Effects of Vitamin D on tumor cell proliferation and migration, tumor initiation and anti-tumor immune response in head and neck squamous cell carcinomas.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) are among the six most common cancers, with a constantly poor prognosis. Vitamin D has been found to have antineoplastic and immunomodulatory properties in various cancers. This study investigated the impact of Vitamin D on the initiation and progression as well as antitumor immune response in HNSCCs, both in vitro and in vivo. METHODS: An immunocompetent, orthotopic oral carcinogenesis mouse model was used to examine the influence of Vitamin D3 substitution on HNSCC initiation and progression in vivo. Tumor immune infiltration was analyzed by immunohistochemistry targeting CD3, CD8, NKR-P1C, FOXP3, and CD163. Two HPV- and two HPV+ HNSCC cell lines were treated with 1,25-dihydroxyvitamin D3 to analyze effects on tumor cell proliferation, migration and transcriptomic changes using RNA-sequencing, differential gene expression and gene set enrichment analysis. RESULTS: Vitamin D3 treatment led to a significant suppression of HNSCC initiation and progression, while also stimulating tumor immune infiltration with CD3+, CD8+ and NKR-P1C+ cells and lowering levels of M2 macrophages and Treg cells in vivo. In vitro experiments showed an inhibition of HNSCC cell proliferation and migration in HPV+ and HPV- cell lines. RNA-sequencing showed significant regulations in IL6 JAK STAT3, hypoxia signaling and immunomodulatory pathways upon Vitamin D3 treatment. CONCLUSION: The findings of our study highlight the promising potential of Vitamin D in the therapeutic repertoire for HNSCC patients given its immune modulating, anti-proliferative and anti-migratory properties. Clinical transferability of those in vitro and in vivo effects should be further validated in clinical trials.

Prediction of lymph node status in patients with surgically treated head and neck squamous cell carcinoma via neck lavage cytology: A pilot study.

BACKGROUND: Neck dissection is a standardized surgical procedure for patients with head and neck squamous cell carcinoma (HNSCC) and plays a critical role in the choice of adjuvant treatment based on histopathological findings. Saline irrigation is routinely performed at the end of surgery. However, this irrigant is not used for diagnostic purposes. METHODS: Intraoperative irrigation of the neck dissection wound was performed in 56 patients with HNSCC (N = 93 neck dissections), and the cytological suspension obtained was processed via the liquid-based cytology (LBC) technique, Papanicolaou staining, and immunocytochemical staining. Microscopic preparations were screened for the presence of tumor cells and classified as positive, borderline, or negative. These results were correlated with the histopathological and clinical data. RESULTS: Neck lavage LBC demonstrated high diagnostic value in detecting lymph node metastases (N+) with extracapsular spread (ECS), with a specificity, sensitivity, negative predictive value, and positive predictive value of 93.1%, 100%, 100%, and 80%, respectively. Tumor cells were detected in 4.8% of N- cases, 20% of N+ cases without ECS, and 100% of N+ cases with ECS. Receiver operating characteristic curve analysis showed an area under the curve of 0.8429 for the prediction of N+ (p < .0001) and 0.9658 for the prediction of N+ with ECS (p < .0001). CONCLUSIONS: Differential lavage cytology can provide valid and rapid information on the lymph node status in patients with HNSCC and showed an excellent correlation with histopathology. Thus, neck lavage LBC may facilitate faster and more reasonable planning of adjuvant treatment and help improve the therapeutic management of patients with HNSCC.

