Coupling and regulation mechanisms of the flavin-dependent halogenase PyrH observed by infrared difference spectroscopy.

Flavin-dependent halogenases are central enzymes in the production of halogenated secondary metabolites in various organisms and they constitute highly promising biocatalysts for regioselective halogenation. The mechanism of these monooxygenases includes formation of hypohalous acid from a reaction of fully reduced flavin with oxygen and halide. The hypohalous acid then diffuses via a tunnel to the substrate-binding site for halogenation of tryptophan and other substrates. Oxidized flavin needs to be reduced for regeneration of the enzyme, which can be performed in vitro by a photoreduction with blue light. Here, we employed this photoreduction to study characteristic structural changes associated with the transition from oxidized to fully reduced flavin in PyrH from Streptomyces rugosporus as a model for tryptophan-5-halogenases. The effect of the presence of bromide and chloride or the absence of any halides on the UV-vis spectrum of the enzyme demonstrated a halide-dependent structure of the flavin-binding pocket. Light-induced FTIR difference spectroscopy was applied and the signals assigned by selective isotope labeling of the protein moiety. The identified structural changes in α-helix and ß-sheet elements were strongly dependent on the presence of bromide, chloride, the substrate tryptophan, and the product 5-chloro-tryptophan, respectively. We identified a clear allosteric coupling in solution at ambient conditions between cofactor-binding site and substrate-binding site that is active in both directions, despite their separation by a tunnel. We suggest that this coupling constitutes a fine-tuned mechanism for the promotion of the enzymatic reaction of flavin-dependent halogenases in dependence of halide and substrate availability.

Diagnostic accuracy of HPV16 early antigen serology for HPV-driven oropharyngeal cancer is independent of age and sex.

A growing proportion of head and neck cancer (HNC), especially oropharyngeal cancer (OPC), is caused by human papillomavirus (HPV). There are several markers for HPV-driven HNC, one being HPV early antigen serology. We aimed to investigate the diagnostic accuracy of HPV serology and its performance across patient characteristics. Data from the VOYAGER consortium was used, which comprises five studies on HNC from North America and Europe. Diagnostic accuracy, that is, sensitivity, specificity, Cohen's kappa and correctly classified proportions of HPV16 E6 serology, was assessed for OPC and other HNC using p16INK4a immunohistochemistry (p16), HPV in situ hybridization (ISH) and HPV PCR as reference methods. Stratified analyses were performed for variables including age, sex, smoking and alcohol use, to test the robustness of diagnostic accuracy. A risk-factor analysis based on serology was conducted, comparing HPV-driven to non-HPV-driven OPC. Overall, HPV serology had a sensitivity of 86.8% (95% CI 85.1-88.3) and specificity of 91.2% (95% CI 88.6-93.4) for HPV-driven OPC using p16 as a reference method. In stratified analyses, diagnostic accuracy remained consistent across sex and different age groups. Sensitivity was lower for heavy smokers (77.7%), OPC without lymph node involvement (74.4%) and the ARCAGE study (66.7%), while specificity decreased for cases with <10 pack-years (72.1%). The risk-factor model included study, year of diagnosis, age, sex, BMI, alcohol use, pack-years, TNM-T and TNM-N stage. HPV serology is a robust biomarker for HPV-driven OPC, and its diagnostic accuracy is independent of age and sex. Future research is suggested on the influence of smoking on HPV antibody levels.

Detection of serum biomarkers of HPV-16 driven oropharynx and oral cavity cancer in Brazil.

