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Simple Summary: PD-L1 expression determines patients' eligibility for immunotherapy. Current sampling does not consider the heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC) within the primary tumor. Moreover, potential differences are not considered when comparing primary tumors and their associated metastases or local recurrences, hereby excluding potential responders to immunotherapy. Here, we investigated the inter-sample heterogeneity of PD-L1 expression by analyzing multiple samples from individual patients. Multisection staining revealed clinically relevant CPS changes, which would have potentially affected treatment decisions in 28.7% (intra-tumoral), 44.4% (tumor vs. metastases), and 61.5% (initial tumor vs. local recurrence) of patients, respectively, compared with single-region staining. Increased CPS in primary tumors and lymph node metastases were associated with improved 5-year overall survival. Our results suggest that multiple tumor sections should be evaluated in HNSCC patients when assessing PD-L1 expression prior to potential immunotherapy, particularly if the initial result was negative. For practical reasons, in many studies PD-L1 expression is measured by combined positive score (CPS) from a single tumor sample. This does not reflect the heterogeneity of PD-L1 expression in head and neck squamous cell carcinoma (HNSCC). We investigated the extent and relevance of PD-L1 expression heterogeneity in HNSCC analyzing primary tumors and recurrences (LRs), as well as metastases. Tumor tissue from 200 HNSCC patients was immunohistochemically stained for PD-L1 and analyzed using image-analysis software QuPath v3.4 with multiple specimens per patient. CPS was ≥20 in 25.6% of primary tumors. Intra-tumoral heterogeneity led to a therapeutically relevant underestimation of PD-L1 expression in 28.7% of patients, when only one specimen per patient was analyzed. Inter-tumoral differences in PD-L1 expression between primary tumors and lymph node metastasis (LNM) or LR occurred in 44.4% and 61.5% (CPS) and in 40.6% and 50% of cases (TPS). Overall survival was increased in patients with CPS ≥ 1 vs. CPS < 1 in primary tumors and LNM (hazard ratio: 0.46 and 0.35; p < 0.005); CPS in LR was not prognostic. Our analysis shows clinically relevant intra- and inter-sample heterogeneity of PD-L1 expression in HNSCC. To account for heterogeneity and improve patient selection for immunotherapy, multiple sample analyses should be performed, particularly in patients with CPS/TPS < 1. [ABSTRACT FROM AUTHOR]

(1) Background Oral squamous cell carcinomas (OSCC) are a common malignancy of the oral cavity and are often diagnosed when they have already spread to the regional lymph nodes. Advanced stages of cancer are characterized by the development of distant metastases. Angiogenesis, a hallmark of cancer, is known to contribute to cancer progression and metastasis. High microvessel density (MVD) has been linked to poor clinical outcomes in various types of cancer. (2) Methods: In this study, we aimed to investigate the spatial heterogeneity of blood vessels by comparing the tumor center and invasion front and to evaluate its prognostic value in OSCC. A total of 71 OSCC patient specimens were collected. The tissue was immunohistochemically stained using CD31 antibody to assess the MVD in the tumor center and the invasion front. Furthermore, the associations between the histopathological parameters, including MVD, disease-free survival (DFS), and overall survival (OS) were computed. (3) Results: In our study, we found a significantly higher presence of blood vessels at the invasion front of OSCCs compared to the tumor center. However, we did not observe any significant differences in MVD between different tumor stages. High intratumoral MVD was shown to be a positive prognostic factor for DFS (p = 0.047). (4) Conclusions: To the best of our knowledge, we were the first to analyze MVD as a prognostic factor by considering its spatial heterogeneity in OSCC. However, further studies are warranted to further elucidate the complexity of microvascular spatial heterogeneity and its influence on prognosis. [ABSTRACT FROM AUTHOR]

Metastatic cervical lymph nodes are a frequent finding in head and neck squamous cell carcinoma (HNSCC). If a non-surgical approach is primarily chosen, a therapy response evaluation of the primary tumor and the affected lymph nodes is necessary in the follow-up. Supplementary contrast-enhanced ultrasound (CEUS) can be used to precisely visualize the microcirculation of the target lesion in the neck, whereby malignant and benign findings differ in their uptake behavior. The same applies to many other solid tumors. For various tumor entities, it has already been shown that therapy monitoring is possible through regular contrast-enhanced sonography of the primary tumor or the affected lymph nodes. Thus, in some cases, maybe in the future, a change in therapy strategy can be achieved at an early stage in the case of non-response or, in the case of therapy success, a de-escalation of subsequent (surgical) measures can be achieved. In this paper, a systematic review of the available studies and a discussion of the potential of therapy monitoring by means of CEUS in HNSCC are presented. [ABSTRACT FROM AUTHOR]