Dual Sec62/Ki67 immunocytochemistry of liquid-based cytological preparations represents a highly valid biomarker for non-invasive detection of head and neck squamous cell carcinomas.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCC) are frequently diagnosed in advanced stages, which limits therapeutic options and results in persistently poor patient outcomes. The aim of this study was to use liquid-based swab cytology (LBC) in combination with dual immunocytochemical detection of migration and proliferation markers Sec62 and Ki67 in order to allow non-invasive early detection of HNSCC as well as to analyse the diagnostic validity of this method for predicting the malignancy of suspicious oral lesions. METHODS: 104 HNSCC patients and 28 control patients, including healthy patients (n = 17), papilloma (n = 1) and leukoplakia patients (n = 10), were included in this study. For all patients, an LBC swab followed by simultaneous immunocytochemical detection of Sec62 and Ki67 was performed. Immunocytochemical as well as cytopathological results were correlated with histological diagnoses and clinical findings. RESULTS: All HNSCC patients (100%) showed dual Sec62/Ki67 positivity, and all control patients except for the papilloma patient were negative for Sec62/Ki67 (96.4%), resulting in a 100% sensitivity and 96.4% specificity of Sec62/Ki67 dual stain for non-invasive detection of HNSCC. The positive predictive value was 99% and the negative predictive value was 100%. Sec62 expression levels showed a positive correlation with tumour de-differentiation (p = 0.0489). CONCLUSION: Simultaneous immunocytochemical detection of Sec62/Ki67 using LBC represents a promising non-invasive and easy-to-apply tool for the early detection of HNSCC in routine clinical practice. This novel technique can help to avoid incisional biopsies and reduce the frequency with which general anaesthesia is used in diagnostic procedures in patients with suspicious oral lesions.

Prognostic impact of intra- and peritumoral immune cell subpopulations in head and neck squamous cell carcinomas - comprehensive analysis of the TCGA-HNSC cohort and immunohistochemical validation on 101 patients.

Background: Due to the expanding role of immune checkpoint inhibition in the treatment of head and neck squamous cell carcinoma, understanding immunological processes in the tumor microevironment (TME) has strong translational importance. Though analytical methods for a comprehensive analysis of the immunological TME have constantly improved and expanded over the past years the prognostic relevance of immune cell composition in head and neck cancer TME largely remains ambiguous with most studies focusing on one or a small subset of immune cells. Methods: The overall survival (OS) of the TCGA-HNSC patient cohort comprising 513 head and neck cancer patients was correlated with a total of 29 different immune metrics including a wide spectrum of immune cell subpopulations as well as immune checkpoint receptors and cytokines using RNAseq based immune deconvolution analyses. The most significant predictors of survival among these 29 immune metrics were validated on a separate HNSCC patient cohort (n=101) using immunohistochemistry: CD3, CD20+CXCR5, CD4+CXCR5, Foxp3 and CD68. Results: Overall immune infiltration irrespective of immune cell composition showed no significant correlation with the patients' overall survival in the TCGA-HNSC cohort. However, when focusing on different immune cell subpopulations, naïve B cells (p=0.0006), follicular T-helper cells (p<0.0001), macrophages (p=0.0042), regulatory T cells (p=0.0306), lymphocytes (p=0.0001), and cytotoxic T cells (p=0.0242) were identified as highly significant predictors of improved patient survival. Using immunohistochemical detection of these immune cells in a second independent validation cohort of 101 HNSCC patients, we confirmed the prognostic relevance of follicular T helper cells, cytotoxic T cells and lymphocytes. In multivariable analysis, HPV negativity and advanced UICC stages were identified as additional prognostic biomarkers associated with poor outcome. Conclusion: Our study highlights the prognostic relevance of the immunological tumor environment in head and neck cancer and demonstrates that a more detailed analysis of immune cell composition and immune cell subtypes is necessary to accurately prognosticate. We observed the highest prognostic relevance for lymphocytes, cytotoxic T cells, and follicular T helper cells, suggesting further investigations focusing on these specific immune cell subpopulations not only as predictors of patient prognosis but also as promising targets of new immunotherapeutic strategies.

Expression of 3q Oncogene SEC62 Predicts Survival in Head and Neck Squamous Cell Carcinoma Patients Treated with Primary Chemoradiation.

