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In this article, we review the chapter on tumors of the larynx, hypopharynx, trachea and parapharyngeal space in the new edition of the WHO book, focusing on the new developments in comparison to the previous edition. Squamous cell carcinoma (SCC) and its variants are by far the most common malignancies at these locations, with very limited new insights. The most important is the introduction of new targeted treatment-checkpoint inhibitors, with a new task for pathologists, who may help to predict the response to treatment by analyzing the expression of targeted proteins in biopsy samples. Precancerous lesions remain a controversial topic and, similarly to other organs, it is acceptable to use the terms "dysplasia" or "squamous intraepithelial lesion" (SIL), but there is a slight difference between low-grade dysplasia and low-grade SIL: in the former, mild atypia must be present, while the latter also includes hyperplastic epithelium without atypia. Two approaches have been proposed: a two-tiered system with low- and high-grade dysplasia/SIL and a three-tiered system with an additional category, carcinoma in situ. We are still searching for reliable diagnostic markers to surpass the subjectivity in biopsy diagnosis, with a few potential candidate markers on the horizon, e.g., stem cell markers. Other tumors are rare at these locations, e.g., hematolymphoid, neuroendocrine and salivary gland neoplasms, and are no longer included in Chapter 3. They must be diagnosed according to criteria described in specific chapters. The same holds true for soft tissue tumors, with the exception of cartilaginous neoplasms, which are still included in Chapter 3., (© 2022. The Author(s).)

Aims: The transcriptional activity of high-risk human papillomaviruses (HR-HPV) within oropharyngeal squamous cell carcinomas (OPSCC) has been linked to improved survival of patients. HR-HPV mRNA silver in situ hybridization (SISH) was evaluated on a cohort of OPSCC and compared with viral HPV DNA tests and p16 expression. Clinical outcomes of HPV-driven OPSCC and non-HPV related OPSCC were also studied., Methods: We evaluated 67 OPSCC and 3 papillomas, obtained from 62 patients, for detection of HR-HPV DNA by PCR tests. The positive samples were additionally studied by the SISH method using three probes of HPV16, HPV18, and HP33, and for p16 expression detected by immunohistochemistry. SISH assays were evaluated for the presence/number and intensity of signals in cancer cells. Prognostic significance of HPV status in our cohort was evaluated with univariate and multivariate statistics., Results: According to the HR-HPV PCR tests, 46 (69%) OPSCC cases were HPV positive, while three papillomas were negative. Of total 46 HPV-positive OPSCCs, 43 cases were also SISH-positive, while p16 overexpression was found in 45 of 46 HPV positive OPSCC cases. In OPSCC specimens, the sensitivity and specificity of the combined SISH probes (HPV16 and 33) were both 100.00%, when compared to HPV PCR. HPV positivity of the tumors appeared significant for predicting progression-free survival, cause specific survival and overall survival in a multivariate setting., Conclusions: The recently developed mRNA SISH methodology can detect HPV-driven OPSCCs without any additional test in 79% of cases. Positive SISH signals enable the visualization of viral transcripts required to recognize clinically relevant HPV infection. However, rare and tiny signals require an experienced pathologist to establish a consensus interpretation of results. The currently applied HR-HPV mRNA SISH analysis may serve as a groundwork for additional studies., (Copyright © 2021 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Chapter 3 "Tumours of the hypopharynx, larynx, trachea, and parapharyngeal space" of the World Health Organization (WHO) Blue Book 2017 "Classification of Head and Neck Tumours" shows a shortened list of entities, especially due to reducing the number of benign and malignant soft tissue tumours, malignant melanoma and some others, which are transferred to more frequently affected regions of the head and neck. The basic concept of the new edition is to assimilate all advances concerning the discussed tumours in a shorter framework, appropriate for daily work. The main emphasis is on the most frequent lesions and tumors originating from the covering squamous epithelium. Laryngeal and hypopharyngeal conventional squamous cell carcinoma (CSCC), its variants and precursor lesions, occupy a major part of the chapter. New data on etiopathogenesis, with the focus on human papillomavirus (HPV) infection, are discussed in relation to the entities of the squamous epithelium. Although only a small fraction of these lesions are HPV-related, further studies are required for evaluation of the potential prognostic and therapeutic benefit of mRNA HPV determination. In contrast to earlier data, laryngeal and hypopharyngeal verrucous SCC, spindle cell SCC and basaloid SCC are not anymore considered as HPV-related tumours. New data on the pathogenesis of spindle cell SCC exhibiting divergent differentiation by epithelial-mesenchymal transition, are also briefly discussed. The most important innovation is brought by the section on precursor lesions, in which a unified two-tier classification, consisting of low- and high-grade dysplasia, is introduced. The proposed two-tier system can also be transformed into a three-tier classification for treatment purposes, with a distinction between carcinoma in situ and high-grade dysplasia. The reviewed morphological criteria of the proposed system are based on the amended Ljubljana classification. The section on laryngeal neuroendocrine carcinomas (NEC) represents a considerable improvement in terminology and classification. NEC are divided into well-, moderate- and poorly-differentiated neuroendocrine carcinoma. The latter is additionally divided into small cell NEC and large cell NEC (LCNEC). It is of extreme importance that LCNEC, which was associated in the WHO 2005 edition with atypical carcinoid/moderately differentiated neuroendocrine carcinoma, grade II, has now been transferred into the group of poorly differentiated NEC, grade III, displaying a specific morphology and poorer prognosis.