Background: Evaluating the tumor microenvironment and its influence on clinical management and therapy response is becoming increasingly important. However, only a few studies deal with the spatial distribution of immune cells within the tumor. This study aimed to describe the topology of immune cells in the microenvironment of oral squamous cell carcinoma (OSCC) sectioned by tumor invasion front and tumor center and to test their prognostic relevance regarding patient survival. Methods: A total of 55 OSCC patient specimens were collected retrospectively. The cancer tissue was immunohistochemically stained using an automated tissue stainer Ventana Benchmark Ultra (Roche) and analyzed using discrete expression marker profiles on immune cells. We investigated CD4+ lymphocytes, CD8+ lymphocytes, CD68+ macrophages, CD163+ macrophages, and M1 macrophages regarding their spatial distribution. Results: The statistical analysis revealed that the quantity and distribution of CD4+ (p = 0.007), CD8+ (p < 0.001), CD68+ (p < 0.001), CD163+ cells (p = 0.004), and M1 (p < 0.001) macrophages were significantly higher at the invasion front compared to the tumor center in all observed cases. However, high and low immune cell counts in the tumor center and invasion front were not associated with overall survival. Conclusion: Our results show two distinct immune microenvironments of the tumor center compared to the invasion front. Future studies are needed to explore how these results can be leveraged to improve patient therapy and outcome. [ABSTRACT FROM AUTHOR]

To improve management of head and neck squamous cell carcinoma patients, we need to increase our understanding of carcinogenesis, to identify biomarkers, and drug targets. This study aimed to identify novel biomarkers by providing transcriptomics profiles of matched primary tumors, lymph node metastasis, and non-malignant tissue of 20 HNSCC patients as well as by bioinformatic analyses of a TCGA HNSCC cohort, comprising 554 patients. We provide cancer cell signaling networks differentially expressed in tumors versus metastases, such as mesenchymal–epithelial transition, and structural integrity networks. As a proof of principle study, we exploited the data sets and performed functional analyses of a novel cytokeratin, cytokeratin24 (cKRT24), which had not been described as biomarker for tumors before. Survival analysis revealed that low cKRT24 expression correlated with poor overall survival in HNSCC. Experimentally, downregulation of cKRT24 in primary tumors, metastases, and HNSCC cell lines was verified on mRNA and protein level. Cloning and ectopic overexpression of cKRT24 not only affected viability and growth of HNSSC cell lines, but also inhibited tumor growth in murine xenograft studies. We conclude that cKRT24 functions as a tumor suppressor in HNSCC, and may serve as an additional prognostic biomarker and novel target to support current HNSCC treatments. [ABSTRACT FROM AUTHOR]

The aim of this study was to examine the prevalence of synchronous upper aerodigestive tract (UAT) tumors within oral squamous cell carcinoma (OSCC) patients, and to specify distinct risk groups who benefit from panendoscopy. Definite clinical and pathohistological characteristics, as well as overall and recurrence-free survival (OAS and RFS, respectively) of OSCC patients with and without synchronous second UAT tumors, carcinomas in situ, or higher-grade dysplasia/metaplasia, were evaluated based on a retrospective population-based cohort study, including alignment with cancer registry data. Out of 727 included OSCC patients, 465 cases (64.0%) received panendoscopy. Among these, 18 UAT tumors were detected, all of which were linked to patients with a positive history of nicotine abuse. Every synchronous UAT tumor was revealed by panendoscopy, which, analyzed as an independent staging procedure, was accompanied by a low complication rate (1.7%). When illuminating the impact of a second UAT tumor in OSCC patients, survival analysis revealed reduced 5-year OAS (63.9% vs 43.5%, p = 0.010) and RFS (57.1% vs 32.4%, p = 0.016) for patients with a second oncology diagnosis of the UAT. Within the limitations of the study, it seems that panendoscopy should be performed in the majority of patients suffering from OSCC, because most of them have a history of smoking and drinking, which correlates with an increased risk of developing synchronous UAT tumors. [ABSTRACT FROM AUTHOR]