Primary chemoradiotherapy (CRT) is an established treatment option for locally advanced head and neck squamous cell carcinomas (HNSCC) usually combining intensity modified radiotherapy with concurrent platinum-based chemotherapy. Though the majority of patients can be cured with this regimen, treatment response is highly heterogeneous and can hardly be predicted. SEC62 represents a metastasis stimulating oncogene that is frequently overexpressed in various cancer entities and is associated with poor outcome. Its role in HNSCC patients undergoing CRT has not been investigated so far. A total of 127 HNSCC patients treated with primary CRT were included in this study. The median follow-up was 5.4 years. Pretherapeutic tissue samples of the primary tumors were used for immunohistochemistry targeting SEC62. SEC62 expression, clinical and histopathological parameters, as well as patient outcome, were correlated in univariate and multivariate survival analyses. High SEC62 expression correlated with a significantly shorter overall survival (p = 0.015) and advanced lymph node metastases (p = 0.024). Further significant predictors of poor overall and progression-free survival included response to therapy (RECIST1.1), nodal status, distant metastases, tobacco consumption, recurrence of disease, and UICC stage. In a multivariate Cox hazard proportional regression analysis, only SEC62 expression (p = 0.046) and response to therapy (p < 0.0001) maintained statistical significance as independent predictors of the patients' overall survival. This study identified SEC62 as an independent prognostic biomarker in HNSCC patients treated with primary CRT. The role of SEC62 as a potential therapeutic target and its interaction with radiation-induced molecular alterations in head and neck cancer cells should further be investigated.

Antagonizing Sec62 function in intracellular Ca2+ homeostasis represents a novel therapeutic strategy for head and neck cancer.

Various cancer types including head and neck squamous cell carcinomas (HNSCC) show a frequent amplification of chromosomal region 3q26 that encodes, among others, for the SEC62 gene. Located in the ER membrane, this translocation protein is known to play a critical role as a potential driver oncogene in cancer development. High SEC62 expression levels were observed in various cancer entities and were associated with a poor outcome and increased metastatic burden. Because of its intracellular localization the SEC62 protein is poorly accessible for therapeutic antibodies, therefore a functional SEC62 knockdown represents the most promising mechanism of a potential antineoplastic targeted therapy. By stimulating the Ca2+ efflux from the ER lumen and thereby increasing cellular stress levels, a functional inhibition of SEC62 bears the potential to limit tumor growth and metastasis formation. In this study, two potential anti-metastatic and -proliferative agents that counteract SEC62 function were investigated in functional in vitro assays by utilizing an immortalized human hypopharyngeal cancer cell line as well as a newly established orthotopic murine in vivo model. Additionally, a CRISPR/Cas9 based SEC62 knockout HNSCC cell line was generated and functionally characterized for its relevance in HNSCC cell proliferation and migration as well as sensitivity to SEC62 targeted therapy in vitro.

Podoplanin expression in lymph node metastases of head and neck cancer and cancer of unknown primary patients.

INTRODUCTION: Head and neck squamous cell carcinomas (HNSCCs) are cancers with generally poor prognosis. Outcomes have not improved in decades, with more than half of the patients presenting with lymph node metastases at the time of diagnosis. A unique subtype of HNSCC, cancer of unknown primary of the head and neck (HNCUP) is associated with a poor outcome. Increased expression of the D2-40 gene (podoplanin) has been described for several human malignancies and has been associated with increased metastatic potential of cancer cells. METHODS: In order to examine the role of podoplanin in lymph node metastasis of HNSCC generally and HNCUP specifically, we evaluated the prognostic impact of podoplanin expression in HNSCC- (n = 68) and HNCUP-associated lymph node metastases (n = 30). The expression of podoplanin was analyzed by immunohistochemical staining of lymph node tissue samples and correlated with clinical and histopathological data. RESULTS: We found a non-significant tendency towards a higher podoplanin expression in HNCUP compared to HNSCC lymph node metastases and a significant correlation between a high podoplanin expression and advanced node-stage classification. Podoplanin expression had no significant impact on overall survival for both groups and did not correlate with human papillomavirus tumor status. CONCLUSION: Taken together, our results suggest that upregulation of podoplanin may be associated with a stimulation of lymphatic metastasis in head and neck cancer.