BACKGROUND: HPV-16 driven oropharynx/oral cavity squamous cell carcinomas prevalence varies globally. We evaluated the presence of HPV-16 ctDNA and HPV-16 E6 antibodies in samples obtained from participants treated at the Instituto do Cancer do Estado de Sao Paulo, ICESP, and from whom tumoral HPV DNA, HPV-16 E6*I mRNA, and p16INK4a status was also accessed. METHODS: HPV was genotyped by PCR-hybridization. All HPV DNA positive and ∼10 % HPV DNA negative cases underwent p16INK4a immunohistochemistry and E6*I RNA testing using a multiplex bead based protocol. HPV-16 ctDNA and anti-E6 antibodies were assessed by ddPCR (digital droplet PCR) and multiplex serology, respectively. RESULTS: The prevalence of HPV-16 in oropharynx carcinoma (OPC) cases was low (8.7 %) when considering solely HPV-16 DNA detection, and even lower (5.2 %) when taken into consideration the concomitant detection of HPV-16 E6*I RNA and/or p16INK4 (HPV-16 attributable fraction - AF). None of the oral cavity cancer (OCC) cases were detected with HPV-16 DNA. HPV-16 ctDNA was more commonly detected than HPV-16 E6 antibodies (29.8 % versus 10.6 %). Both serum biomarkers attained 100 % sensitivity of detecting HPV-16 AF OPC, however the specificity of the HPV-16 anti-E6 biomarker was higher compared to ctDNA (93.2 % versus 75.0 %). Finally, when both HPV-16 ctDNA and anti-E6 biomarkers were considered together, the sensitivity and specificity for HPV-16 OPC detection was 100 % and about 70 %, respectively, independently of analyzing HPV-16 DNA positive or HPV-16 AF tumors. CONCLUSIONS: Our findings corroborate that serum biomarkers are highly sensitive and specific biomarkers for detection of HPV-associated OPC.

Predictors for Survival of Patients with Squamous Cell Carcinoma of Unknown Primary in the Head and Neck Region.

BACKGROUND: Human papillomavirus (HPV) status is the most important predictor of survival in oropharyngeal squamous cell carcinoma (OPSCC). In patients with cervical lymph node metastases of squamous cell carcinoma of unknown origin (CUPHNSCC), much less is known. METHODS: We assessed a consecutive cohort of CUPHNSCC diagnosed from 2000-2018 for HPV DNA, mRNA, p16INK4a (p16) expression, and risk factors to identify prognostic classification markers. RESULTS: In 32/103 (31%) CUPHNSCC, p16 was overexpressed, and high-risk HPV DNA was detected in 18/32 (56.3%). This was mostly consistent with mRNA detection. In recursive partitioning analysis, CUPHNSCC patients were classified into three risk groups according to performance status (ECOG) and p16. Principal component analysis suggests a negative correlation of p16, HPV DNA, and gender in relation to ECOG, as well as a correlation between N stage, extranodal extension, and tobacco/alcohol consumption. CONCLUSIONS: Despite obvious differences, CUPHNSCC shares similarities in risk profile with OPSCC. However, the detection of p16 alone appears to be more suitable for the classification of CUPHNSCC than for OPSCC and, in combination with ECOG, allows stratification into three risk groups. In the future, additional factors besides p16 and ECOG may become important in larger studies or cases with special risk profiles.

Detection of stage I HPV-driven oropharyngeal cancer in asymptomatic individuals in the Hamburg City Health Study using HPV16 E6 serology - A proof-of-concept study.

Background: The lack of detectable precancerous lesions poses challenges to the early detection of human papillomavirus-driven oropharyngeal cancer (HPV-OPC). Antibodies against HPV16 early proteins, especially E6, are uniquely sensitive and specific biomarkers detectable years prior to HPV-OPC diagnosis. Thus, HPV16 early protein serology warrants clinical investigation for HPV-OPC screening. Methods: Using multiplex serology, we analyzed HPV16 serum antibodies of the first 5000 participants (n=4,424 sera, recruited 2016-2017) of the Hamburg City Health Study, a population-based prospective cohort (45-74 years). Participants seropositive for HPV16 E6 and at least one additional early protein (E1, E2, E7) were considered at high risk for HPV-OPC development and invited to six-monthly non-invasive head and neck follow-up (FU) examinations (visual inspection, endoscopy, ultrasonography, performed 2019-2020). Participants with suspicious lesions were examined by magnetic resonance imaging and panendoscopy with biopsy. Histologically confirmed OPC cases were treated according to standard of care. Findings: In total, 35 out of 4,424 study participants (0·8%, 95% confidence interval (CI) 0·6-1·1%) were HPV16 E6 seropositive. Among these, eleven (0·3%, 95%CI 0·1-0·5%) were considered at high risk for HPV-OPC of which nine were successfully re-contacted and invited to regular clinical FU examinations. Two males and one female were diagnosed with stage I HPV-OPC within 1·3 years of clinical FU (3-4 years after initial blood draw), representing one diagnosis of prevalent advanced disease, one incident diagnosis of advanced disease, and one incident diagnosis of early disease. The remaining participants showed no detectable signs of cancer, and undergo regular examinations (median clinical FU: 1·0 years, median total FU from blood draw to last clinical FU visit: 4·7 years). Interpretation: HPV16 early antibodies allowed identifying three asymptomatic stage I HPV-OPC patients, out of eleven participants considered at high risk. However, two of the three cases already showed signs of advanced disease at diagnosis. Targeting multiple early proteins may considerably improve the positive predictive value of HPV16 serology and may have clinical utility for HPV-OPC screening. Funding: This work was funded by DKFZ and UKE intramural funding.

