Human papillomavirus in laryngeal and hypopharyngeal lymphoepithelial carcinoma.

The involvement of human papillomavirus (HPV) in laryngeal and hypopharyngeal lymphoepithelial carcinoma was investigated in a series of ten cases (seven laryngeal and three hypopharyngeal), retrieved from the files of three tertiary hospitals in the 2000-2017 period, through polymerase chain reaction with SPF10 primers and INNO-LiPA HPV Genotyping Extra II (Innogenetics). Epstein-Barr virus (EBV) was tested in all cases with in situ hybridization INFORM EBER Probe (Ventana Medical Systems). p16 and p53 expression were immunohistochemically analyzed. Calculated annual incidence was 0.013/100,000, and prevalence was 0.2% of laryngeal and hypopharyngeal carcinomas. All cases were EBV negative. HPV was detected in five cases, three of which also overexpressed p16. HPV16 was detected in four cases, and HPV58 in one case. Five cases were HPV negative, only one of these five overexpressed p16. No recurrence was observed in nine cases during follow-up. The 5-year disease-specific-survival rate was 100%. Mean overall survival was 87 months. Lymphoepithelial carcinoma of the larynx and hypopharynx are not related to EBV. Simultaneous HPV+/p16+ is consistent with HPV causation in a fraction of laryngeal and hypopharyngeal lymphoepithelial carcinomas.

Differential Diagnosis in Neuroendocrine Neoplasms of the Larynx.

The differential diagnosis of neuroendocrine neoplasms of the larynx is broad and includes lesions of epithelial, mesenchymal, and neuroectodermal origin. These lesions have overlapping clinical and pathologic aspects and must be carefully considered in the differential diagnosis of laryngeal neoplasms. The prognosis and treatment are also different among these tumor types, which necessitates making these distinctions clinically. The current literature was reviewed to provide updated information regarding the epithelial-derived tumors, including carcinoid, atypical carcinoid, small cell neuroendocrine carcinomas, large cell neuroendocrine carcinoma, and squamous cell carcinoma with neuroendocrine component. These tumors are compared and contrasted with non-epithelial-derived tumors such as paraganglioma and nonmucosal tumors, such as medullary thyroid carcinoma. The morphologic and cytologic features are discussed, along with helpful immunohistochemical and ancillary investigations.

How phenotype guides management of non-conventional squamous cell carcinomas of the larynx?

Although the majority of laryngeal malignancies are the conventional squamous cell carcinomas (SCC), a wide variety of malignant epithelial tumors can affect the larynx. Current treatment guidelines are designed to guide clinicians in management of conventional laryngeal SCC. Less is known about the biological behavior and responsiveness to therapy and overall outcomes of other malignant epithelial lesions. Because a spectrum of disease biology is represented by these rare phenotypes, an understanding of the basic biology can help direct management to optimize clinical outcome in this group of patients. This review provides a critical analysis of literature relating to the diagnosis, management, and outcome of patients with non-conventional squamous malignant epithelial neoplasms of the larynx. Particular attention is paid to features which are at variance with the conventional SCC and how these impact on management of these rare tumors.

Laryngeal Squamous Intraepithelial Lesions: An Updated Review on Etiology, Classification, Molecular Changes, and Treatment.

Laryngeal carcinogenesis is a multistep process, characterized by an accumulation of genetic changes associated with architectural and cytologic alterations, ranging from squamous hyperplasia to carcinoma in situ and encompassed by the terminology of squamous intraepithelial lesions (SILs). The etiology, classification, genetic changes, and malignant progression of these lesions are reviewed. Tobacco remains the principal etiological factor with gastroesophageal reflux disease recently considered as a possible factor. In contrast, there is little evidence that microbiological agents, especially human papillomavirus infection, are frequently involved in laryngeal carcinogenesis and probably <10% of SILs are driven by biologically active human papillomavirus infection. Light microscopy, despite a degree of subjectivity, remains the mainstay of accurate diagnosis, prognosis, and guidance for a patient's treatment. The currently used classifications, the dysplasia system, squamous intraepithelial neoplasia, and the Ljubljana classification, reflect different standpoints on this important topic. The modified Ljubljana classification, with good interobserver agreement, could be considered as a proposal for a unified classification of laryngeal SILs. This review also briefly discusses recently discovered genetic changes, such as CDKN2A and CTNNB1 genes, and chromosome instability of chromosomes 1 and 7; however, none of these can at present improve histologic diagnosis. Malignant progression of precursor lesions varies from 2% to 74%, according to different studies. Cold-steel microinstruments, CO2 laser, and radiotherapy are used to treat the different grades of precursor lesions. There is as yet no worldwide agreement on the treatment of high-grade lesions and carcinoma in situ.

