European Archives of Oto-Rhino-Laryngology (2021) 278:1717–1722 https://doi.org/10.1007/s00405-020-06403-y REVIEW ARTICLE European Laryngological Society position paper on laryngeal dysplasia Part I: aetiology and pathological classification Edward Odell1 · Hans Edmund Eckel2 · Ricard Simo3 · Miquel Quer4 · Vinidh Paleri5 · Jens Peter Klussmann6 · Marc Remacle7 · Elisabeth Sjögren8 · Cesare Piazza9 Received: 16 March 2020 / Accepted: 24 September 2020 / Published online: 13 October 2020 © The Author(s) 2020 Abstract Purpose of review To give an overview of the current knowledge regarding the aetiology, epidemiology, and classification of laryngeal dysplasia (LD) and to highlight the contributions of recent literature. As most cases of dysplasia occur at the glottic level and data on diagnosis and management are almost exclusively from this location, laryngeal dysplasia in this position paper is taken to be synonymous with dysplasia of the vocal folds. Summary LD has long been recognized as a precursor lesion to laryngeal squamous cell carcinoma (SCC). Tobacco and alcohol consumption are the two single most important etiological factors for the development of LD. There is currently insufficient evidence to support a role of reflux. Although varying levels of human papillomavirus have been identified in LD, its causal role is still uncertain, and there are data suggesting that it may be limited. Dysplasia has a varying presentation including leukoplakia, erythroleukoplakia, mucosal reddening or thickening with exophytic, “tumor-like” alterations. About 50% of leukoplakic lesions will contain some form of dysplasia. It has become clear that the traditionally accepted molecular pathways to cancer, involving accumulated mutations in a specific order, do not apply to LD. Although the molecular nature of the progression of LD to SCC is still unclear, it can be concluded that the risk of malignant transformation does rise with increasing grade of dysplasia, but not predictably so. Consequently, grading systems are inherently troubled by the weak correlation between the degree of the dysplasia and the risk of malignant transformation. The best data on LD grading and outcomes come from the Ljubljana group, forming the basis for the World Health Organization classification published in 2017. Keywords Carcinoma in situ · Dysplasia · Laryngeal intraepithelial neoplasia · Laryngeal carcinoma 2 * Elisabeth Sjögren Department of Oto‑Rhino‑Laryngology, Klagenfurt General evsjogren@lumc.nl Hospital, Feschnigstr. 11, Klagenfurt, Austria 3 Edward Odell Department of Otorhinolaryngology Head and Neck Surgery, edward.odell@kcl.ac.uk Guy’s and St Thomas’ Hospital NHS Foundation Trust, London, UK Ricard Simo 4 ricardsimo1@icloud.com Department of Otorhinolaryngology and Head and Neck Surgery, Hospital de la Santa Creu i Sant Pau, Universitat Miquel Quer Autònoma de Barcelona, Barcelona, Spain MQuer@santpau.cat 5 Head and Neck Unit, Royal Marsden Hospital, London, UK Vinidh Paleri 6 Vinidh.Paleri@rmh.nhs.uk Department of Otorhinolaryngology, Head and Neck Surgery, Medical Faculty, Cologne, Germany Jens Peter Klussmann 7 jens.klussmann@uk‑koeln.de Department of Otorhinolaryngology, Head and Neck Surgery, CH Luxembourg, Luxembourg, Belgium Marc Remacle 8 remacle.marc@chl.lu Department of Otorhinolaryngology, Head and Neck Surgery, Leiden University Medical Center, Leiden, The Netherlands Cesare Piazza 9 cesare.piazza@unibs.it Department of Otorhinolaryngology‑ Head and Neck Surgery, ASST Spedali Civili of Brescia, University 1 Head and Neck Pathology, King’s College London, Guy’s of Brescia, Brescia, Italy Hospital, London SE1 9RT, UK 13 Vol.:(0123456789) 1718 European Archives of Oto-Rhino-Laryngology (2021) 278:1717–1722 Aetiology LD and found HPV 6/11 in 50% of patients with low-grade dysplasia and in 88% of patients with high-grade dyspla- Carcinogenesis is traditionally considered to be a multi- sia [10]. No HPV high-risk types were detected by these year, multistep, multipath disease of progressive genetic authors when screened for HPV 16 and 18 [10]. Waters damage but this view is challenged for cancers arising et al. investigated precancerous lesions of the larynx for in dysplasia [1, 2]. Aetiological factors involved in the HPV high-risk types and only 1 out of 15 resulted positive genesis and progression of laryngeal dysplasia (LD) and by in situ hybridization technique [12]. squamous cell carcinoma (SCC) include environmental