Sex-specific aspects in patients with oropharyngeal squamous cell carcinoma: a bicentric cohort study.

BACKGROUND: Oropharyngeal squamous cell carcinoma (OPSCC) is the only subgroup of head neck cancer that presents with an increased incidence. Gender-specific studies in other cancer entities have revealed differences in treatment response and prognosis. However, only limited data in OPSCC according to gender and human papillomavirus (HPV) status exist. Therefore, we aimed to investigate sex-specific differences in OPSCC and how these may be distributed in relation to HPV and other risk factors. METHODS: This retrospective, bicentric study included 1629 patients with OPSCC diagnosed between 1992 and 2020. We formed subgroups based on TNM status, American Joint Cancer Committee 8th edition (AJCC8), HPV status, treatment modality (surgery (± radio(chemo)therapy (RCT) vs. definitive RCT) and patient-related risk factors and investigated gender differences and their impact on patients survival via descriptive-,uni- and multivariate analysis. RESULTS: With the exception of alcohol abuse, no significant differences were found in risk factors between men and women. Females presented with better OS than males in the subgroup T1-2, N + , independent of risk factors (p = 0.008). Males demonstrated significant stratification through all AJCC8 stages (all p < 0.050). In contrast, women were lacking significance between stage II and III (p = 0.992). With regard to therapy (surgery (± R(C)T) - vs. definitive RCT) women treated with surgery had better OS than men in the whole cohort (p = 0.008). Similar results were detected in the HPV-negative OPSCC sub-cohort (p = 0.042) and in high-risk groups (AJCC8 stage III and IV with M0, p = 0.003). CONCLUSION: Sex-specific differences in OPSCC represent a health disparity, particularly according to staging and treatment, which need to be addressed in future studies.

PD-L1 Expression and a High Tumor Infiltrate of CD8+ Lymphocytes Predict Outcome in Patients with Oropharyngeal Squamous Cells Carcinoma.

Carcinogenesis of human papillomavirus (HPV)-related (+) oropharyngeal squamous cell carcinoma (OPSCC) differs from HPV-negative (-) OPSCC. HPV-related immune-escape-mechanism could be responsible for the development and progression of HPV+ tumors and an immunophenotype different from HPV- OPSCC is expected. The purpose of this study was to analyze the expression of programmed cell death protein 1 ligand 1 (PD-L1) and its prognostic relevance in relation to CD8+ tumor infiltrating lymphocytes (TILs) and the major histocompatibility complex (MHC) I expression in OPSCC. We quantified PD-L1 expression on tumor cells (TC) and macrophages and MHC I expression in association to CD8+ TILs by immunohistochemistry on tissue microarray derived from 171 HPV+/-OPSCC. HPV-status was determined by p16INK4a immunohistochemistry/HPV-DNA detection. Presence of CD8+ TILs, PD-L1 expression on TC, and a more frequent loss of MHC I in HPV+ compared to HPV- OPSCC was detected. A high amount of CD8+ TILs in the whole cohort and in HPV+ OPSCC and PD-L1 expression on TC in HPV- OPSCC was associated with favorable overall survival. There was a trend for an improved outcome according to PD-L1 expression (macrophages) in HPV+ OPSCC without reaching statistical significance. CD8+ TILs and PD-L1-expression have prognostic impact in OPSCC and might present useful biomarkers for predicting clinical outcome and personalized therapy concepts.

LAG-3, TIM-3 and VISTA Expression on Tumor-Infiltrating Lymphocytes in Oropharyngeal Squamous Cell Carcinoma-Potential Biomarkers for Targeted Therapy Concepts.