High-risk human papillomavirus is transcriptionally active in a subset of sinonasal squamous cell carcinomas.

It has been reported that high-risk human papillomavirus (HPV) is a causative agent of a subgroup of oropharyngeal carcinomas. In these tumors, the presence of the transcriptionally active HPV has been proved through the identification of HPV E6 or E7 messenger RNA (mRNA) transcripts. The aim of the study was to assess the HPV-active transcription in a series of sinonasal carcinomas, in correlation with the HPV DNA identification and the p16 immunohistochemistry. Seventy patients with squamous cell carcinomas of the sinonasal tract were included in the survey. The main clinicopathological characteristics were recorded. All tumors were investigated for HPV through the HPV DNA detection by PCR, using the SPF10 primers and by in situ hybridization, using the high-risk GenPoint probe (Dako, Glostrup, Denmark). HPV16 E7 mRNA transcripts detection was performed by RT-PCR in 27 cases. The immunostaining for p16 was performed in all cases. Fourteen carcinomas (20%) were positive for high-risk HPV by PCR: 13 HPV16 and one HPV35. In situ hybridization showed a dotted nuclear positivity in all these cases. HPV16 E7 mRNA was detected in seven tumors harboring HPV16; in the remaining HPV-positive cases, RNA did not reach the quality for analysis. Strong, diffuse positivity for p16 was observed only in the HPV-positive cases. The 14 HPV-positive squamous cell carcinomas were non-keratinizing or scarcely keratinizing tumors. No significant differences were found in terms of gender, age, or staging at diagnosis between HPV-positive and HPV-negative tumors. However, differences in disease-free survival and overall survival between both groups of patients were significant (P=0.004 and P=0.028, respectively). In conclusion, we have shown that HPV is the etiological agent of a subset of sinonasal carcinomas demonstrating the transcriptionally active HPV in these tumors. Immunostaining for p16 can be used as a surrogate marker to identify these tumors.

Evaluation of a new grading system for laryngeal squamous intraepithelial lesions--a proposed unified classification.

AIMS: To verify the applicability, reproducibility and predictive value of a proposed unified classification (amended Ljubljana classification) for laryngeal squamous intraepithelial lesions (SILs). METHODS AND RESULTS: Six internationally recognized experts and three pathologists from Ljubljana contributed to this study by evaluating a set of laryngeal SILs using the new system: low-grade SIL, high-grade SIL, and carcinoma in situ (CIS). The overall agreement among reviewers was good. Overall unweighted and weighted κ-values and 95% confidence intervals were 0.75 (0.65-0.84) and 0.80 (0.71-0.87), respectively. The results were stratified between the international reviewers and the Ljubljana pathologists. The former had good overall agreement, and the latter had very good agreement. Kaplan-Meier survival curves showed a significant difference (P < 0.0001) between patients with low-grade and high-grade SILs; 19 of 1204 patients with low-grade SILs and 30 of 240 patients with high-grade SILs progressed to malignancy in 2-15 years and in 2-26 years, respectively. CONCLUSIONS: The proposed modification to the Ljubljana classification provides clear morphological criteria for defining the prognostic groups. The criteria facilitate better interobserver agreement than previous systems, and the retrospective follow-up study demonstrates a highly significant difference in the risk of malignant progression between low-grade and high-grade SILs.