An increase in the incidence of oropharyngeal squamous cell carcinoma (OPSCC) was observed in several population-based registries and has been attributed to human papillomavirus (HPV) infection. In the present study, we aimed to assess the contribution of HPV infection to the burden of mucosal head and neck squamous cell carcinoma (HNSCC) in Slovenia. For this purpose, data from the nationwide Cancer Registry of Slovenia for cases diagnosed between 1983 and 2009 were analyzed to determine time trends of age-adjusted incidence rates and survival in terms of annual percentage change (APC) for HNSCC in potentially HPV-related and HPV-unrelated sites. In addition, determination of p16 protein, HPV DNA and E6/E7 mRNA was performed in a cohort of OPSCC patients identified from the prospective database for the years 2007-2008. In total, 2,862 cases of HNSCC in potentially HPV-related sites and 7,006 cases in potentially HPV-unrelated sites were identified with decreased incidence observed over the time period in both groups (-0.58; 95 % CI -1.28 to -0.13 and -0.90; 95 % CI -1.23 to -0.57). Regardless of the group, incidence trends for both genders showed a significant decrease in men and increase in women. In a cohort of 99 OPSCC patients diagnosed between 2007 and 2008, 20 (20.2 %) patients had HPV positive tumors and exhibited a superior outcome compared to HPV-negative patients. In conclusion, results of the epidemiologic and histopathologic study confirmed that HPV infection had no major impact on the incidence trends in the Slovenian patients with HNSCC and, specifically, OPSCC during the studied period.

Background: The purpose of this study was to assess the efficacy and toxicity of docetaxel, cisplatin/5-fluorouracil (TPF) induction chemotherapy and concomitant immunochemoradiotherapy with cetuximab and cisplatin in unresectable head and neck carcinoma., Methods: Treatment consisted of TPF induction chemotherapy (docetaxel 75 mg/m(2) day 2; cisplatin, 75 mg/m(2) day 2; and 5-fluorouracil 750 mg/m(2) days 1-4; 4 cycles), followed by radiotherapy (RT) and concomitant weekly cetuximab, (250 mg/m(2), after a loading dose of 400 mg/m(2)) and cisplatin (30 mg/m(2))., Results: Twenty-five of 30 patients completed 4 cycles of induction chemotherapy. Six or more concomitant infusions of cisplatin and cetuximab were administered in 13 of 25 and 18 of 25 patients, respectively. The 2-year locoregional control, disease-free survival (DFS), and overall survival (OS) were 47%, 47%, and 50%, respectively. Patients with grade ≥ 2 skin reaction to cetuximab had a superior outcome., Conclusion: The tested regimen was effective; however, cetuximab and low-dose cisplatin after induction TPF increased the treatment toxicity. A grade ≥ 2 skin rash correlated with improved efficacy., (© 2013 Wiley Periodicals, Inc.)