Treatment of metastasized or recurrent oral (OSCC) and oropharyngeal (OPSCC) squamous cell carcinoma remains challenging. Targeted antibody-based therapy inter alia for PD-1 / PD-L1 axis shows promising results, but whether PD-L1 expression varies between the subentities remains unclear. The expression pattern of PD-L1 (EPR19759 antibody, Abcam, Berlin, Germany) and p16 (CINtech® Histology Kit, Ventana, Oro Valley, USA) was determined immunohistochemically and analyzed by HALO™ Image Analysis Software (Indica Lab, Albuquerque, USA). For PD-L1, combined positivity score (CPS), tumor proportion score (TPS) and histoscore, were assessed and results correlated with epidemiological data. In total, 161 patients (OSCC: n = 78, OPSCC: n = 83) were included. A mean of 43.6% (±34.0%) of the specimen showed increased PD-L1 expression that did not differ quantitatively between subentities (TPS: p = 0.159, CPS: p = 0.078), but qualitatively (histoscore: p = 0.003). In the mean follow-up period (45.6 months), contrary to age (p = 0.006) and advanced T-Status (p = 0.018), PD-L1 expression did not correlate with overall (OS, p = 0.191) and recurrence free survival (RFS: p = 0.193) in both subentities. No correlation of p16 and PD-L1 expression was found (p = 0.844). PD-L1 is differentially expressed between OSCC and OPSCC, however without influence on OS. Furthermore, p16 status was not related to PD-L1 expression. This may have implications for future (immune) therapeutical approaches for oral cancer. [ABSTRACT FROM AUTHOR]

Background: Pembrolizumab and cetuximab are antibodies under investigation in head and neck squamous cell carcinoma (HNSCC) either as single agents or combined with cisplatin and other chemotherapeutic drugs, e.g., 5-fluorouracil and/or docetaxel. However, also the combination of both antibodies may have potential in recurrent/metastatic (R/M) HNSCC, in particular in cisplatin-resistant or -refractory cases or patients with comorbid disease, e.g. patients with impaired renal function. Methods: To clarify potential benefit that may result from such combination, we used the FLAVINO assay, a short-time ex vivo assay to compare responsiveness of HNSCC to pembrolizumab, cetuximab and both combined regarding colony formation of epithelial cells of biopsy-derived tumor samples and their cytokine production within three days either without or with stimulation with 10 ng/mL interferon gamma (IFN-γ). Vascular endothelial growth factor A (VEGF), monocyte chemoattractant protein 1 (MCP-1 or CCL2), interleukin 6 (IL-6), IL-8, IFN-γ, and interferon gamma-induced protein 10 (IP-10 or CXCL10) in supernatants were measured by ELISA. Results: We detected huge heterogeneity in response to cetuximab, pembrolizumab and both combined with and without IFN-γ stimulation. Moreover, we detected a link between IFN-γ induced IP-10 release and improved outcome in those HNSCC patients who were capable to respond to IFN-γ and pembrolizumab, cetuximab and both combined with a further increase in IP-10 production. We derived an "IP-10 score" that independent from clinical characteristics of HNSCC patients and therapy regimens applied was able to predict their outcome. Conclusions: The heterogeneity in the ex vivo response of cetuximab, pembrolizumab and both combined with and without IFN-γ stimulation identifies subgroups of HNSCC patients with deviating OS. [ABSTRACT FROM AUTHOR]

Squamous cell carcinomas of the head and neck are the subject of numerous current studies, especially in view of the increasing incidence of tumors induced by human papillomavirus (HPV) and the latest changes to the TNM classification of oropharyngeal squamous cell carcinoma (OPSCC). In addition to HPV status, the presence of extranodal extension of lymph node metastases represents an important risk and prognostic factor, which has now been integrated into the staging algorithm of the eighth edition of TNM classification for HPV-negative OPSCC. In the past numerous studies had shown a lack of prognostic significance of extranodal extension in HPV-associated tumors. However, extranodal extension–as a possible risk factor even in HPV-positive OPSCC–remains an important subject of current studies, which are now particularly characterized by high numbers of cases. In this paper, diagnostic methods and the prognostic significance of extranodal extension in surgically treated HPV-positive OPSCC are presented and discussed based on relevant literature, and the results of current publications are summarized. Further development of diagnostic criteria and procedures as well as international standardization of clinical diagnostics of extranodal extension should be encouraged. Several studies demonstrate that extranodal extension results in worse survival outcomes even in HPV-positive tumors, in contrast to results of previous studies. Consequently, whether the prognostic significance of extranodal extension is not actually relevant to outcome and the staging algorithm of HPV-positive OPSCC should be questioned and further analyzed. [ABSTRACT FROM AUTHOR]