Molecular diagnostic alterations in squamous cell carcinoma of the head and neck and potential diagnostic applications.

Head and neck squamous cell carcinoma (HNSCC) is a common malignancy that continues to be difficult to treat and cure. In many organ systems and tumor types, there have been significant advances in the understanding of the molecular basis for tumorigenesis, disease progression and genetic implications for therapeutics. Although tumorigenesis pathways and the molecular etiologies of HNSCC have been extensively studied, there are still very few diagnostic clinical applications used in practice today. This review discusses current clinically applicable molecular markers, including viral detection of Epstein-Barr virus and human papillomavirus, and molecular targets that are used in diagnosis and management of HNSCC. The common oncogenes EGFR, RAS, CCND1, BRAF, and PIK3CA and tumor suppressor genes p53, CDKN2A and NOTCH are discussed for their associations with HNSCC. Discussion of markers with potential future applications is also included, with a focus on molecular alterations associated with targeted therapy resistance.

Expression of Ep-CAM, but not of E48, associates with nodal involvement in advanced squamous cell carcinomas of the larynx.

AIMS: To evaluate epithelial cell adhesion molecule (Ep-CAM) and E48 expression, and their relationship with histological differentiation and nodal metastasis, in laryngeal squamous cell carcinomas (SCC). METHODS AND RESULTS: The expression of Ep-CAM and E48 was investigated using immunohistochemistry in a series of 66 SCC (stages 3 and 4) and their adjacent non-neoplastic epithelia. Ep-CAM expression increased with the progression from normal squamous epithelium to SCC. It was detected in 96% of carcinomas and high levels of Ep-CAM expression (50% or more positive cells) were associated with poorer differentiation (P = 0.003) and the presence of lymph node metastases (P = 0.001). E48 expression was characteristically strong and diffuse in non-neoplastic squamous epithelium, and decreased with progression to SCC. Poorly differentiated (grade 4) tumours had lower proportions of E48-positive cells than well- to moderately- differentiated cases (P < 0.001). CONCLUSIONS: Expression of both Ep-CAM and E48 correlated with cell differentiation, although in inverse fashion. In particular, the association between high levels of Ep-CAM expression and high frequency of nodal metastases suggests that Ep-CAM plays a role in the development of lymph node metastases in SCC of the larynx.

Sinonasal tumors: a clinicopathologic update of selected tumors.

The sinonasal cavities show a wide variety of neoplasms of epithelial, mesenchymal, neural/neuroectodermal or hematopoietic origin. The differential diagnosis for these tumors may be difficult due to overlapping morphologies, variable patterns in ancillary studies, and potentially confusing terminology. In this report, an updated review of the spectrum of neoplasia is provided, using the World Health Organization 2005 classification as a guide. Classic tumors that are generally limited to the sinonasal tract are described and new information regarding molecular pathogenesis is reviewed. Also new entities that have the sinonasal tract as a site of predilection, such as sinonasal renal cell-like adenocarcinoma and NUT midline carcinoma are highlighted.

Methylthioadenosine phosphorylase inactivation depends on gene deletion in laryngeal squamous cell carcinoma.

AIMS: Methylthioadenosine phosphorylase (MTAP) is an essential enzyme for the methionine and adenosine salvage pathway in normal cells, frequently inactivated in many different human cancers. MTAP status could be important for tumour cell sensitivity to adjuvant chemotherapy. To our knowledge, there have been no reports to date on MTAP status in laryngeal carcinoma. METHODS AND RESULTS: A series of 31 laryngeal squamous cell carcinomas was investigated for MTAP mRNA expression using reverse transcription and quantitative polymerase chain reaction (qPCR), as well as for MTAP gene deletion and/or promoter hypermethylation using qPCR and methylation-specific PCR, respectively. Low MTAP mRNA expression was found in 32% of cases, and was associated with MTAP gene deletion (in 70%; P<0.001) but not with MTAP promoter hypermethylation, indicating that, in this tumour, gene deletion is the main mechanism for MTAP inactivation. Neither low mRNA expression nor gene deletion was associated with any of the clinicopathological parameters investigated. CONCLUSION: Given the significance of MTAP status for cell sensitivity to different chemotherapeutic regimens, our results suggest that determination of MTAP inactivation should be taken into consideration in managing laryngeal squamous cell carcinomas.