Pagliuca et al. analyzed 30 samples with histologically factors (carcinogen exposure), genetic changes, epigenetic proven LD from smokers and non-smokers by polymerase aberrations and immune escape. Tobacco and alcohol con- chain reaction (PCR). HPV-DNA was not detected in any sumption are the most important single factors, implicated (0%) of the samples [11]. Though the association between in 75% of all head and neck SCC (HNSSC) and have a LD and HPV is well-known, the correct prevalence should multiplicative combined effect [2]. Additionally, the role be confirmed in larger, prospective series of patients evalu- of occupational factors have also been assessed [3]. From ated by multiple DNA and RNA testing, because DNA-PCR a recent review of the literature, the authors conclude that alone might overestimates the proportion of virus driven there is insufficient evidence to support a causal role of LD [13]. reflux in laryngeal SCC, mainly because of the confound- In other entities such as cervical carcinoma, HPV-associ- ing effect of tobacco and alcohol consumption and the ated premalignant lesions are well documented, whereas in inaccuracies in the diagnosis of reflux [4]. Human papil- oropharyngeal SCC this phenomenon is lacking [14]. Only lomavirus (HPV) infection is now considered a potential a few studies with small numbers of patients have investi- aetiological agent and will be covered separately in a fol- gated the role of HPV in premalignant lesions of the lar- lowing section. Precancerous laryngeal lesions occur with ynx and detection of high-risk types was lacking. There is a an estimated annual incidence in the United States of 10.2 fundamental difference between HPV-DNA detection in the lesions per 100,000 males and 2.1 lesions per 100,000 healthy population, LD and laryngeal cancer, and the rates females [5]. The current annual incidence of LD in Europe of biological activity of HPV-DNA often remains unclear. is not known. Higher rates of HPV detection seem more likely to be related to transient HPV infections and future studies are necessary to clarify the impact of HPV in pathogenesis. The role of HPV in LD Clinical presentation More than 200 different HPV genotypes have been distin- guished and those can be divided into low-, intermediate-, LD usually occur at the level of the vocal cords. In the supra- and high-risk types. Recurrent laryngeal papillomatosis is glottis and/or subglottis it is usually asymptomatic and diag- primarily caused by low-risk HPV types 6 and 11. The risk nosed only incidentally during examinations performed for of malignant transformation in laryngeal papillomatosis other reasons. The guiding symptom of LD, like for any is reported to be 1–7% [6]. In the paediatric population other pathology at the level of the vocal cords, is an altered earlier onset of papillomatosis and pulmonary disease are voice production. Since this symptom is unspecific and can predictors of dysplastic transformation whereas in adults be observed in all pathological alterations of the vocal cords, the risk is higher for patients with later onset [7]. In a it simply indicates the need for a proper laryngeal evalua- large German series, the incidence of airway carcinoma tion, to be performed by an otolaryngologist using at least in recurrent respiratory papillomatosis (RRP) was 4% [8]. a transnasal (or transoral) fiberoptic instrument. Pain, swal- The rate for dysplasia (mild to moderate) was 7.5% for the lowing disorders or airway problems are rarely encountered juvenile-onset RRP, 12.1% for adult-onset RRP and 19.7% in LD. At laryngoscopy, leukoplakia, erythroleukoplakia, for single papillomas. Most dysplasias were located at the hyperkeratotic lesions, mucosal reddening or thickening glottic region [8]. HPV-DNA integration in RRP can be with exophytic tumour-like alterations may all be found in related to malignant transformation [9]. Rates of dysplasia LD and no single clinical diagnosis should be considered in laryngeal papillomatosis vary from 5 to 28% [7, 8, 10], pathognomonic for LD. However, that being said, LD fre- whereas prevalence of HPV in low- and high-grade dyspla- quently presents as laryngeal leukoplakia or hyperkerato- sia varies between 0 to 83% [10, 11]. HPV types that have sis. In a comprehensive review of the literature, Isenberg been detected are 6, 11, 16, 18, 33, and 57. Davids et al. et al. found no dysplasia in 53.6% out of 2188 laryngeal investigated 24 patients with