Tumor growth and survival requires a particularly effective immunosuppressant tumor microenvironment (TME) to escape destruction by the immune system. While immunosuppressive checkpoint markers like programmed cell death 1 ligand (PD-L1) are already being targeted in clinical practice, lymphocyte-activation-protein 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) and V-domain Ig suppressor of T cell activation (VISTA) inhibitors are currently under investigation in clinical trials. Reliable findings on the expression status of those immune checkpoint inhibitors on tumor-infiltrating lymphocytes (TILs) in the TME of oropharyngeal squamous cell carcinoma (OPSCC) are lacking. This work aims to describe the expression of LAG-3, TIM-3, and VISTA expression in the TME of OPSCC. We created a tissue microarray of paraffin-embedded tumor tissue of 241 OPSCC. Expression of the immune checkpoint protein LAG-3, TIM-3, and VISTA in OPSCC was evaluated using immunohistochemistry and results were correlated with CD8+ T-cell inflammation and human papillomavirus (HPV)-status. 73 OPSCC stained positive for LAG-3 (31%; HPV+:44%; HPV-:26%, p = 0.006), 122 OPSCC stained positive for TIM-3 (51%; HPV+:70%; HPV-:44%, p < 0.001) and 168 OPSCC (70%; HPV+:75%; HPV-:68%, p = 0.313) for VISTA. CD8+ T-cells were significantly associated with LAG-3, TIM-3 and VISTA expression (p < 0.001, p < 0.001, p = 0.007). Immune checkpoint therapy targeting LAG-3, TIM-3, and/or VISTA could be a promising treatment strategy especially in HPV-related OPSCC. Future clinical trials investigating the efficacy of a checkpoint blockade in consideration of LAG-3, TIM-3, and VISTA expression are required.

Upregulation of AKR1C1 and AKR1C3 expression in OPSCC with integrated HPV16 and HPV-negative tumors is an indicator of poor prognosis.

Different studies have shown that HPV16-positive OPSCC can be subdivided based on integration status (integrated, episomal and mixed forms). Because we showed that integration neither affects the levels of viral genes, nor those of virally disrupted human genes, a genome-wide screen was performed to identify human genes which expression is influenced by viral integration and have clinical relevance. Thirty-three fresh-frozen HPV-16 positive OPSCC samples with known integration status were analyzed by mRNA expression profiling. Among the genes of interest, Aldo-keto-reductases 1C1 and 1C3 (AKR1C1, AKR1C3) were upregulated in tumors with viral integration. Additionally, 141 OPSCC, including 48 HPV-positive cases, were used to validate protein expression by immunohistochemistry. Results were correlated with clinical and histopathological data. Non-hierarchical clustering resulted in two main groups differing in mRNA expression patterns, which interestingly corresponded with viral integration status. In OPSCC with integrated viral DNA, often metabolic pathways were deregulated with frequent upregulation of AKR1C1 and AKR1C3 transcripts. Survival analysis of 141 additionally immunostained OPSCC showed unfavorable survival rates for tumors with upregulation of AKR1C1 or AKR1C3 (both p <0.0001), both in HPV-positive (p ≤0.001) and -negative (p ≤0.017) tumors. OPSCC with integrated HPV16 show upregulation of AKR1C1 and AKR1C3 expression, which strongly correlates with poor survival rates. Also in HPV-negative tumors, upregulation of these proteins correlates with unfavorable outcome. Deregulated AKR1C expression has also been observed in other tumors, making these genes promising candidates to indicate prognosis. In addition, the availability of inhibitors of these gene products may be utilized for drug treatment.

HPV16 increases the number of migratory cancer stem cells and modulates their miRNA expression profile in oropharyngeal cancer.

Human papillomavirus type 16 (HPV16) is a major risk for development of oropharyngeal squamous-cell-carcinoma (OPSCC). Although HPV+ OPSCC metastasize faster than HPV- tumors, they have a better prognosis. The molecular and cellular alterations underlying this pathobiology of HPV+ OPSCC remain elusive. In this study, we examined whether expression of HPV16-E6E7 targets the number of migratory and stationary cancer stem cells (CSC). Furthermore, we wanted to elucidate if aberrantly expressed miRNAs in migratory CSC may be responsible for progression of OPSCCs and whether they may serve as potential novel biomarkers for increased potential of metastasis. Our studies revealed that HPV16-E6E7 expression leads to an increase in the number of stationary (CD44high /EpCAMhigh ) stem cells in primary keratinocyte cultures. Most importantly, expression of E6E7 in the cell line H357 increased the migratory (CD44high /EpCAMlow ) CSC pool. This increase in migratory CSCs could also be confirmed in HPV+ OPSCC. Differentially expressed miRNAs from HPV16-E6E7 positive CD44high /EpCAMlow CSCs were validated by RT-qPCR and in situ hybridization on HPV16+ OPSCCs. These experiments led to the identification of miR-3194-5p, which is upregulated in primary HPV16+ OPSCC and matched metastasis. MiR-1281 was also found to be highly expressed in HPV+ and HPV- metastasis. As inhibition of this miRNA led to a markedly reduction of CD44high /EpCAMlow cells, it may prove to be a promising drug target. Taken together, our findings highlight the capability of HPV16 to modify the phenotype of infected stem cells and that miR-1281 and miR3194-5p may represent promising targets to block metastatic spread of OPSCC.