Cytology-based Cancer Surgery of the Head and Neck (CyCaS-HN): a prospective, randomized, controlled clinical trial.

PURPOSE: Liquid-based cytology (LBC) is routinely used in gynecology but is rarely applied in head and neck oncology though many suspicious lesions are easily accessible. While several studies have evaluated the potential use of LBC for early detection and molecular characterization of head and neck squamous cell carcinomas (HNSCCs), no study investigated its potential role in surgical management and therapy planning so far. METHODS: Twenty-five patients with cT1-2 squamous cell carcinomas of the oral cavity and oropharynx were prospectively enrolled in this study and were randomized to two treatment arms: in the control arm, a diagnostic panendoscopy with incisional biopsy was followed by a second operation with transoral tumor resection ± neck dissection and tracheostomy. In the intervention arm, patients underwent LBC diagnostics and in case of a positive result received one single operation with panendoscopy and incisional biopsy for confirmation of LBC result by rapid section histology followed by transoral tumor resection ± neck dissection and tracheostomy in the same session. RESULTS: Time between clinical diagnosis and definitive surgical treatment was significantly shorter in the intervention group compared with the control group (p < 0.0001). Additionally, time of hospitalization (p < 0.0001) and cumulative operation time (p = 0.062) were shorter in the intervention group. No significant differences in overall, progression-free, and disease-specific survival were observed. CONCLUSION: Cytology-based cancer surgery is a promising therapeutic strategy that can potentially be considered for a well-defined group of early-stage HNSCC patients and help to avoid repetitive general anesthesia, shorten the diagnosis-to-treatment interval and spare operation as well as hospitalization time.

HPV Status as Prognostic Biomarker in Head and Neck Cancer-Which Method Fits the Best for Outcome Prediction?

The incidence of human papillomavirus (HPV)-related head and neck cancer (HNSCC) is rising globally, presenting challenges for optimized clinical management. To date, it remains unclear which biomarker best reflects HPV-driven carcinogenesis, a process that is associated with better therapeutic response and outcome compared to tobacco/alcohol-induced cancers. Six potential HPV surrogate biomarkers were analyzed using FFPE tissue samples from 153 HNSCC patients (n = 78 oropharyngeal cancer (OPSCC), n = 35 laryngeal cancer, n = 23 hypopharyngeal cancer, n = 17 oral cavity cancer): p16, CyclinD1, pRb, dual immunohistochemical staining of p16 and Ki67, HPV-DNA-PCR, and HPV-DNA-in situ hybridization (ISH). Biomarkers were analyzed for correlation with one another, tumor subsite, and patient survival. P16-IHC alone showed the best performance for discriminating between good (high expression) vs poor outcome (low expression; p = 0.0030) in OPSCC patients. Additionally, HPV-DNA-ISH (p = 0.0039), HPV-DNA-PCR (p = 0.0113), and p16-Ki67 dual stain (p = 0.0047) were significantly associated with prognosis in uni- and multivariable analysis for oropharyngeal cancer. In the non-OPSCC group, however, none of the aforementioned surrogate markers was prognostic. Taken together, P16-IHC as a single biomarker displays the best diagnostic accuracy for prognosis stratification in OPSCC patients with a direct detection of HPV-DNA by PCR or ISH as well as p16-Ki67 dual stain as potential alternatives.

Expression of SEC62 Oncogene in Benign, Malignant and Borderline Melanocytic Tumors-Unmasking the Wolf in Sheep's Clothing?