laryngeal papillomatosis and leukoplakia biopsies [15]. Mild and/or moderate dysplasia and carcinoma in situ were present in 33.5% and 15.2% of 13 European Archives of Oto-Rhino-Laryngology (2021) 278:1717–1722 1719 biopsies, respectively. SCC developed in 3.7%, 10.1%, and which the pathologists can disagree. Conversely, a grading 18.1% of patients whose initial biopsies demonstrated no system that only had one category would be completely dysplasia, mild to moderate dysplasia, or severe dysplasia reproducible, but useless clinically. It is also important and carcinoma in situ. to remember that it is possible to be reproducibly wrong. Disagreement between pathologists provides more infor- mation about the correct degree of dysplasia than agree- Classification ment [17], so that for published studies in oral dysplasia having multiple observers is considered the gold standard Clinicians often do not understand why dysplasia grading method. Integration of different observations is usually cannot be easily standardised. The reasons lie in the very done by consensus, involving a third observer if required, varied histological appearances, confounding non-specific as described by Speight et al. [18]. changes such as inflammation, lack of understanding of the The best data on LD grading and outcome comes from the underlying process and the difficulty of proving validity of Ljubljana group who have published over many years, now grading schemes through long term outcome studies. Meth- with over 30 years follow-up data [19]. Unfortunately, during ods for grading LD were initially influenced heavily by those that period their grading system has changed its terminol- used in the cervix, where dysplasia progresses from the basal ogy and its difficult definitions have led to it being adopted layer to the surface. However, this is inappropriate in all in only a few centres. Its excellent evidence base has led upper aerodigestive tract mucosa where the mucosa kerati- to this system forming the framework of the World Health nises and changes are driven by tobacco carcinogens and not Organization (WHO) system published in 2017 [20]. This HPV integration in the basal layers. Application of cervical divides LD into only two categories of low-grade and high dysplasia scoring systems significantly under-grades laryn- grade dysplasia. The WHO book indicates that a category geal dysplasia. However, these old concepts remain in both of carcinoma in situ may still be used but the criteria, ‘com- oral and laryngeal dysplasia grading systems. plete loss of stratification and/or severe cytological atypia There is no agreement on the nature of the spectrum of and atypical mitoses’ allow considerable flexibility in their changes seen in precursor lesions, some considering all as application. This additional category allows pathologists to dysplastic, while others accepting hyperplasia as an initial provide a diagnosis of malignancy rather than dysplasia and stage in carcinogenesis. A particular problem is the defini- may be helpful in centres where radiotherapy (RT) is used tion of carcinoma in situ, and whether it can be identified for these most severe changes. Table 1 shows the evolution and merits a separate designation. of the WHO grading system for LD. The reference standard for any dysplasia grading system Recent grading systems places emphasis on architectural must be the clinical outcome. Unfortunately outcome studies changes but still define the difference between dysplasia are difficult to perform and require long follow up periods. grades primarily in terms of the thickness of the epithelium For this reason many published papers concentrate either involved, up to half the thickness being considered low- on case control series or on interobserver agreement. Case grade provided the upper layers have normal maturation. control studies have merit but suffer the problem that the The effect of this definition is to ensure that hyperplasia, tissue being examined microscopically is not the area that inflammatory and reactive changes are safely grouped into became malignant. Outcome studies can improve on this by the low-grade lesions. However, it leaves most truly dysplas- including multiple biopsy samples over many years. tic lesions in a single group without identifying the high- Published papers on interobserver agreement often sug- est risk ones and cannot clearly differentiate low-risk from gest that dysplasia grading is inaccurate and