Viral load, gene expression and mapping of viral integration sites in HPV16-associated HNSCC cell lines.

HPV-related HNSCC generally have a better prognosis than HPV-negative HNSCC. However, a subgroup of HPV-positive tumors with poor prognosis has been recognized, particularly related to smoking, EGFR overexpression and chromosomal instability. Viral integration into the host genome might contribute to carcinogenesis, as is shown for cervical carcinomas. Therefore, all HPV16-positive HNSCC cell lines currently available have been carefully analyzed for viral and host genome parameters. The viral integration status, viral load, viral gene expression and the presence of aneusomies was evaluated in the cell lines UD-SCC-2, UM-SCC-047, UM-SCC-104, UPCI:SCC090, UPCI:SCC152, UPCI:SCC154 and 93VU147T. HPV integration was examined using FISH, APOT-PCR and DIPS-PCR. Viral load and the expression of the viral genes E2, E6 and E7 were determined via quantitative PCR. All cell lines showed integration-specific staining patterns and signals indicating transcriptional activity using FISH. APOT- and DIPS-PCR identified integration-derived fusion products in six cell lines and only episomal products for UM-SCC-104. Despite the observed differences in viral load and the number of viral integration sites, this did not relate to the identified viral oncogene expression. Furthermore, cell lines exhibited EGFR expression and aneusomy (except UPCI:SCC154). In conclusion, all HPV16-positive HNSCC cell lines showed integrated and/or episomal viral DNA that is transcriptionally active, although viral oncogene expression was independent of viral copy number and the number of viral integration sites. Because these cell lines also contain EGFR expression and aneusomy, which are parameters of poor prognosis, they should be considered suitable model systems for the development of new antiviral therapies.

Methylation status of HPV16 E2-binding sites classifies subtypes of HPV-associated oropharyngeal cancers.

BACKGROUND: The human papillomavirus (HPV) E2 protein is a transcriptional repressor of the oncogenes E6/E7 and loss of E2 function is considered a key step in carcinogenesis. Integration of HPV into the host genome may disrupt the E2 gene. Furthermore, methylation of CpG dinucleotides in E2-binding sites (E2BSs) in the HPV upstream regulatory region may interfere with transcriptional repression of E6 and E7 by E2. The authors hypothesized that the CpG methylation status of E2BS identifies subtypes of HPV type 16 (HPV16)-associated oropharyngeal squamous cell cancers (OPSCC) in association with E2 gene integrity and viral integration. METHODS: Methylation of 10 CpG dinucleotides within the upstream regulatory region, encompassing E2BSs 1, 2, 3, and 4, was quantitatively analyzed by bisulfite pyrosequencing in 57 HPV16-associated OPSCC cases. E2 status was analyzed by gene amplification and quantitative real-time reverse transcriptase-polymerase chain reaction. Viral integration was determined by integration-specific polymerase chain reaction methods. RESULTS: Three subgroups with differential methylation at E2BS3 and E2BS 4 were identified: 1) complete methylation (>80%) associated with the presence of integrated HPV genomes with an intact E2 gene; 2) intermediate methylation levels (20%-80%) with predominantly episomal HPV genomes with intact E2; and 3) no methylation (<20%) with a disrupted E2 gene. Patients with high methylation levels tended to have a worse 5-year overall survival compared with patients with intermediate methylation (hazard ratio, 3.23; 95% confidence interval, 1.13-9.24 [P = .06]). CONCLUSIONS: Methylation of E2BS3 and E2BS4 in OPSCC is associated with E2 integrity and viral physical status. It might explain deregulated viral oncogene expression in the presence of E2. The prognostic significance of E2BS methylation for patients with HPV-associated OPSCC needs to be analyzed further.