Absolute Risk of Oropharyngeal Cancer After an HPV16-E6 Serology Test and Potential Implications for Screening: Results From the Human Papillomavirus Cancer Cohort Consortium.

PURPOSE: Seropositivity for the HPV16-E6 oncoprotein is a promising marker for early detection of oropharyngeal cancer (OPC), but the absolute risk of OPC after a positive or negative test is unknown. METHODS: We constructed an OPC risk prediction model that integrates (1) relative odds of OPC for HPV16-E6 serostatus and cigarette smoking from the human papillomavirus (HPV) Cancer Cohort Consortium (HPVC3), (2) US population risk factor data from the National Health Interview Survey, and (3) US sex-specific population rates of OPC and mortality. RESULTS: The nine HPVC3 cohorts included 365 participants with OPC with up to 10 years between blood draw and diagnosis and 5,794 controls. The estimated 10-year OPC risk for HPV16-E6 seropositive males at age 50 years was 17.4% (95% CI, 12.4 to 28.6) and at age 60 years was 27.1% (95% CI, 19.2 to 45.4). Corresponding 5-year risk estimates were 7.3% and 14.4%, respectively. For HPV16-E6 seropositive females, 10-year risk estimates were 3.6% (95% CI, 2.5 to 5.9) at age 50 years and 5.5% (95% CI, 3.8 to 9.2) at age 60 years and 5-year risk estimates were 1.5% and 2.7%, respectively. Over 30 years, after a seropositive result at age 50 years, an estimated 49.9% of males and 13.3% of females would develop OPC. By contrast, 10-year risks among HPV16-E6 seronegative people were very low, ranging from 0.01% to 0.25% depending on age, sex, and smoking status. CONCLUSION: We estimate that a substantial proportion of HPV16-E6 seropositive individuals will develop OPC, with 10-year risks of 17%-27% for males and 4%-6% for females age 50-60 years in the United States. This high level of risk may warrant periodic, minimally invasive surveillance after a positive HPV16-E6 serology test, particularly for males in high-incidence regions. However, an appropriate clinical protocol for surveillance remains to be established.

Prevalence of Transcriptionally Active HPV Infection in Tumor-Free Oropharyngeal Tissue of OPSCC-Patients.

Objectives: The natural history of HPV-related oropharyngeal squamous cell carcinoma (OPSCC) is still largely unknown. Since reports of second primary tumors (SPTs) in patients with HPV-related OPSCCs are increasing, a multifocal HPV infection, hinting a «virus-induced field effect¼, has been hypothesized. This study aimed to investigate the HPV-prevalence in normal appearing oropharyngeal tissue in patients with OPSCCs. Materials and Methods: 49 OPSCC patients undergoing panendoscopy were prospectively enrolled. Tumor specimens and biopsies of normal appearing oropharyngeal tissue adjacent to and distant from the index OPSCC underwent histopathological examination, p16INK4A immunohistochemical staining, HPV DNA and mRNA-detection. Patient characteristics and follow-up data on SPTs were obtained. Results: 26 of 49 (53%) OPSCC were positive for HPV DNA and p16INK4A. HPV mRNA was detected in 23 of 26 (88%) of these tumor samples. HPV DNA was detected in 36% adjacent mucosa and in 17% distant mucosa samples and only in patients with an HPV-related index OPSCC. HPV mRNA could not be detected in tumor-free distant and adjacent mucosa samples. No evidence of association between HPV detection in normal appearing mucosa and development of second primary tumors was found. Conclusions: HPV was detectable but not transcriptionally active in adjacent/distant tumor-free oropharyngeal tissue. This suggests that a multifocal HPV infection, hinting a «virus-induced fielcd cancerization¼, may not be pertaining to HPV-related OPSCC.