The establishment of novel biomarkers in liquid biopsies of cancer patients has come more into focus in prognostic and diagnostic research efforts. Due to their prognostic relevance disseminated tumor cells or circulating tumor cells are the subject of intensive research and are discussed as early diagnostic indicators for treatment failure and the formation of micrometastases. A potential association of this early-systemic tumor component with poor prognosis of cancer patients could be already demonstrated for various entities including breast, colon, lung, melanoma, ovarian and prostate cancers. Thus, the detection of circulating tumor cells seems to be also applicable for minimal-invasive monitoring of therapy progress in head and neck cancer patients. A major problem of the use in clinical routine is that circulating tumor cells could not be detected by modern imaging techniques. To overcome these limitations highly sensitive detection methods and techniques for their molecular characterization are urgently needed allowing mechanistic understanding and targeting of circulating tumor cells. Especially the medical application of nanotechnology (nanomedical methods) has made valuable contributions to the field. Here, we want to provide a comprehensive overview on (nanomedical) detection methods for circulating tumor cells and discuss their merits, pitfalls and future perspectives especially for head and neck solid squamous cell carcinoma (HNSCC) patients. [ABSTRACT FROM AUTHOR]

Background: Human papillomavirus (HPV)-related head and neck squamous cell carcinoma represents an important subgroup of head and neck cancer, but HPV occurs also in the less common neuroendocrine carcinomas (NEC). The PD-1/PD-L1 pathway appears to be activated in pulmonary NEC and correlates with a higher mutation burden, but the potential of NEC to respond to checkpoint inhibitors is unknown to a large extent.Objectives: To determine the HPV status of NEC of the head and neck region and to investigate the expression of PD-1 and its ligands PD-L1 and PD-L2.Methods: Surgical tumor samples from 2006 to 2017 were analyzed. HPV status was determined by p16 immunohistochemistry (IHC) and multiplex PCR. IHC using the Cologne Score was performed for PD-1, PD-L1, and PD-L2.Results: Seven NEC tumor samples were analyzed, three of them showed HPV type 18. Expression of PD-1 and PD-L1 differed widely and showed no correlation to HPV status. IHC showed an overexpression of PD-L2 in most of the patients.Conclusions and Significance: A multicentric analysis of NEC is needed to further evaluate the role of HPV as well as immunocheckpoints with regard to inflammatory immune response in genesis and clinical course of this rare tumor entity. Biomarkers for selection of novel treatment regimens, including immunotherapeutic approaches, are warranted. [ABSTRACT FROM AUTHOR]

Recognizing the prognostic power differentiating HPV-associated oropharyngeal squamous cell cancer (OPSCC) from OPSCC with other causes, the UICC Cancer Staging Manual 8th edition realizes significant changes from the 7th edition. Purpose of this study was to evaluate the differences of prognostic impact between the 7th and the latest edition of TNM Classification as well as to examine risk factors like extranodal extension (ENE) and lymph node ratio (LNR) for HPV-mediated OPSCC. The study includes 255 patients with OPSCC and initial diagnosis between 2008 and 2015. HPV status was determined according to p16 immunohistochemistry (IHC) and all patients were classified as defined by 7th and 8th edition of UICC. Prognostic influence of ENE and LNR was analyzed for patients with HPV-mediated OPSCC. 41.2% of the OPSCC were p16-positive. Implementation of the 8th edition of the UICC lead to a better differentiation between the respective stages. Regarding HPV-positive OPSCC, Kaplan-Meier survival curves showed a significantly better overall survival (OS) for patients with a LNR ≤10% as well as for patients with negative ENE status (p = 0.004, p = 0.008). 8th edition of UICC achieves to differentiate properly between the UICC stages. However, the staging rule of ignoring ENE in HPV-mediated OPSCC should be further analyzed. Moreover LNR might be a possible additional prognostic factor – especially regarding HPV-positive tumors. [ABSTRACT FROM AUTHOR]

To evaluate the utility of lymph node ratio (LNR) as a potential prognostic predictor and to test whether LNR may be useful as a potential selection criterion for adjuvant treatment in patients with oropharyngeal squamous cell carcinoma (OPSSC). This retrospective study included 384 patients with regionally metastasized OPSCC who underwent primary surgery with or without adjuvant therapy from 1980 to 2010. LNR was calculated as the ratio of positive lymph nodes to the total number of lymph nodes removed during neck dissection. Statistical analysis using a Cox regression model was carried out. The 5-year disease-specific survival (DSS) was 73 %. An individual LNR peak at 0.1 was closest to the median of 0.0909, and both were set as cut-off values. Patients in the group greater than median had a hazard ratio (HR) of 2.472 for a DSS event; this was close to an HR of 2.513 for LNR >0.1. In multivariate analysis, LNR showed a markedly stronger HR with regard to survival in comparison with the grouped pN classification. The covariate treatment modality did not meet the assumption of the Cox regression, and it was therefore not possible to comment reasonably on the issue of whether LNR could be a potential selection criterion for adjuvant treatment. Lymph node ratio is in itself a valuable additional prognostic factor for risk stratification. According to the current results, the most valuable LNR for OPSSC is expected to be located in the range from 0.09 to 0.1. Further investigations in large prospective trials will be required to allow evidence-based recommendations for treatment decisions based on the LNR. [ABSTRACT FROM AUTHOR]