Management of neck metastases of unknown primary origin united in two European centers.

Combined analysis of diagnostic and therapeutic management of neck metastases of carcinoma of unknown primary origin ('true CUP') in two European tertiary referral centers (University Medical Centers of Maastricht, NL and Cologne, D) to contribute to the ongoing discussion on management in CUP. Retrospective analysis of 29 (Maastricht) and 22 (Cologne) true cervical CUP syndrome patients (squamous cell carcinoma). The diagnostic and therapeutic approaches were correlated with clinical follow-up data and HPV status. In total, 48 out of 51 true CUP patients received postsurgical adjuvant radiotherapy. In eight patients from Cologne, this was combined with concomitant platin-based chemotherapy. Neither in Cologne nor in Maastricht, radiotherapy of the pharyngeal mucosa was commonly performed (n = 6, 12.5 %) The percentage of patients who were irradiated ipsilaterally or bilaterally did not differ between both institutes (N = 21/27 in Maastricht vs. 11/21 in Cologne), nor did the 5-year overall survival differ significantly. Oncogenic HPV was only found in 4 out of 51 CUPs (7, 8 %). Therefore, no relation with overall and recurrence-free survival could be detected. No occult primary tumors were revealed during follow-up despite de-escalation of therapy by abandoning irradiation of the pharyngeal mucosa in both institutes. There were no significant differences between ipsilateral and bilaterally irradiated patients regarding overall and recurrence-free survival. The occurrence of distant metastases was more often noticed in ipsilaterally treated patients as compared to bilaterally radiated patients (8 vs. 2, p = 0.099). Those patients all had been classified N2b or higher. International guidelines still are not unified and there is an urgent need for a consented therapeutic regimen. Comparison of two international strategies on the management of CUP patients is presented and further research is recommended regarding the role of radiotherapy of the pharyngeal axis, the value of unilateral and bilateral radiotherapy and the role of concomitant or induction chemotherapy in CUP patients, particularly in N2b or higher-staged neck disease. The prevalence and role of HPV in true CUP after thorough diagnostic work-up seem limited in our case series, particularly when compared to the role in oropharyngeal carcinomas.

Expression of podoplanin and prognosis in oropharyngeal cancer.

It has been shown that podoplanin expression is associated with carcinoma of the aerodigestive tract. Recent studies indicate that podoplanin may serve as a prognostic biomarker in oral carcinoma. In order to provide evidence on the role of podoplanin in oropharyngeal squamous cell carcinoma, we evaluated the prognostic impact of podoplanin in these patients. We analyzed formalin-fixed tissue samples from 107 consecutive patients with oropharyngeal squamous cell carcinoma. HPV typing and immunohistochemical staining for both p16 and podoplanin were performed. Expression of podoplanin was seen in 38.3% of all cases. We found no correlation of the podoplanin scores with either p16 expression or with HPV status. There was no significant correlation of podoplanin expression with the staging variables T, N, M, and tumor grading. Podoplanin expression did neither influence the 5-year overall survival nor the 5-year disease-free survival. Concluding, we could not find a prognostic role of podoplanin expression neither in the HPV-positive cases nor in the HPV-negative cases. It appears that podoplanin is not expressed as often in oropharyngeal cancer compared to oral cancer. We could not show any relation of lymph node metastases and podoplanin expression in this homogenous cohort of tumors.

P16(INK4A) immunostaining is a strong indicator for high-risk-HPV-associated oropharyngeal carcinomas and dysplasias, but is unreliable to predict low-risk-HPV-infection in head and neck papillomas and laryngeal dysplasias.