Detection of Circulating HPV16 DNA as a Biomarker for Cervical Cancer by a Bead-Based HPV Genotyping Assay.

Human papillomavirus (HPV) circulating tumor DNA (HPV ctDNA) was proposed as a biomarker for the detection and disease monitoring of HPV-related cancers. One hundred eighty plasma samples obtained from women diagnosed with HPV16-positive cervical cancer (CC) (n = 100), HPV16-positive premalignant lesions (cervical intraepithelial neoplasia grade 3 [CIN3]) (n = 20), and HPV DNA-negative controls (n = 60) were randomly selected from the archives for evaluating the performance of a bead-based HPV genotyping assay (E7 type-specific multiplex genotyping assay [E7-MPG]) in detecting HPV16 ctDNA. The performance of the E7-MPG was compared with those of DNA detection by droplet digital PCR (ddPCR) and detection of HPV16 E6 antibodies evaluated in an independent study. Internal controls to assess DNA quality were included in the molecular assays, i.e., beta-globin and ESR1, respectively. The sensitivity and specificity of E7-MPG and/or E6 antibodies to detect HPV16-positive CCs were evaluated. HPV16 ctDNA was detected using the E7-MPG in 42.3% of all plasma samples and in 74.7% of plasma samples from HPV16-positive CC cases. The validation of E7-MPG data by ddPCR showed that the sensitivity of the E7-MPG test for HPV16-positive CC detection was higher than that of ddPCR (74.7% versus 63.1%; P < 0.001). When both HPV16 ctDNA and E6 antibodies were considered, the sensitivity for HPV16-positive CC detection increased from 74.7% to 86.1%, while the specificity was unchanged at 97.8%. The performance of E7-MPG for the detection of HPV16 ctDNA appears to be at least as sensitive as that of ddPCR, offering an additional tool for ctDNA detection of HPV16-positive CC. The use of an additional blood marker of HPV infection, such as E6 antibodies, further improved the detection of CC. IMPORTANCE The validity of HPV ctDNA as a marker of HPV-driven cancers has been previously reported. Herein we validated an alternative to ddPCR for HPV16 ctDNA detection, using a bead-based HPV genotyping assay that offers the potential advantage of reducing the cost of clinical management due to the multiplex capability of the test, thus facilitating its use in clinical settings. In addition, we analyzed HPV ctDNA in the context of E6 antibodies as an additional HPV marker. The HPV16 ctDNA biomarker appeared to be highly specific and, to a lesser extent, sensitive for the detection of CC, mainly indicated for those at an advanced tumor stage. In this proof-of-principle study, E6 antibodies were mainly detected in early tumor stages of CC, while HPV ctDNA was mainly positive at advanced tumor stages.

Germline determinants of humoral immune response to HPV-16 protect against oropharyngeal cancer.

Although several oropharyngeal cancer (OPC) susceptibility loci have been identified, most previous studies lacked detailed information on human papillomavirus (HPV) status. We conduct a genome-wide analysis by HPV16 serology status in 4,002 oral cancer cases (OPC and oral cavity cancer (OCC)) and 5,256 controls. We detect four susceptibility loci pointing to a distinct genetic predisposition by HPV status. Our most notable finding in the HLA region, that is now confirmed to be specific of HPV(+)OPC risk, reveal two independent loci with strong protective effects, one refining the previously reported HLA class II haplotype association. Antibody levels against HPV16 viral proteins strongly implicate the protective HLA variants as major determinants of humoral response against L1 capsid protein or E6 oncoprotein suggesting a natural immune response against HPV(+)OPC promoted by HLA variants. This indicates that therapeutic vaccines that target E6 and attenuate viral response after established HPV infections might protect against HPV(+)OPC.

Survival advantage in patients with human papillomavirus-driven oropharyngeal cancer and variation by demographic characteristics and serologic response: Findings from Head and Neck 5000.