Activated growth signaling pathway expression in Ewing sarcoma and clinical outcome.

BACKGROUND: In Ewing sarcoma (EWS) most of the research on signaling pathways has been performed on cell lines or animal models. The objective of the current study was to determine the relation between clinical outcome and the expression of proteins involved in active growth signaling pathways. METHODS: A paraffin-embedded microarray of 45 human primary EWS tissue specimens was stained with the antibodies against c-KIT, AKT, p-AKT, p-mTOR, IGF-1R, IGFBP-3, MAPK, p27(KIP1) , and p70S6 kinase. Immunohistochemical staining was correlated with patient overall survival (OS). RESULTS: In the univariate analysis 3 variables showed statistical significance to predict survival: presence of metastasis, p-mTOR, and p27(KIP1). A positive stain for p-mTOR (hazard ratio of 4.74 [95% CI (57, 121)]) was significantly (log-rank test with a P = 0.029) associated with better OS. Also, a positive stain for p27(KIP1) (hazard ratio of 6.87 [95% CI (77, 136)] was significantly (log-rank test with a P = 0.009) associated with better OS. Multivariate analysis showed metastasis (HR: 4.3; 95% CI: 0.99, 19; P = 0.05), p-mTOR (HR: 4.8 with 95% CI: 0.6, 38; P = 0.13) and p27 (HR: 5.3; 95% CI: 1.37, 20; P = 0.01) as independent prognostic factors of outcome. CONCLUSIONS: In our series, p-mTOR and p27(KIP1) protein overexpression were independently associated with better survival.

Current diagnostic strategies for undifferentiated tumours of the nasal cavities and paranasal sinuses.

Several malignant tumours occurring in the sinonasal tract may present with an undifferentiated morphology. Overall, these lesions pose significant diagnostic difficulties for the surgical pathologist, especially in limited biopsy material, but their correct classification is becoming increasingly important for an appropriate treatment strategy. This review deals with the criteria for differential diagnosis of these neoplasms, with emphasis on recent advances in immunohistochemistry and molecular biology, as well as with previous progress in electron microscopy. Through careful microscopic examination of haematoxylin and eosin-stained sections, in the light of clinical information and imaging data, a list of differential diagnoses can be made and an appropriate panel of antibodies can be chosen to further categorize the tumour. An initial panel including cytokeratins, synaptophysin, S100 protein, desmin and CD45 may allow the classification of most lesions or may help to narrow the list of differential diagnoses. Further refinement can be obtained through second-line markers, including in-situ hybridization for Epstein-Barr virus, other neuroendocrine markers, melanocytic markers, myogenin, CD99, other lymphocyte markers, and CD138 and light chains. Finally, molecular analysis can further assist in the recognition of specific entities such as nuclear protein in testis midline carcinoma, Ewing's sarcoma/peripheral neuroectodermal tumour, alveolar rhadbomyosarcoma, and poorly differentiated synovial sarcoma.

Primary mucin-producing tumours of the salivary glands: a clinicopathological and morphometric study.

AIMS: To determine clinicopathological and morphometric features that discriminate between mucin-producing primary salivary gland carcinomas. MATERIALS AND RESULTS: Fifteen mucin-producing tumours were stratified into five colloid carcinomas (CCs), four mucinous cystadenocarcinomas (MCAs), three mucin-rich salivary duct carcinomas (SDCs) and three mucin-rich mucoepidermoid carcinomas (MECs). The mean patient age was 70, 58, 43 and 63 years for CC, MCA, SDC and MEC, respectively. Eleven of 15 patients were female. The majority of CC cases originated from major salivary glands; MCA showed a predilection for the minor salivary glands. No disease-related mortality was observed in the CC group; one patient died in the MCA group, and one in the SDC group. Receiver-operating characteristic curve analysis revealed an optimal cut-off point of 17% of the tumour cells in contact with stroma that best distinguished between the CC and MCA. Histomorphometric measurements revealed that CC was best differentiated from MCA by smaller nuclear size and more regular chromatin. CONCLUSIONS: Strict morphological criteria of CC coupled with assessment of the tumour cell/stroma relationship and the nuclear features facilitate discrimination between mucinous tumours of salivary gland.