Human papillomavirus (HPV) is a risk factor for the development of benign and malignant mucosal head and neck lesions. P16(INK4A) is often used as a surrogate marker for HPV-infection, although there is still controversy with respect its reliability. Our aim was to determine if p16(INK4A) overexpression can accurately predict both high-risk and low-risk-HPV-presence in (pre)malignant and benign head and neck lesions. P16(INK4A) immunohistochemistry was performed on paraffin-embedded tissue sections of 162 oropharyngeal squamous cell carcinomas (OPSCC), 14 tonsillar and 23 laryngeal dysplasias, and 20 tonsillar and 27 laryngeal papillomas. PCR, enzyme-immunoassay and FISH analysis were used to assess HPV-presence and type. Of the 162 OPSCC and 14 tonsillar dysplasias, 51 (31%) and 10 (71%) were HPV16-positive, respectively. All tonsillar papillomas were HPV-negative and four laryngeal dysplasias and 26 laryngeal papillomas were positive for HPV6 or -11. P16(INK4A) immunohistochemistry revealed a strong nuclear and cytoplasmic staining in 50 out of 51 HPV16-positive and 5 out of 111 HPV-negative OPSCC (p < 0.0001) and in all HPV16-positive tonsillar dysplasias, whereas highly variable staining patterns were detected in the papillomas and laryngeal dysplasias, irrespective of the HPV-status. In addition, the latter lesions generally showed a higher nuclear than cytoplasmic p16(INK4A) immunostaining intensity. In conclusion, our data show that strong nuclear and cytoplasmic p16(INK4A) overexpression is a reliable surrogate indicator for HPV16 in OPSCC and (adjacent) dysplasias. For HPV6 or -11-positive and HPV-negative benign and premalignant lesions of the tonsil and larynx, however, p16(INK4A) immunostaining is highly variable and cannot be recommended to predict HPV-presence.

Chromosome stability in tonsillar squamous cell carcinoma is associated with HPV16 integration and indicates a favorable prognosis.

Tonsillar squamous cell carcinoma (TSCC) is frequently associated with human papillomavirus (HPV) and chromosome instability. Data from cellular model systems are, however, controversial concerning a relation between HPV and chromosome instability development. Here we studied this association in 77 primary TSCC with known clinical outcome and cell cycle protein expression profiles. Thirty-two tumors (42%) showed HPV16-integration. All 77 cases were analyzed by fluorescence in situ hybridization using chromosome 1- and 7-specific centromere DNA probes to detect chromosome instability, indicated by the presence of chromosome imbalances and/or polyploidization for these chromosomes. In addition, eight HPV-positive dysplasias, seven of which were adjacent to a carcinoma, were analyzed. Disomy for chromosome 1 and 7 was present in 29 out of 77 TSCC (38%), of which 19 were HPV16-positive (p = 0.002). Aneusomy was observed in the remaining 48 TSCC, of which 13 were HPV-positive. Aneusomies correlated significantly with tobacco- and alcohol consumption (p = 0.001 and p = 0.016, respectively) and a higher T-stage (p = 0.018). Both HPV-positivity and chromosome disomy were significantly associated with a favorable disease-free survival (p = 0.001 and p = 0.025, respectively). Particularly in the HPV16-positive group chromosome instability is a very strong indicator for an unfavorable prognosis (p = 0.032). In the dysplasias an identical HPV and chromosome copy number status was identified as in the adjacent tumors. We conclude that HPV-positive TSCC and their precursor lesions are more often genetically stable than HPV-negative lesions and that these tumors are associated with a favorable prognosis. Chromosome instability is an indicator for unfavorable prognosis, particularly in the HPV-positive patient group.

Prognostic value of proliferating cell nuclear antigen in parotid gland cancer.

Although cell proliferation is related to tumour aggressiveness and prognosis, there are few studies describing the expression of proliferative markers in salivary gland cancer. Our aim was to assess the long-term prognostic value of the proliferating cell nuclear antigen (PCNA) in a large group of histologically different salivary gland cancers. We analysed the expression of PCNA in 159 patients with parotid gland cancer by means of immunohistochemistry. The mean follow-up time was 56.6 months. A high expression of PCNA showed a significant correlation to the patients' pathological lymph node stage (p = 0.004). A high PCNA expression significantly indicated a poor 5-year disease-free (p = 0.046) and overall survival rate (p = 0.018). The PCNA expression was the only prognostic factor for a worse 5-year disease-free and overall survival in acinic cell carcinomas (p = 0.004, p = 0.022). The correlation between PCNA expression and survival probabilities of salivary gland cancer might make proliferation markers helpful tools in patient follow-up, prognosis and targeted therapy in salivary gland cancer in future.