BACKGROUND: Patients with human papillomavirus (HPV)-driven oropharyngeal cancer (OPC) experience better survival than those with HPV-negative OPC. It is unclear whether this benefit varies by demographic characteristics and serologic response. METHODS: Records from 1411 patients with OPC who had HPV serology data were analyzed. HPV status was based on HPV type 16 (HPV16) E6 serology. Participants were followed for a median of 5.9 years, and Cox proportional hazards models were used to estimate hazard ratios (HRs). The association between HPV status and overall survival was analyzed by age group, sex, smoking status, tumor site, HPV antibody levels, and HPV antibody pattern. Models were adjusted for age, sex, smoking status, and comorbidity. RESULTS: For the overall association between HPV status and survival, the fully adjusted HR was 0.43 (95% CI, 0.33-0.56). The HR was 0.19 (95% CI, 0.10-0.35) for participants aged ≤54 years, 0.38 (95% CI, 0.25-0.56) for those aged 55 to 64 years, and 0.73 (95% CI, 0.47-1.13) for those aged ≥65 years (P for interaction = .023). There was no clear evidence for an interaction by sex, smoking status, or tumor site. Survival did not differ according to E6 antibody levels in those who were seropositive. All seropositivity patterns were associated with increased survival compared with a pattern of seronegativity for all antibodies. Patients who are positive for E1, E2, E6, and E7 may experience better survival. CONCLUSIONS: HPV status confers a survival advantage across all groups. This survival advantage is more marked for younger patients. The HPV antibody pattern, but not the antibody level, may also affect survival.

Site-by-site tracking of signal transduction in an azidophenylalanine-labeled bacteriophytochrome with step-scan FTIR spectroscopy.

Signal propagation in photosensory proteins is a complex and multidimensional event. Unraveling such mechanisms site-specifically in real time is an eligible but a challenging goal. Here, we elucidate the site-specific events in a red-light sensing phytochrome using the unnatural amino acid azidophenylalanine, vibrationally distinguishable from all other protein signals. In canonical phytochromes, signal transduction starts with isomerization of an excited bilin chromophore, initiating a multitude of processes in the photosensory unit of the protein, which eventually control the biochemical activity of the output domain, nanometers away from the chromophore. By implementing the label in prime protein locations and running two-color step-scan FTIR spectroscopy on the Deinococcus radiodurans bacteriophytochrome, we track the signal propagation at three specific sites in the photosensory unit. We show that a structurally switchable hairpin extension, a so-called tongue region, responds to the photoconversion already in microseconds and finalizes its structural changes concomitant with the chromophore, in milliseconds. In contrast, kinetics from the other two label positions indicate that the site-specific changes deviate from the chromophore actions, even though the labels locate in the chromophore vicinity. Several other sites for labeling resulted in impaired photoswitching, low structural stability, or no changes in the difference spectrum, which provides additional information on the inner dynamics of the photosensory unit. Our work enlightens the multidimensionality of the structural changes of proteins under action. The study also shows that the signaling mechanism of phytochromes is accessible in a time-resolved and site-specific approach by azido probes and demonstrates challenges in using these labels.

Detection of HPV16/18 E6 Oncoproteins in Head and Neck Squamous Cell Carcinoma Using a Protein Immunochromatographic Assay.