SEC62 oncogene located at chromosomal region 3q26 encodes for a transmembrane protein of the endoplasmic reticulum (ER) and is expressed at high levels in numerous human malignancies. SEC62 overexpression has been associated with worse prognosis and high risk for lymphatic and distant metastases in head and neck cancer, cervical cancer, hepatocellular cancer, and lung cancer. However, its role in the development and tumor biology of melanocytic lesions has not been investigated so far. An immunohistochemical study including 209 patients with melanocytic lesions (malignant melanoma (MM), n = 93; melanoma metastases (MET), n = 28; Spitz nevi (SN), n = 29; blue nevi (BN), n = 21; congenital nevi (CN), n = 38) was conducted and SEC62 expression was correlated with clinical data including patient survival and histopathological characteristics. SN showed the highest SEC62 expression levels followed by MET, MM, CN, and BN. High SEC62 expression correlated with a shorter overall and progression-free survival in MM patients. Additionally, high Sec62 levels correlated significantly with higher tumor size (T stage), the presence of tumor ulceration, and the presence of lymph node as well as distant metastases. Strikingly, SEC62 expression showed a strong correlation with Clark level. Taken together, these data demonstrate that SEC62 is a promising prognostic marker in MM and has the potential to predict biological behavior and clinical aggressiveness of melanocytic lesions.

Augmented reality-enhanced navigation in endoscopic sinus surgery: A prospective, randomized, controlled clinical trial.

OBJECTIVE: Endoscopic sinus surgery represents the gold standard for surgical treatment of chronic sinus diseases. Thereby, navigation systems can be of distinct use. In our study, we tested the recently developed KARL STORZ NAV1 SinusTracker navigation software that incorporates elements of augmented reality (AR) to provide a better preoperative planning and guidance during the surgical procedure. METHODS: One hundred patients with chronic sinus disease were operated on using either a conventional navigation software (n = 52, non-AR, control group) or a navigation software incorporating AR elements (n = 48, AR, intervention group). Incidence of postoperative complications, duration of surgery, surgeon-reported benefit from the navigation system and patient-reported postoperative rehabilitation were assessed. RESULTS: The surgeons reported a higher benefit during surgery, used the navigation system for more surgical steps and spent longer time with preoperative image analysis when using the AR system as compared with the non-AR system. No significant differences were seen in terms of postoperative complications, target registration error, operation time and postoperative rehabilitation. CONCLUSION: The AR enhanced navigation software shows a high acceptance by sinus surgeons in different stages of surgical training and offers potential benefits during surgery without affecting the duration of the operation or the incidence of postoperative complications. LEVEL OF EVIDENCE: 1b.

Vitamin D deficiency in head and neck cancer patients - prevalence, prognostic value and impact on immune function.

Vitamin D deficiency is frequently observed in human cancer patients and a prognostic relevance could be shown for some entities. Additionally, it is known that vitamin D can stimulate the patients' antitumor immunity. However, valid epidemiological data for head and neck squamous cell carcinoma (HNSCC) patients are sparse and functional studies on a possible connection between vitamin D and the patients' immune system are missing. 25-OH vitamin D serum levels were analyzed in 231 HNSCC patients and 232 healthy controls and correlated with clinical data and patient survival. Intra- and peritumoral infiltration with T-cell, NK-cell and macrophage populations was analyzed in 102 HNSCC patients by immunohistochemistry. In 11 HNSCC patients, NK-cells were isolated before and after vitamin D substitution and analyzed for their cytotoxic activity directed against a HNSCC cell line. Vitamin D serum levels were significantly lower in HNSCC patients compared with healthy controls. Low vitamin D levels were associated with lymphatic metastasis and a negative HPV status and were a significant predictor of poor overall survival. HNSCC patients with severe vitamin D deficiency showed significantly altered intra- and peritumoral immune cell infiltrate levels. After vitamin D substitution, the patients' NK cells showed a significant rise in cytotoxic activity. Taken together, we could show that Vitamin D deficiency is highly prevalent in HNSCC patients and is a predictor of poor survival. Vitamin D substitution used as an adjuvant in immune therapies such as cetuximab and nivolumab treatment could support antitumorigenic immune responses, thus contributing to the improvement of the patients' prognosis in the context of a multimodal therapy.