Association between verrucous carcinoma (VC) of the head and neck and human papillomaviruses (HPV) is highly controversial. Previous prevalence studies focused mostly on α-PV, while little is known about other PV genera. Our aim was to investigate the prevalence of a broad spectrum of HPV in VC of the head and neck using sensitive and specific molecular assays. Formalin-fixed, paraffin-embedded samples of 30 VC and 30 location-matched normal tissue samples were analysed, by using six different polymerase chain reaction-based methods targeting DNA of at least 87 HPV types from α-PV, β-PV, γ-PV and μ-PV genera, and immunohistochemistry against p16 protein. α-PV, γ-PV and μ-PV were not detected. β-PV DNA was detected in 5/30 VC (16.7%) and in 18/30 normal tissue samples (60.0%): HPV-19, -24 and -36 were identified in VC, and HPV-5, -9, -12, -23, -24, -38, -47, -49 and -96 in normal tissue, whereas HPV type was not determined in 2/5 cases of VC and in 6/18 normal tissue samples. p16 expression was detected in a subset of samples and was higher in VC than in normal tissue. However, the reaction was predominantly cytoplasmic and only occasionally nuclear, and the extent of staining did not exceed 75%. Our results indicate that α-PV, γ-PV and μ-PV are not associated with aetiopathogenesis of VC of the head and neck. β-PV DNA in a subset of VC and normal tissue might reflect incidental colonization, but its potential biological significance needs further investigation., (© 2013 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

The current state in the field of classifying oral and laryngeal precursor lesions, as proposed in the WHO 2005 Blue Book is not ideal. The results of various inter-observer studies have shown that the currently used grading systems, with different basic concepts and different terminology, cannot continue to be reliably used in the future. The different etiology of cervical and head and neck precursor lesions requires a classification designed to cater to the specificities of the head and neck region. Trying to harmonize different classifications of the oral and laryngeal precursor lesions, we have proposed four crucial steps to set up a unified classification of squamous intraepithelial lesions (SILs): (a) the classification should contain two grades, low-grade and high-grade lesions and, specifically for the larynx, an additional grade-carcinoma in situ (CIS) which must be separated from high-grade laryngeal SILs; (b) the terminology should be unified; our preference is for the term SIL over squamous intraepithelial neoplasia; (c) all leading morphological criteria for low- and high-grade lesions, as well as for CIS, should be clearly defined; (d) agreement between clinicians and pathologists should be achieved on the most appropriate choice of treatment of different grades of SILs in separate head and neck areas.

Lysosomal proteases cathepsins B and L (CB, CL) and their endogenous inhibitors stefins A and B (SA, SB) are associated with tumor cell invasion and metastasis. The purpose of this study was to determine the immunohistochemical (IHC) localization of these parameters in tissue sections of 65 patients with operable head and neck squamous cell carcinoma (HNSCC) and to evaluate the prognostic significance of the observed IHC reactions. In SCC cells, a dot-like staining pattern for CB and CL was especially polarized in the perinuclear area and for stefins the characteristic IHC pattern was a diffuse cytoplasmic reaction. Higher SA immunoreactivity scores were found prognostically advantageous in univariate survival analysis [locoregional control (LRC), P = 0.003; disease-free survival (DFS), P = 0.023; disease-specific survival (DSS), P = 0.030] and appeared significant for predicting LRC (P = 0.019) in a multivariate setting. Among node-positive extracapsular extension-negative patients, SA positivity correlated with a favorable outcome (LRC, P = 0.094; DFS, P = 0.013; DSS, P = 0.012). In conclusion, SA immunoreactivity in tumor cells was related to a favorable prognosis. In the neck node-positive extracapsular extension-negative subgroup, SA immunoreactivity scores can be used to identify patients at increased risk for disease relapse.

Introduction: The Abbott RealTime is a novel real-time PCR assay designed for concurrent individual genotyping of HPV16 and HPV18 and pooled detection of 12 HPV genotypes: HPV31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68 in cervical swab specimens. In this study, the performance of RealTime for detecting HPV in formalin-fixed, paraffin-embedded tissue specimens of head and neck cancers was compared to the Innogenetics INNO-LiPA assay, which allows identification of 28 HPVs, including all 14 covered by RealTime., Methods: A total of 60 FFPE tissue specimens obtained from the same number of patients with histologically confirmed cancer of the oral cavity or oropharynx were included in the study. Following DNA extraction using a Qiagen DNA Mini Kit, RealTime and INNO-LiPA were performed on all samples, as instructed by the manufacturers., Results: A 136-bp fragment of human beta-globin serving as an internal control in RealTime was successfully amplified from all 60 tissue samples included in the study. RealTime and INNO-LiPA showed 100% agreement and detected HPV DNA in 5/60 (8.3%) of the cancer samples, which all contained genotype HPV16., Conclusions: RealTime assay is a reliable, sensitive, and specific diagnostic tool for the detection and partial genotyping of targeted HPV genotypes in FFPE tissue specimens of oral and oropharyngeal cancer.