Nuclear translocation of ß-catenin and decreased expression of epithelial cadherin in human papillomavirus-positive tonsillar cancer: an early event in human papillomavirus-related tumour progression?

AIMS: High-risk human papillomaviruses (HPVs) constitute an important risk factor for tonsillar cancer. This study describes changes in cell adhesion molecules during metastasis of HPV-related and HPV-unrelated tonsillar carcinomas. METHODS AND RESULTS: We examined 48 primary tonsillar carcinoma samples (25 HPV-16 DNA-positive, 23 HPV-16 DNA-negative) and their respective lymph node metastases for their HPV status and for the expression of p16, epithelial cadherin (E-cadherin), ß-catenin, and vimentin. A positive HPV-specific polymerase chain reaction finding correlated significantly with p16 overexpression in both primary tumours and their metastases (P<0.0001 for both). In HPV-unrelated carcinomas, the expression of E-cadherin was significantly lower in metastases than in primary tumours (P<0.001). In contrast, the expression of nuclear ß-catenin was significantly higher in metastases than in primary tumours (P=0.016). In HPV-related carcinomas, nuclear localization of ß-catenin expression was already apparent in primary tumours (P=0.030). The expression of vimentin significantly correlated with the grading of the primary tumour (P=0.021). CONCLUSIONS: Our data indicate that the down-regulation of E-cadherin and the up-regulation of nuclear ß-catenin expression might be crucial steps during tumour progression of tonsillar carcinomas, being already present in primary tumours in HPV-driven carcinomas, but becoming apparent in HPV-unrelated tumours later in the process of metastasis.

Causes and Consequences of HPV Integration in Head and Neck Squamous Cell Carcinomas: State of the Art.

A constantly increasing incidence in high-risk Human Papillomaviruses (HPV)s driven head and neck squamous cell carcinomas (HNSCC)s, especially of oropharyngeal origin, is being observed. During persistent infections, viral DNA integration into the host genome may occur. Studies are examining if the physical status of the virus (episomal vs. integration) affects carcinogenesis and eventually has further-reaching consequences on disease progression and outcome. Here, we review the literature of the most recent five years focusing on the impact of HPV integration in HNSCCs, covering aspects of detection techniques used (from PCR up to NGS approaches), integration loci identified, and associations with genomic and clinical data. The consequences of HPV integration in the human genome, including the methylation status and deregulation of genes involved in cell signaling pathways, immune evasion, and response to therapy, are also summarized.

Comprehensive Analysis of VEGFR2 Expression in HPV-Positive and -Negative OPSCC Reveals Differing VEGFR2 Expression Patterns.

VEGF signaling regulated by the vascular endothelial growth factor receptor 2 (VEGFR2) plays a decisive role in tumor angiogenesis, initiation and progression in several tumors including HNSCC. However, the impact of HPV-status on the expression of VEGFR2 in OPSCC has not yet been investigated, although HPV oncoproteins E6 and E7 induce VEGF-expression. In a series of 56 OPSCC with known HPV-status, VEGFR2 expression patterns were analyzed both in blood vessels from tumor-free and tumor-containing regions and within tumor cells by immunohistochemistry using densitometry. Differences in subcellular colocalization of VEGFR2 with endothelial, tumor and stem cell markers were determined by double-immunofluorescence imaging. Immunohistochemical results were correlated with clinicopathological data. HPV-infection induces significant downregulation of VEGFR2 in cancer cells compared to HPV-negative tumor cells (p = 0.012). However, with respect to blood vessel supply, the intensity of VEGFR2 staining differed only in HPV-positive OPSCC and was upregulated in the blood vessels of tumor-containing regions (p < 0.0001). These results may suggest different routes of VEGFR2 signaling depending on the HPV-status of the OPSCC. While in HPV-positive OPSCC, VEGFR2 might be associated with increased angiogenesis, in HPV-negative tumors, an autocrine loop might regulate tumor cell survival and invasion.