of limited clini- non-risk lesions [21]. If follow-up studies using a 3/4 grade cal value of limited clinical value. This negative impression system published since 2000 are compared, it is striking that arises largely through analysis using the kappa statistic, within any individual study there is not a good correlation which is inappropriate for grading continuum [16]. Using between the risk of malignant transformation and the degree this analysis for dysplasia grading, it is almost impossi- of dysplasia, but that the mean values show good correla- ble to prove better than ‘moderate’ agreement between tion in metanalysis [22]. It can be concluded that the risk of pathologists. malignant transformation does rise with increasing grade of The histological changes of dysplasia form a continu- dysplasia, but unpredictably so. ous spectrum and it is not expected that pathologists will From the clinical point of view, it is natural to look to the be able to agree on an exact grade of dysplasia in every published data to identify risk of malignant transformation. case. Despite this, data from interobserver agreement stud- However, transformation will only occur in a minority of ies are used to promote binary rather than 3 or 5 grade even high-risk lesions and may take many years to develop. scales. It is evident that if there are more grades of dyspla- sia, then there are more boundaries between grades over 13 1720 European Archives of Oto-Rhino-Laryngology (2021) 278:1717–1722 Table 1  Comparison of grading systems for LD, after WHO 2017 [20], with corrected levels. The published version limits low-grade dysplasia to the lower third, rather than the lower half of the epithelium Level of abnormal WHO 2005 Classifica- SIN Classification [35] Ljubljana Classification Amended Lju- WHO 2017 [20] maturation (WHO tion [34] [33] bljana classifica- 2005) tion [19] Squamous hyperplasia Squamous hyperplasia Squamous hyperplasia Low grade SIL Low grade dysplasia Lower 1/3 Mild dysplasia SIN 1 Basal/parabasal hyper- plasia 1/3 to 1/2 Moderate dysplasia SIN 1 or 2 Atypical hyperplasia High grade SIL Upper 1/2–3/4 SIN 2 High grade dysplasia * Full thickness Severe dysplasia Carcinoma in situ Carcinoma in situ Carcinoma in situ SIN squamous intraepithelial neoplasia, SIL intraepithelial lesion * A grade of carcinoma in situ may be used if a three-tiered system is preferred Lessons learned from oral dysplasia interdependent both on each other and on the normal cells for some growth factors, nutrients, and survival signalling. This It has not proved possible to define a single epithelial dyspla- evolutionary model of dysplasia is established in Barrett’s sia grading system applicable to all HN sites [20], because oesophagus [28] and the mouth [29–31] and has a number of the nature of the diseases causing dysplasia at different sites important consequences for our understanding of the develop- vary, as do the histological changes. For the oral cavity, the ment of cancer. WHO system continues to recommend a three grade division Dysplasia, at least in the mouth, is not a process that is into mild, moderate, and severe dysplasia and considers a progressing to cancer but, rather, the result of random muta- two grade system unproven [20]. Carcinoma in situ is con- tions and chromosomal changes that generate primed cells sidered synonymous with severe dysplasia. susceptible to further genetic changes. In a seminal study, Dysplasia in larynx differs from other upper aerodigestive Makarev et al. performed pathway analysis to identify which tract sites such as mouth for many reasons including expo- of the many cancer-related signalling pathways were involved sure to tobacco carcinogens in only the vapour phase, and in oral cancers and precursor lesions [32]. They showed that induction of four times as many mutations per cell in larynx cancer was associated with activating mutations in one of five than oral cavity [23]. In addition there are a number of oral main pathways, the AKT/mTOR, RAS/RAF/MAPK, JAK/ potentially malignant disorders that are not necessarily asso- STAT, WNT or TGF beta pathways. When individual path- ciated with smoking. Oral lesions also show a greater range ways were examined in cancers and precursor lesions, it was of cytological atypia in low-grade dysplasia than laryngeal striking that the latter showed inhibition of almost all pathways lesions [24] and severe dysplasia can affect only the lower studied, while the cancers