Aims: To investigate the expression of microRNAs miR-21, miR-31, miR-203, miR-125a-5p and miR-125b and proteins phosphatase and tensin homologue (PTEN) and p63 in verrucous carcinoma (VC) of the head and neck., Methods and Results: Thirty cases of VC, 50 cases of conventional squamous cell carcinoma (SCC) and 30 samples of normal epithelium of the head and neck were included. Real-time polymerase chain reaction and immunohistochemistry were used to analyse the expression of microRNAs and proteins, respectively. In comparison to normal epithelium, miR-21 was overexpressed in both VC and SCC and miR-31 was overexpressed in VC and in well- and moderately differentiated SCC. Levels of miR-203 were elevated in VC but unaltered or reduced in SCC, and levels of miR-125a-5p and miR-125b were reduced in VC but unaltered in SCC. PTEN was down-regulated in both VC and SCC, whereas p63 was down-regulated in VC but up-regulated in SCC. Differential expression of p63 in VC correlated inversely with the expression of miR-21 and miR-203., Conclusions: Differences between VC, SCC and normal epithelium in expression profiles of investigated molecules indicate their association with the pathogenesis and clinicopathological characteristics of VC. Our results suggest that some microRNAs and proteins, particularly miR-125b, miR-203 and p63, might be useful in the diagnosis of VC., (© 2012 Blackwell Publishing Ltd.)

Verrucous carcinoma (VC) is a variant of squamous cell carcinoma (SCC), characterised by its inability to metastasize. In contrast, hybrid carcinomas, composed of VC and foci of conventional SCC, harbour a metastatic potential. Correct pathohistological diagnosis is therefore crucial for the choice of treatment. There is mounting evidence that desmosomes are involved in several aspects of carcinogenesis. Previous studies have shown an altered expression of desmosomal components in conventional SCC, which was associated with tumour behaviour, but no data have been found on desmosomes in VC. We therefore analysed the expression of desmosomal components in biopsy samples of 21 cases of VC and 5 cases of hybrid carcinoma of the head and neck in comparison to 23 cases of conventional SCC and 47 samples of normal squamous epithelium of similar localisation, using immunohistochemistry and real-time reverse-transcription polymerase chain reaction. We found that the expression patterns of desmosomal components in VC were fairly similar to those in normal epithelium but differed significantly from those in conventional SCC. Immunohistochemical reactions against desmosomal components disclosed the foci of SCC in hybrid carcinomas. In conclusion, we believe that expression patterns of desmosomal components in VC are consistent with its less aggressive behaviour. Differential expression of desmosomal components between VC and SCC makes some desmosomal components potentially useful in the diagnostics of VC, especially for the detection of hybrid carcinoma.

MicroRNAs are small, noncoding RNAs that regulate gene expression by posttranscriptional regulation of target genes. miR-200 family and miR-205 have been shown experimentally to regulate epithelial-mesenchymal transition. As epithelial-mesenchymal transition is the postulated pathogenetic mechanism in spindle cell carcinoma, we analyzed the expression of these microRNAs in spindle cell carcinoma of the head and neck in comparison to conventional squamous cell carcinoma of similar location and stage. We also analyzed the expression of classic and desmosomal cadherins, which are believed to be important targets during epithelial-mesenchymal transition. Forty-five cases of spindle cell carcinoma and 45 cases of squamous cell carcinoma of the head and neck were analyzed using real-time polymerase chain reaction for microRNAs, and immunohistochemistry for classic cadherins (E- and N-cadherins) and desmosomal cadherins. We found a significant down-regulation of the miR-200 family and miR-205, loss of desmosomal cadherins, and an altered expression of classic cadherins in spindle cell carcinoma in comparison to squamous cell carcinoma. Down-regulation of the miR-200 family and miR-205 strongly supports the postulated role of epithelial-mesenchymal transition in spindle cell carcinoma. These microRNAs act on transcription repressors that were also up-regulated in our cases of spindle cell carcinoma, both on mRNA and on protein levels. The result is not only an altered expression of classic cadherins in adherens junctions but also a complete loss of desmosomal cadherins., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Spindle cell carcinoma (SpCC) is a biphasic tumor composed of squamous cell carcinoma (SCC) and malignant spindle cells. There is mounting evidence that epithelial-mesenchymal transition (EMT) plays an important role in the pathogenesis of SpCC. Transcription repression has recently emerged as a fundamental mechanism triggering EMT in experimental models. Our aim is to analyze the expression of transcription repressors Snail, Slug, Twist, and SIP1 in SpCC of the head and neck in comparison to SCC, matched for location and stage. Thirty cases of SpCC and 30 cases of SCC of the head and neck were included. Snail, Slug, Twist, and SIP1 expression was analyzed on mRNA and protein levels, using real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. By RT-PCR, we found upregulation of mRNA for transcription factors Snail, Slug, Twist, and SIP1 in SpCC when compared to SCC. This upregulation was statistically significant for Slug, Twist, and SIP1 but nonsignificant for Snail. Immunohistochemistry was performed for Snail, Slug, and SIP1 and demonstrated a positive reaction for Slug and SIP1 in all cases and for Snail in two thirds of SpCC cases. Our finding of upregulation of all four tested transcription factors supports the hypothesis that EMT plays an important role in the pathogenesis of SpCC of the head and neck.