Genetic signatures of HPV-related and unrelated oropharyngeal carcinoma and their prognostic implications.

PURPOSE: Patients with human papillomavirus (HPV)-containing oropharyngeal squamous cell carcinomas (OSCC) have a better prognosis than patients with HPV-negative OSCC. This may be attributed to different genetic pathways promoting cancer. EXPERIMENTAL DESIGN: We used comparative genomic hybridization to identify critical genetic changes in 60 selected OSCC, 28 of which were associated with HPV-16 as determined by HPV-specific PCR and fluorescence in situ hybridization analysis and positive p16(INK4A) immunostaining. The results were correlated with HPV status and clinical data from patients. RESULTS: Two thirds of OSCC harbored gain at 3q26.3-qter irrespective of HPV status. In HPV-negative tumors this alteration was associated with advanced tumor stage (P=0.013). In comparison with HPV-related OSCC, the HPV-negative tumors harbored: (a) a higher number of chromosomal alterations and amplifications (P=0.03 and 0.039, respectively); (b) significantly more losses at 3p, 5q, 9p, 15q, and 18q, and gains/amplifications at 11q13 (P=0.002, 0.03; <0.001, 0.02, 0.004, and 0.001, respectively); and (c) less often 16q losses and Xp gains (P=0.02 and 0.03). Survival analysis revealed a significantly better disease-free survival for HPV-related OSCC (P=0.02), whereas chromosome amplification was an unfavorable prognostic indicator for disease-free and overall survival (P=0.01 and 0.05, respectively). Interestingly, 16q loss, predominantly identified in HPV-related OSCC, was a strong indicator of favorable outcome (overall survival, P=0.008; disease-free survival, P=0.01) and none of these patients had a tumor recurrence. CONCLUSIONS: Genetic signatures of HPV-related and HPV-unrelated OSCC are different and most likely underlie differences in tumor development and progression. In addition, distinct chromosomal alterations have prognostic significance.

Viral Integration Analysis Reveals Likely Common Clonal Origin of Bilateral HPV16-Positive, p16-Positive Tonsil Tumors.

Infections with high-risk human papilloma viruses (HPV) are responsible for a significant number of oropharyngeal squamous cell carcinoma (OPSCC), with infection rates currently rising at epidemic rates in the western world. Synchronous bilateral HPV+ tumors of both tonsils are a very rare event whose understanding, however, could provide important insights into virus-driven tumor development and progression and whether such integration events are of clonal origin. In this study we analyzed a single case of a bilateral tonsillar p16+ HPV+OPSCC. The viral integration status of the various tumor samples was determined by integration-specific PCR methods and sequencing, which identified viral insertion sites and affected host genes. Integration events were further confirmed by transcript analysis. Analysis of the tumors revealed common viral integration events involving the CD36 gene, as well as a unique event in the LAMA3 gene which resulted in loss of LAMA3 exon one in both tissues that had lost the complex viral LAMA3 integration event. In addition, there were several integration events into intergenic regions. This suggests a common origin but individual evolution of the tumors, supporting the single-clone hypothesis of bilateral tumor development. This hypothesis is further supported by the fact that the two cellular genes LAMA3 and CD36 as targets of viral integration are involved in cell migration and ECM-receptor interactions, which provides a possible mechanism for clonal migration from one tonsil to another.

Tumor-associated B cells and humoral immune response in head and neck squamous cell carcinoma.