High numbers of PDCD1 (PD-1)-positive T cells and B2M mutations in microsatellite-unstable colorectal cancer.

DNA mismatch repair (MMR)-deficient cancers accumulate high numbers of coding microsatellite mutations, which lead to the generation of highly immunogenic frameshift peptide (FSP) neoantigens. MMR-deficient cells can grow out to clinically manifest cancers either if they evade immune cell attack or if local T-cells get exhausted. Therefore, a subset of MSI cancer patients responds particularly well to treatment with immune checkpoint inhibitors. We analyzed whether immune evasion in MMR-deficient cancer mediated by loss of HLA class I or II antigens is related to local immune cell activation status. Microsatellites located in Beta2-microglobulin (B2M) and the HLA class II-regulatory genes RFX5 and CIITA were analyzed for mutations in MMR-deficient colorectal cancers (n = 53). The results were related to CD3-positive and PDCD1 (PD-1)-positive T-cell infiltration. PDCD1 (PD-1)-positive T-cell counts were significantly higher in B2M-mutant compared to B2M-wild type tumors (median: 22.2 cells per 0.25 mm2 vs. 2.0 cells per 0.25 mm2, Wilcoxon test p = 0.002). Increasing PDCD1 (PD-1)-positive T-cell infiltration was significantly related to an increased likelihood of B2M mutations (OR = 1.81). HLA class II antigen expression status was significantly associated with enhanced overall T-cell infiltration, but not related to PDCD1 (PD-1)-positive T-cells. These results suggest that immune evasion mediated by B2M mutation-induced loss of HLA class I antigen expression predominantly occurs in an environment of activated PDCD1 (PD-1)-positive T cell infiltration. If B2M mutations interfere with anti-PDCD1 (PD-1)/CD274 (PD-L1) therapy success, we predict that resistance towards anti-PDCD1 (PD-1) therapy may - counterintuitively - be particularly common in patients with MMR-deficient cancers that show high PDCD1 (PD-1)-positive T cell infiltration.

Microsatellite instability in pulmonary adenocarcinomas: a comprehensive study of 480 cases.

A major molecular pathway of genetic instability in cancer is DNA mismatch repair deficiency, leading to accumulation of numerous mutations at repetitive DNA sequence stretches (microsatellites), known as high-level microsatellite instability (MSI-H). In colorectal cancer, MSI-H tumors show a clinical behavior different from microsatellite-stable (MSS) tumors. Data about the prevalence of MSI among non-small cell lung cancer (NSCLC) are conflicting, and clinical relevance of MSI is largely unknown. We analyzed a series of 480 pulmonary adenocarcinomas (ADC) for MSI using a sensitive mononucleotide marker panel (BAT25, BAT26, and CAT25). Positive cases were further analyzed by immunohistochemical staining for DNA mismatch repair proteins. Results were correlated with clinicopathological variables. MSI-H was detected in 4/480 (0.8 %) cases. In none of these, a background of Lynch syndrome was found. Three of the patients developed a metachronous carcinoma (esophagus, pancreas, and kidney). All MSI-H cases were stage I and occurred in smokers/ex-smokers. Mutations were found in EGFR (n = 2), KRAS (n = 1), or BRAF (n = 1). MSI-H neoplasms had a higher proliferative activity (38.7 %) than MSS neoplasms (28.3 %). Mean overall survival for MSS and MSI-H cases was 64.8 (CI 60.4-69.1) and 47.1 (CI 21-73.2) months, respectively. When specific mononucleotide marker panels are applied, the MSI-H phenotype is rare and predominantly found in early stage ADC of smokers. However, the frequency of MSI-H is in the range of other relevant molecular alterations. In the era of precision therapy, associations with distinct clinicopathological variables merit further investigation.