Laryngeal Dysplasia: Persisting Dilemmas, Disagreements and Unsolved Problems-A Short Review.

We present the historical review and current state of the histopathological classifications and terminology of laryngeal precursor lesions. Attention to recent genetic findings is also presented; although in need of additional confirmation, these raise possibility for early detection of patients at risk of dysplasia progression. Although a number of identified genetic alterations with a promising diagnostic and prognostic value are emerging, none of the known genetic alterations can be currently implemented in clinical practice as a completely reliable diagnostic and/or prognostic marker. Regarding the terminology of precursor lesions, dysplasia remains the most frequently used term, but squamous intraepithelial lesion can be used as a synonym as well. Histological findings, in spite of certain degree of subjectivity, remain at present the most reliable method for an accurate diagnosis. The current 2017 WHO classification seems to successfully stratify risk of malignant progression, with a significantly different risk of malignant progression between low-grade dysplasia and high-grade dysplasia. In case of pronounced architectural disorders, severe cellular and nuclear atypias, and an increased number of mitoses, also atypical form, the high-grade dysplasia and carcinoma in situ can be separated. The Slovenian tertiary centers have a policy of surgical removal of high-grade SILs and life-long close follow-up. Radiotherapy is reserved for more pronounced intraepithelial lesions classified as carcinoma in situ and invasive cancer. Such a distinction can facilitate clinical decision to use radiotherapy if complete surgical removal is not possible.

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.

Transcription factors Snail, Slug, Twist, and SIP1 in spindle cell carcinoma of the head and neck.

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.

Silent sinus syndrome: combined sinus surgery and orbital reconstruction - report of 15 cases.

BACKGROUND: Silent sinus syndrome (SSS) is defined as spontaneous, painless enophthalmos, hypoglobus with orbital floor resorption and maxillary sinus collapse on the ipsilateral side. Different methods of orbital floor reconstruction have been proposed. AIMS/OBJECTIVES: The purpose was to analyse the results of combined endoscopic sinus surgery (ESS) and reconstruction using orbital floor implant of 15 patients with SSS and to present recent histological findings. MATERIALS AND METHODS: Retrospective case review of 15 patients with SSS treated in clinic between 2007 and 2017. RESULTS: Eleven women and four men presented with unilateral, spontaneous enophthalmos. Averaged duration of enophthalmos was 10.7 months. On affected side, mean enophthalmos was 2.6 mm and hypoglobus 2.7 mm. Computed tomography imaging (CT) imaging showed maxillary sinus opacification on the affected side in every case, and the orbital floor was displaced downwards in all cases. In total, 13 patients underwent simultaneous ESS and rebuilding of orbital floor with a titanium implant. Statistical analysis confirmed significant differences for pre- and postoperative measure of enophthalmos and hypoglobus. CONCLUSION AND SIGNIFICANCE: Implementation of titanium implants is the reliable method of reconstruction that allows good aesthetic result, shorter time of procedure with an excellent long-term outcome and satisfactory patient's tolerance.

Lymphomas of the head and neck region: an update.

The field of haematopathology is rapidly evolving and for the non-specialized pathologist receiving a specimen with the possibility of a lymphoid malignancy may be a daunting experience. The coincidence of the publication, in 2017, of the WHO monographies on head and neck and haematopoietic and lymphoid tumours prompted us to write this review. Although not substantially different from lymphomas elsewhere, lymphomas presenting in this region pose some specific problems and these are central to the review. In addition, differences in subtype frequency and morphological variations within the same entity are discussed. The difficulty in diagnosis related to some specimens led us to briefly mention common subtypes of systemic lymphomas presenting in the head and neck region.