OBJECTIVES/HYPOTHESIS: The accurate diagnostic assessment of clinically relevant human papillomavirus (HPV) infections in patients with head and neck squamous cell carcinoma represents an urgent unmet medical need. The aim of this study was to determine feasibility, accuracy, and clinical significance of HPV16/18 E6 oncoprotein detection on cytological specimens from oropharyngeal squamous cell carcinoma (OPSCC) and neck lymph node metastasis of SCC from unknown primary tumor (CUP) via a protein immunochromatographic assay. STUDY DESIGN: Cross-sectional study. METHODS: Cytological specimens from primary tumor and neck metastases were collected from 34 patients with OPSCC or CUP and applied to a lateral flow format test that detects HPV16 and HPV18 E6 oncoproteins. E6 oncoprotein positivity or negativity in these specimens was compared to the specimens' "HPV-driven" reference status, defined by presence of HPV-DNA in combination with p16INK4a overexpression and/or HPV E6 seropositivity. RESULTS: Eighteen of 29 OPSCC (62%) and three of five CUP (60%) were HPV-driven according to our reference method. The E6 oncoprotein lateral flow test had a sensitivity of 94% (95% CI: 70%-100%) and a specificity of 100% (95% CI: 66%-100%) on primary tumor, and a sensitivity of 88% (95% CI: 64%-99%) and a specificity of 100% (95% CI: 74%-100%) on neck metastases. Test agreement between the E6 lateral flow test and the clinical reference method, HPV-DNA plus p16INK4a was excellent, both for primary lesion and neck metastases. CONCLUSIONS: We found the detection of HPV16/18 E6 oncoproteins to be a feasible, highly reliable, and low-invasive method to assess "HPV-driven" status in OPSCC and CUP. LEVEL OF EVIDENCE: II Laryngoscope, 131:1042-1048, 2021.

Unraveling most abundant mutational signatures in head and neck cancer.

Genomic alterations are a driving force in the multistep process of head and neck cancer (HNC) and result from the interaction of exogenous environmental exposures and endogenous cellular processes. Each of these processes leaves a characteristic pattern of mutations on the tumor genome providing the unique opportunity to decipher specific signatures of mutational processes operative during HNC pathogenesis and to address their prognostic value. Computational analysis of whole exome sequencing data of the HIPO-HNC (Heidelberg Center for Personalized Oncology-head and neck cancer) (n = 83) and TCGA-HNSC (The Cancer Genome Atlas-Head and Neck Squamous Cell Carcinoma) (n = 506) cohorts revealed five common mutational signatures (Catalogue of Somatic Mutations in Cancer [COSMIC] Signatures 1, 2, 3, 13 and 16) and demonstrated their significant association with etiological risk factors (tobacco, alcohol and HPV16). Unsupervised hierarchical clustering identified four clusters (A, B, C1 and C2) of which Subcluster C2 was enriched for cases with a higher frequency of signature 16 mutations. Tumors of Subcluster C2 had significantly lower p16INK4A expression accompanied by homozygous CDKN2A deletion in almost one half of cases. Survival analysis revealed an unfavorable prognosis for patients with tumors characterized by a higher mutation burden attributed to signature 16 as well as cases in Subcluster C2. Finally, a LASSO-Cox regression model was applied to prioritize clinically relevant signatures and to establish a prognostic risk score for head and neck squamous cell carcinoma patients. In conclusion, our study provides a proof of concept that computational analysis of somatic mutational signatures is not only a powerful tool to decipher environmental and intrinsic processes in the pathogenesis of HNC, but could also pave the way to establish reliable prognostic patterns.

HPV driven squamous cell head and neck cancer of unknown primary is likely to be HPV driven squamous cell oropharyngeal cancer.

OBJECTIVES: To compare risk factors and survival in people with oropharyngeal cancer (OPC) and cancer unknown primary (CUP). MATERIALS AND METHODS: We recruited 5511 people with head and neck cancer between 2011 and 2014. We collected data on age, gender, smoking, sexual behaviour, treatment intent, stage, co-morbidity, p16 protein overexpression and biological samples. We assessed human papillomavirus (HPV) status using serological response and p16 immunohistochemistry. We followed up participants to identify those who had died. We used Cox proportional hazards regression models to estimate survival and adjust for confounders. RESULTS: Of the 4843 people with squamous cell cancer 196 had CUP - a prevalence of 4.0% (95% CI 3.5% to 4.6%). Of those people with OPC and CUP 69% (1150/1668) and 60% (106/178) respectively had HPV driven tumours. People with HPV driven tumours were likely to be younger, male, non-smokers, with higher stage disease, a history of oral sex and less co-morbidity. People with HPV negative CUP and HPV driven CUP had the survival of people with a stage II/III HPV negative OPC and a stage I/II HPV driven OPC respectively. The adjusted hazard ratio for HPV driven OPC and CUP compared with HPV negative OPC and CUP was 0.46 (95% CI 0.35 to 0.59) and 0.34 (95% CI 0.14 to 0.82) respectively. CONCLUSION: HPV driven CUP is likely to be HPV driven OPC. Identifying effective methods of detecting occult OPC could improve CUP management and allow the detection of early lesions in high risk groups.