Conclusion: In pT2 floor of mouth cancer (FOMC), a standardized neck dissection (ND) should be carried out, due to the high risk of occult metastases. In cases of pT1 carcinomas with a clinically negative neck using high imaging standards and expertise a tight 'wait and watch' strategy can be used. Objective: To report on the oncologic results with primary surgical treatment of pT1/pT2 FOMC and to examine the benefit of elective ND in cN0. Methods: This was a retrospective study design including 216 patients who were treated between 1980 and 2010 for pT1/2 FOMC. The 5-year disease-specific survival (DSS), local and (loco)regional control based on the T and N classification and depth of infiltration were investigated. Results: DSS was 79.5% and (loco)regional control was 89.8%. DSS in patients staged pN0 was significantly higher, at 84.6%, than in those staged pN+, at 60%. ND was performed in 165 patients, and adjuvant therapy was administered in 124 patients. Ninety patients (41.7%) were staged cN0, and 53.3% of them underwent elective ND. The risk of occult metastases was 4.8% in pT1 and 28.6% in pT2. A depth of infiltration of ≥ 5 mm showed a markedly higher relative frequency of occult neck metastases and pN+ status. [ABSTRACT FROM AUTHOR]

Highlights from the article: Corrigendum to "Staging of oropharyngeal squamous cell carcinoma of the head and neck: Prognostic features and power of the 8th edition of the UICC staging manual" [Eur. J. Surg. Oncol.

Simple Summary: Growth factor expression is a negative prognostic factor in head and neck squamous cell carcinoma (HNSCC). Targeted therapy has a limited effect on the treatment of advanced stages due to evolving resistance mechanisms. The aim of this study was to assess the distribution of growth factor receptors in oropharyngeal squamous cell cancer (OPSCC) and evaluate their role in the context of the human papillomavirus status, prognosis and possible relevance for targeted therapy. Tissue microarrays of 78 primary OPSCC, 35 related lymph node metastasis, 6 distant metastasis and 9 recurrent tumors were manufactured to evaluate the expression of human epidermal growth factor receptor (EGFR/erbB/Her)1–4 and c-Met by immunohistochemistry. EGFR and c-Met are relevant negative prognostic factors especially in noxae-induced OPSCC. Thus, dual targeting of EGFR and c-Met could be a promising prospective target in OPSCC treatment. Frequent coexpression of assessed receptors represents a possible intrinsic resistance mechanism in targeted therapy. This study aimed to assess the distribution of growth factor receptors in oropharyngeal squamous cell cancer (OPSCC) and evaluate their role in the context of human papillomavirus (HPV) status, prognosis and potential relevance for targeted therapy. The protein expression of human epidermal growth factor receptor (Her)1–4 and c-Met were retrospectively assessed using semiquantitative immunohistochemistry on tissue microarrays and analyzed for correlations as well as differences in the clinicopathological criteria. Her1–4 and c-met were overexpressed compared to normal mucosa in 46%, 4%, 17%, 27% and 23%, respectively. Interestingly, most receptors were coexpressed. Her1 and c-Met were inversely correlated with p16 (p = 0.04; p = 0.02). Her2 and c-Met were associated with high tobacco consumption (p = 0.016; p = 0.04). High EGFR, Her3, Her4 and c-Met expression were associated with worse overall and disease-free survival (p ≤ 0.05). Furthermore, EGFR and c-Met expression showed raised hazard ratios of 2.53 (p = 0.02; 95% CI 1.24–5.18) and 2.45 (p = 0.02; 95% CI 1.13–5.35), respectively. Her4 was expressed less in distant metastases than in corresponding primary tumors and was correlated to a higher T category. EGFR and c-Met are relevant negative prognostic factors in OPSCC, independent of known clinicopathological parameters. We suggest dual targeting of EGFR and c-Met as a promising strategy for OPSCC treatment. [ABSTRACT FROM AUTHOR]