B lymphocytes are important players in immune responses to cancer. However, their composition and function in head and neck squamous cell carcinoma (HNSCC) has not been well described. Here, we analyzed B cell subsets in HNSCC (n = 38), non-cancerous mucosa (n = 14) and peripheral blood from HNSCC patients (n = 38) and healthy controls (n = 20) by flow cytometry. Intratumoral B cells contained high percentages of activated (CD86+), antigen-presenting (CD86+/CD21-) and memory B cells (IgD-/CD27+). T follicular helper cells (CD4+/CXCR5+/CD45RA-/CCR7-) as key components of tertiary lymphoid structures and plasma cells made up high percentages of the lymphocyte infiltrate. Percentages of regulatory B cell varied depending on the regulatory phenotype. Analysis of humoral immune responses against 23 tumor-associated antigens (TAA) showed reactivity against at least one antigen in 56% of HNSCC patients. Reactivity was less frequent in human papillomavirus associated (HPV+) patients and healthy controls compared to HPV negative (HPV-) HNSCC. Likewise, patients with early stage HNSCC or MHC-I loss on tumor cells had low TAA responses. Patients with TAA responses showed CD4+ dominated T cell infiltration compared to mainly CD8+ T cells in tumors without detected TAA response. To summarize, our data demonstrates different immune infiltration patterns in relation to serological TAA response detection and the presence of B cell subpopulations in HNSCC that can engage in tumor promoting and antitumor activity. In view of increasing use of immunotherapeutic approaches, it will be important to include B cells into comprehensive phenotypic and functional analyses of tumor-associated lymphocytes.

Characterization of tumor-associated T-lymphocyte subsets and immune checkpoint molecules in head and neck squamous cell carcinoma.

The composition of tumor-infiltrating lymphocytes (TIL) reflects biology and immunogenicity of cancer. Here, we characterize T-cell subsets and expression of immune checkpoint molecules in head and neck squamous cell carcinoma (HNSCC). We analyzed TIL subsets in primary tumors (n = 34), blood (peripheral blood mononuclear cells (PBMC); n = 34) and non-cancerous mucosa (n = 7) of 34 treatment-naïve HNSCC patients and PBMC of 15 healthy controls. Flow cytometry analyses revealed a highly variable T-cell infiltration mainly of an effector memory phenotype (CD45RA-/CCR7-). Naïve T cells (CD45RA+/CCR7+) were decreased in the microenvironment compared to PBMC of patients, while regulatory T cells (CD4+/CD25+/CD127low and CD4+/CD39+) were elevated. Furthermore, we performed digital image analyses of entire cross sections of HNSCC to define the 'Immunoscore' (CD3+ and CD8+ cell infiltration in tumor core and invasive margin) and quantified MHC class I expression on tumor cells by immunohistochemistry. Immune checkpoint molecules cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death 1 (PD-1) and programmed cell death 1 ligand 1 (PD-L1) were increased in TILs compared to peripheral T cells in flow-cytometric analysis. Human papillomavirus (HPV) positive tumors showed higher numbers of TILs, but a similar composition of T-cell subsets and checkpoint molecule expression compared to HPV negative tumors. Taken together, the tumor microenvironment of HNSCC is characterized by a strong infiltration of regulatory T cells and high checkpoint molecule expression on T-cell subsets. In view of increasingly used immunotherapies, a detailed knowledge of TILs and checkpoint molecule expression on TILs is of high translational relevance.

High glucose uptake unexpectedly is accompanied by high levels of the mitochondrial ß-F1-ATPase subunit in head and neck squamous cell carcinoma.

A hallmark of solid tumors is the consumption of large amounts of glucose and production of lactate, also known as Warburg-like metabolism. This metabolic phenotype is typical for aggressive tumor growth, and can be visualized by 18F-fluorodeoxyglucose (18F-FDG) uptake detected by positron emission tomography (PET). High 18F-FDG uptake inversely correlates with survival and goes along with reduced expression of the catalytic beta-subunit of the H+-ATP synthase (ß-F1-ATPase) in several tumor entities analyzed so far.For this study we characterized a series of 15 head and neck squamous cell carcinoma (HNSCC) by (i) determining 18F-FDG-uptake; (ii) quantitative expression analysis of ß-F1-ATPase (Complex V), NDUF-S1 (Complex I) and COX1 (Complex IV) of the mitochondrial electron transport chain (ETC), as well as Hsp60 (mitochondrial mass) and GAPDH (glycolysis) in tumor cells; (iii) sequencing of the mtDNA of representative tumor samples.Whereas high 18F-FDG-uptake also correlates with poor prognosis in HNSCC, it surprisingly is accompanied by high levels of ß-F1-ATPase, but not by any of the other analyzed proteins.In conclusion, we here describe a completely new phenotype of metabolic adaptation possibly enabling those tumors with highest levels of ß-F1-ATPase to rapidly proliferate even in hypoxic zones, which are typical for HNSCC.