Epstein-Barr virus and human papillomavirus serum antibodies define the viral status of nasopharyngeal carcinoma in a low endemic country.

Epstein-Barr virus (EBV) causes nasopharyngeal carcinoma (NPC) in endemic regions, where almost every tumor is EBV-positive. In Western populations, NPC is rare, and human papillomavirus infection (HPV) has been suggested as another viral cause. We validated multiplex serology with molecular tumor markers, to define EBV-positive, HPV-positive and EBV-/HPV-negative NPCs in the United Kingdom, and analyzed survival differences between those groups. Sera from NPC cases (n = 98) and age- and sex-matched controls (n = 142) from the Head and Neck 5000 clinical cohort study were analyzed. IgA and IgG serum antibodies against 13 EBV antigens were measured and compared with EBER in situ hybridization (EBER-ISH) data of 41 NPC tumors (29 EBER-ISH positive, 12 negative). IgG antibodies to EBV LF2 correctly diagnosed EBV-positive NPCs in 28 of 29 cases, while all EBER-ISH negative NPCs were seronegative to LF2 IgG (specificity = 100%, sensitivity = 97%). HPV early antigen serology was compared to HPV molecular markers (p16 expression, HPV DNA and RNA) available for 41 NPCs (13 positive, 28 negative). Serology matched molecular HPV markers in all but one case (specificity = 100%, sensitivity = 92%). EBV and HPV infections were mutually exclusive. Overall, 67% of the analyzed NPCs were defined as EBV-positive, 18% as HPV-positive and 14% as EBV/HPV-negative. There was no statistical evidence of a difference in survival between the three groups. These data provide evidence that both, EBV-positive and HPV-positive NPCs are present in a low incidence country, and that EBV and HPV serum antibodies correlate with the viral status of the tumor.

Antibody Responses to Cancer Antigens Identify Patients with a Poor Prognosis among HPV-Positive and HPV-Negative Head and Neck Squamous Cell Carcinoma Patients.

PURPOSE: The identification of high-risk patients within human papillomavirus (HPV)-positive and -negative head and neck squamous cell carcinoma (HNSCC) is needed for improved treatment and surveillance strategies. In this study, we set out to discover antibody responses (AR) with prognostic impact in HNSCC stratified by HPV status. EXPERIMENTAL DESIGN: A fluorescent bead-based multiplex serology assay on 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens, and 8 oncogenes) and 29 HPV antigens was performed in samples of 362 patients with HNSCC from five independent cohorts (153 HPV positive, 209 HPV negative). A multivariable Cox proportional hazard model with bootstrapping (M = 1000) was used for validation of prognostic antibody responses. RESULTS: Antibody response to any of the cancer antigens was found in 257 of 362 patients (71%). In HPV-negative patients, antibody responses to c-myc, MAGE-A1, -A4, and Rhodopsin E2 (combined as ARhigh risk) were significantly associated with shorter overall survival. In HPV-positive patients, antibody responses to IMP-1 were discovered as a negative prognostic factor. ARhigh risk (HR = 1.76) and antibody responses to IMP-1 (HR = 3.28) were confirmed as independent markers for a poor prognosis in a multivariable Cox proportional hazard model with bootstrapping (M = 1000). CONCLUSIONS: We identified antibody responses to cancer antigens that associate with a dismal prognosis in patients with HNSCC beyond HPV-positive status. ARhigh risk may be used to detect HPV-negative patients with an extraordinarily bad prognosis. Most importantly, antibody response to IMP-1 may serve as a marker for a subgroup of HPV-positive patients who present with a poor prognosis similar to that in HPV-negative patients.

Absence of disruptive TP53 mutations in high-risk human papillomavirus-driven neck squamous cell carcinoma of unknown primary.