showed pathway activation. The third of the epithelium. only pathways showing activation in dysplastic lesions were Reviewing the outcome data for the Ljubljana/WHO 2017 those associated with apoptosis and cell death. system shows the overall risk for even a high-grade lesion Interestingly, 20 of 68 precursor lesions had carcinoma-like in the larynx is relatively low when compared this with the genetic changes but, even in this minority, only 7 progressed equivalent situation in the oral cavity. Oral severe dysplasia to cancer in a median period of six years. Twenty-eight other has more than three times the risk of malignant transforma- precursors progressed to cancer without developing cancer- tion at 15 years as compared to laryngeal high grade dyspla- like molecular changes first. These workers and others work- sia [19, 20, 25, 26]. ing on exome sequencing have all concluded that there is no The molecular nature of oral lesions is much better under- predictable genetic pathway to carcinoma in the oral cavity. stood than laryngeal lesions. For the oral cavity, the traditional Clinicians need to develop an understanding that dysplastic hypothetical pathways to cancer involving accumulated muta- changes are not the start of a relentless progression to cancer. tions [27] in a specific order are wrong. We now understand that the genetically damaged cells in dysplastic oral epithelium accumulate genetic changes randomly and do not form a sin- Conclusions gle clone that spreads laterally to colonise the tissue. Rather, numerous clones of cells coexist within a dysplastic epithe- LD has long been recognized as a precursor lesion to laryn- lium, each with different types of mutation, gene inactiva- geal SCC. Tobacco and alcohol consumption are still to be tion profile, and chromosomal damage. Clones are probably considered the single most important etiological factors for 13 European Archives of Oto-Rhino-Laryngology (2021) 278:1717–1722 1721 the development of LD, while the role of acidic reflux and occupational exposure? Scand J Work Environ Health 40(3):315– HPV need to be investigated in deeper details. Dysplasia 322. https​://doi.org/10.5271/sjweh​.3403 4. Coca-Pelaz A, Rodrigo JP, Takes RP, Silver CE, Paccagnella D, may have varying clinical presentations including leukopla- Rinaldo A, Hinni ML, Ferlito A (2013) Relationship between kia, erythroleukoplakia, mucosal reddening or thickening reflux and laryngeal cancer. 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Huebbers CU, Preuss SF, Kolligs J, Vent J, Stenner M, Wieland U, Silling S, Drebber U, Speel EJ, Klussmann JP (2013) Inte- Funding No funding was obtained for this review. gration of HPV6 and downregulation of AKR1C3 expression mark malignant transformation in a patient with juvenile-onset Compliance with ethical standards laryngeal papillomatosis. PLoS ONE 8(2):e57207. https​://doi. org/10.1371/journ​al.pone.00572​07 Conflict of interest All authors declare that he/she has no conflict of 10. Davids T, Muller S, Wise JC, Johns MM 3rd, Klein A (2014) interest. Laryngeal papillomatosis associated dysplasia in the adult popu- lation: an update on prevalence and HPV subtyping. Ann Otol- Ethical approval This review did not include individual patient data RhinolLaryngol 123(6):402–408. https​://doi.org/10.1177/00034​ and was conducted in accordance with the ethical standards of the insti- 89414​52684​8 tutional and/or national research committee and with the 1964 Helsinki 11. Pagliuca G, Martellucci S, Degener AM, Pierangeli A, Greco A, declaration and its later amendments or comparable ethical standards. Fusconi M, De Virgilio A, Gallipoli C, de Vincentiis M, Gallo A (2014) Role of human papillomavirus in the pathogenesis of laryngeal dysplasia. Otolaryngol Head Neck Surg 150(6):1018– Open Access This article is licensed under a Creative Commons Attri- 1023. https​://doi.org/10.1177/01945​99814​52574​9 bution 4.0 International License, which permits use, sharing, adapta- 12. Waters HH, Seth R, Hoschar AP, Benninger MS (2010) Does tion, distribution and reproduction in any medium or format, as long HPV have a presence in diffuse high grade pre-malignant lesions as you give appropriate credit to the original author(s) and the source, of the larynx? Laryngoscope 120(Suppl 4):S201. https​://doi. provide a link to the Creative Commons licence, and indicate if changes org/10.1002/lary.21668​ were made. 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