Spindle cell carcinoma (SpCC) is a biphasic tumor composed of squamous cell carcinoma (SCC) and a malignant spindle cell component. There is mounting evidence that SpCC is a monoclonal neoplasm originating from a stem cell giving rise to both components. We tested the hypothesis that spindle cell phenotype might be related to the cadherin-catenin complex, which forms adherens junctions between cells. We analyzed the immunohistochemical expression of E- and N-cadherin, alpha-, beta- and gamma-catenin, and Snail-1, a transcription repressor of E-cadherin, in 30 cases of SpCC, and 30 cases of SCC of the head and neck. In SpCC, cadherin and catenin expression was similar in the SCC component, whereas in the spindle cell component, loss of E-cadherin and neo-expression of N-cadherin was found in 19 cases, loss of cadherins in seven, and their co-expression in four cases. Catenin expression were altered in 18 SpCCs. Snail-1 was found in 19 SpCC cases. In SCC, E-cadherin and catenins were expressed in all cases, and N-cadherin focally in five cases. Snail-1 was observed in the stroma. To summarize, in SpCC, there is an altered expression of the cadherin-catenin complex, associated with morphological transition from epithelial to spindle cell phenotype. These features are reminiscent of epithelial-mesenchymal transition (EMT). Our study thus indicates that EMT might play an important role in the pathogenesis of SpCC. This conclusion is further supported by our finding of Snail-1 expression, a potent inducer of EMT, in more than half SpCC cases.

Loss of heterozygosity (LOH) and microsatellite instability (MSI) have been recognized as important events in squamous cell carcinoma of the head and neck (HNSCC), suggesting involvement of both suppressor and mutator pathways. We analyzed 153 HNSCC with 8 Bethesda reference panel markers and 14 microsatellite markers selected from chromosomal regions known to harbor either tumor-suppressor genes or oncogenes. A combination of multiplex fluorescence-based polymerase chain reaction and automatic fragment analysis was performed. LOH was observed in 78% of all tumors. 2% to 17% LOH frequency was observed with Bethesda reference panel markers comparing to higher 8% to 48% LOH in chromosomal areas 3p, 9p, 11q, and 17p. LOH of 11q14.3 correlated with tumor grade. The proportions of high- and low-MSI tumors were 3% and 10%, respectively, but no mutation was identified in MLH1 and MSH2 mismatch repair genes. These results indicate the dominant role of the suppressor in comparison with the mutator pathway in HNSCC carcinogenesis.

To assess the potential involvement of putative tumor suppressors or metastasis suppressors on chromosome 16q in head and neck squamous cell carcinoma (HNSCC), we have examined 42 primary HNSCCs for loss of heterozygosity (LOH) at 16q and correlated these findings with the occurrence of cervical nodal metastases and other clinical parameters. Seven of the 42 (17%) HNSCCs examined displayed LOH at chromosome 16q24. Three of the seven HNSCCs showed LOH at all of the informative loci analyzed along the chromosome arm, whereas the other four showed only loss of a subset of markers. When LOH at 16q was correlated with clinical parameters, there was no significant correlation with age, sex, clinical stage, T stage, N stage or survival. However, there was a correlation between LOH at chromosome 16q24 and involvement of cervical lymph nodes. Of the seven HNSCCs that had lost heterozygosity at 16q24, six had local metastases to lymph nodes indicating that LOH at 16q24 may have predictive value for the metastatic potential of HNSCCs.

A rare case of intramuscular haemangioma of the middle scalene muscle occurring in a young patient is described and its surgical treatment discussed.