BACKGROUND: To enforce the evidence for causality between high-risk human papillomavirus (hrHPV) infections and neck squamous cell carcinoma from unknown primary (NSCCUP) and provide biological basis for treatment de-intensification, we searched for TP53 mutations in association with HPV status. METHODS: TP53 mutations were searched for by amplification of exons 4 to 10. RESULTS: Of the 70 NSCCUP, 27 (39%) harbored HPV infection. TP53 sequencing resulted in the identification of 19 patients harboring single mutations including 16 disruptive alterations (84%). The association of TP53 mutations and HPV could be evaluated in 48 NSCCUP including those with disruptive mutation in any exon (n = 16) and those without mutations but with complete sequence of exons 4 to 9 (n = 32): no disruptive mutations were found in the 17 HPV-driven NSCCUP but in 16 of the 31 non-HPV-driven NSCCUP (P = .0002). CONCLUSION: In a fraction of cases, NSCCUP is an HPV-driven entity harboring wild-type TP53 gene or nondisruptive TP53 mutations. HPV-driven NSCCUP might benefit from treatment de-intensification.

Patterns of antibody responses to nonviral cancer antigens in head and neck squamous cell carcinoma patients differ by human papillomavirus status.

There have been hints that nonviral cancer antigens are differentially expressed in human papillomavirus (HPV)-positive and HPV-negative head and neck squamous cell carcinoma (HNSCC). Antibody responses (AR) to cancer antigens may be used to indirectly determine cancer antigen expression in the tumor using a noninvasive and tissue-saving liquid biopsy. Here, we set out to characterize AR to a panel of nonviral cancer antigens in HPV-positive and HPV-negative HNSCC patients. A fluorescent microbead multiplex serology to 29 cancer antigens (16 cancer-testis antigens, 5 cancer-retina antigens and 8 oncogenes) and 29 HPV-antigens was performed in 382 HNSCC patients from five independent cohorts (153 HPV-positive and 209 HPV-negative). AR to any of the cancer antigens were found in 272/382 patients (72%). The ten most frequent AR were CT47, cTAGE5a, c-myc, LAGE-1, MAGE-A1, -A3, -A4, NY-ESO-1, SpanX-a1 and p53. AR to MAGE-A3, MAGE-A9 and p53 were found at significantly different prevalences by HPV status. An analysis of AR mean fluorescent intensity values uncovered remarkably different AR clusters by HPV status. To identify optimal antigen selections covering a maximum of patients with ≤10 AR, multiobjective optimization revealed distinct antigen selections by HPV status. We identified that AR to nonviral antigens differ by HPV status indicating differential antigen expression. Multiplex serology may be used to characterize antigen expression using serum or plasma as a tissue-sparing liquid biopsy. Cancer antigen panels should address the distinct antigen repertoire of HPV-positive and HPV-negative HNSCC.

Somatic mutations and promotor methylation of the ryanodine receptor 2 is a common event in the pathogenesis of head and neck cancer.

Genomic sequencing projects unraveled the mutational landscape of head and neck squamous cell carcinoma (HNSCC) and provided a comprehensive catalog of somatic mutations. However, the limited number of significant cancer-related genes obtained so far only partially explains the biological complexity of HNSCC and hampers the development of novel diagnostic biomarkers and therapeutic targets. We pursued a multiscale omics approach based on whole-exome sequencing, global DNA methylation and gene expression profiling data derived from tumor samples of the HIPO-HNC cohort (n = 87), and confirmed new findings with datasets from The Cancer Genome Atlas (TCGA). Promoter methylation was confirmed by MassARRAY analysis and protein expression was assessed by immunohistochemistry and immunofluorescence staining. We discovered a set of cancer-related genes with frequent somatic mutations and high frequency of promoter methylation. This included the ryanodine receptor 2 (RYR2), which showed variable promoter methylation and expression in both tumor samples and cell lines. Immunohistochemical staining of tissue sections unraveled a gradual loss of RYR2 expression from normal mucosa via dysplastic lesion to invasive cancer and indicated that reduced RYR2 expression in adjacent tissue and precancerous lesions might serve as risk factor for unfavorable prognosis and upcoming malignant conversion. In summary, our data indicate that impaired RYR2 function by either somatic mutation or epigenetic silencing is a common event in HNSCC pathogenesis. Detection of RYR2 expression and/or promoter methylation might enable risk assessment for malignant conversion of dysplastic lesions.