcinogenesis, which may occur in the loss of TGFb-mediated inhibition, thereby facilitating progression of precancerous lesions to SCC. 38 The study of ceramide expression in laryngeal carcinogenesis is related to the process of apoptosis. Chi et al. have reported that the expression of ceramide decreases gradually from healthy tissue to tissue with SCC and concluded that a reduction of ceramide expression might contribute to laryngeal carcinogenesis. 45 Promising biomarkers for improving cancer detection include minichromosome maintenance proteins (Mcm-2-7), which assemble in the prereplication complex and are essential for DNA replication in eucaryotic cells. All six proteins are abundant throughout the cell cycle, being broken down rapidly on differentiation and more slowly in quiescence. 41 Chatrath et al. found that Mcm-2 is expressed within the most superficial surface layer in cases of laryngeal CIS and SCC and with minimal expression in basal-parabasal (abnormal) and atypical hyperplasia. The authors suggest that Mcm-2 would be a good biomarker for distinguishing premalignant from malignant lesions. 41 Quantification of cellular DNA by image or flow cytometry has achieved acceptance as an objective and reproducible component in diagnostic pathology. Several studies of laryngeal SILs have shown that a proportion of these lesions show abnormal DNA content and that the incidence of this finding correlates with the degree of SILs Bračko has additionally noted that lack of abnormal DNA does not exclude malignant alteration, since malignant tumours exhibit minimal chromosomal abnormalities resulting in DNA changes, which are below the threshold of sensitivity of measurement with the use of image analysis or flow cytometry. 174 In three studies of SILs in the head and neck region, an increase in polysomy of both chromosomes 7 and 17 was found, and these findings also correlated with progressive grades of SILs. 31,177,178 In 2004, Kim and co-workers performed a study of quantitative PCR for genes specific to mitochondrial abundance in a spectrum of dysplastic head and neck lesions. 179 Their study shows that mitochondrial DNA is directly proportional to histopathological grade. Therefore, high mitochondrial content may be another marker of genetic alteration, a measure of relative DNA injury, and a surrogate measure of histopathological grade. 179 Treatment and prognosis The standard for management of laryngeal SILs is excisional biopsy using stripping microflap excision or laser ablation. As expected, superficial and, often, smaller lesions are better suited for cold dissection. 180 For suspicious aberrations, the removal of entire lesions, which must be properly oriented and prepared for serial sections, is required in order to exclude invasive SCC. Vocal fold submucosal infusion of a

12 650 N Gale et al SH/BPH SH/BPH 1.1% (12/1089) years, mean 7.9 ATYH 9.5% (17/179) 2 24 years, mean 6.2 ATYH Years Figure 6. Percentage of progression of squamous hyperplasia (SH) basal-parabasal hyperplasia (BPH) and atypical hyperplasia (ATYH) of 1268 patients to carcinoma, from 1979 to 2004, in Ljubljana, Slovenia. physiological solution facilitates the excision of the mucous membrane. 8 Cases of simple or basal parabasal cell hyperplasia do not require further treatment after excisional biopsy. The removal of possible detrimental factors is always strongly advised. When a recurrent or a new lesion appears, another excision is mandatory. 8,23 Treatment of potentially malignant SILs and CIS remains controversial. Endoscopic microsurgery and CO 2 laser are the treatments of choice. 109, Surgical removal includes microflap excision and chordectomy. According to many authors, a CO 2 laser offers considerable advantages over traditional techniques, including an easier procedure with the absence of bleeding, diminution of oedema and complete recovery of voice. 23 However, repeated use of laser treatment may induce scarring similar to that of stripping, and the voice outcome may be compromised more than with simple excision, especially after repeat procedures. 185 For surgical removal of atypical hyperplasia and or CIS, stripping is the treatment of choice, accompanied by lifelong close follow-up. CIS is usually additionally treated by surgical procedure or radiotherapy. However, in cases in which free margins are reliably confirmed, no further treatment is needed. Radiation therapy should be used only in cases of CIS. 23 It is not without risk, such as the possibility of postradiation complications and new primary tumour induction. 186 Radiation therapy is also indicated in the case of recurrence of CIS after vocal cord stripping and when stripping is not possible. Fein et al. have described good results of combined surgery and radiation. 187 Table 1. Progression of laryngeal squamous intraepithelial lesions to carcinoma according to different studies Squamous hyperplasia keratosis without atypia Mild dysplasia, SIN I, LIN I, basal-parabasal hyperplasia Moderate dysplasia, SIN II, LIN II Severe dysplasia, SIN III, LIN III, Atypical hyperplasia, CIS* Total Hellquist et al. (1982) (2%) 3 24 (12.5%) 4 39 (10.2%) (5.6%) Olde Kalter et al. (1987) (6%) (24%) 4 8 (50%) (19.3%) Velasco et al. (1987) (2.2%) 6 31 (19.3%) 1 5* (20%) 8 86 (9.3%) Sllamniku et al. (1989) (2.9%) (7.3%) 4 23 (17.4%) (27.7%) (6.7%) Blackwell et al. (1995) (0%) 3 26 (11.5%) 5 15 (33.3%) 4 9* (44.4%) (21.4%) Plch et al. (1998) (0%) 0 63 (0%) 1 25 (4%) 4 7* (57.1%) (4.2%) Gallo et al. (2001) (4.1%) 4 56 (7.1%) 6 28 (21.4%) 3 32 (9.3%) (7.3%) Ricci et al. (2003) (2.3%) 2 44 (4.5%) 5 36 (13.9%) 3 21 (14.3%) (6.4%) Crissman et al. (1993) (1.9%) (17.8%) (11.4%) (9.3%) Present study (1.1%) * (9.5%) (2.3%) CIS, carcinoma in situ; LIN, laryngeal intraepithelial neoplasia; SIN, squamous intraepithelial neoplasia. *CIS is not included.