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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.

Overall survival by HPV-status. Overall survival stratified by HPV-status based on A) molecular HPV-status, B) seropositivity to HPV-16 E6, C) seropositivity to any high-risk HPV-types and D) by molecular HPV-status if available and seropositivity to any high-risk HPV-types if molecular HPV-status was unavailable. Overall survival was significantly longer regardless of the definition of HPV-status (p

Patient characteristics (non-oropharyngeal 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.

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., United States Department of Health & Human Services National Institutes of Health (NIH) - USA NIH National Cancer Institute (NCI) 5U01CA195603-02 U01CA202979, Division of Cancer Epidemiology and Genetics, US NCI, VicHealth, Canadian Institutes of Health Research (CIHR), Cancer Council Victoria, National Health and Medical Research Council (NHMRC) of Australia 209057 396414 1074383, National Cancer Institute P30 CA016056, Einstein Cancer Research Center CA013330, United States Department of Health & Human Services, National Institutes of Health (NIH) - USA, NIH National Heart Lung & Blood Institute (NHLBI) 75N92021D00001 75N92021D00002 75N92021D00003 75N92021D00004 75N92021D00005

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.

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. peerReviewed

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.

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.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.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).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.This work was funded by DKFZ and UKE intramural funding.

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., Genetic susceptibility loci for oropharyngeal cancer have been reported but these studies have not always examined human papillomavirus (HPV) status. Here, the authors perform genome-wide analysis taking into account HPV16 serology status and report two independent loci in the HLA region, suggesting the protective role of HLA variants against HPV infection.

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 (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.

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.

Phytochrome proteins regulate many photoresponses of plants and microorganisms. Light absorption causes isomerization of the biliverdin chromophore, which triggers a series of structural changes to activate the signaling domains of the protein. However, the structural changes are elusive, and therefore the molecular mechanism of signal transduction remains poorly understood. Here, we apply two-color step-scan infrared spectroscopy to the bacteriophytochrome from Deinococcus radiodurans. We show by recordings in H2O and D2O that the hydrogen bonds to the biliverdin D-ring carbonyl become disordered in the first intermediate (Lumi-R) forming a dynamic microenvironment, then completely detach in the second intermediate (Meta-R), and finally reform in the signaling state (Pfr). The spectra reveal via isotope labeling that the refolding of the conserved “PHY-tongue” region occurs with the last transition between Meta-R and Pfr. Additional changes in the protein backbone are detected already within microseconds in Lumi-R. Aided by molecular dynamics simulations, we find that a strictly conserved salt bridge between an arginine of the PHY tongue and an aspartate of the chromophore binding domains is broken in Lumi-R and the arginine is recruited to the D-ring C═O. This rationalizes how isomerization of the chromophore is linked to the global structural rearrangement in the sensory receptor. Our findings advance the structural understanding of phytochrome photoactivation. peerReviewed

Plant cryptochromes are light receptors in land plants and algae with very diverse functions such as circadian timing and lifecycle progression. The receptor consists of a photolyase homology region (PHR) binding the flavin chromophore and a C-terminal extension (CCT) responsible for signaling. The reputed signaling state, the flavin neutral radical, is formed by a femtosecond electron transfer and microsecond proton transfer to the excited, oxidized flavin. Subsequently, a 500 μs loss of β-sheet structure ∼25 Å away from flavin was resolved and suggested to be part of the signal conduction to the CCT. Here, we performed time-resolved, step-scan Fourier transform IR spectroscopy on the PHR of the plant cryptochrome pCRY (formerly CPH1) from Chlamydomonas reinhardtii. In a mutant lacking the proton donor aspartic acid 396 only the flavin anion radical is formed, but we observed the loss of β-sheet structure with a time constant of 1.3 ms, similar to the 500 μs of the wild type. This finding implies that the anion radical may be considered signaling-competent. In the steady state, a variation of external pH up to 8.3 did not have any effect on the difference spectra including the protonated state of Asp396. However, we detected the prominent loss of β-sheet structure by illumination only in the presence of adenosine triphosphate (ATP). We conclude that the bound ATP stabilizes these light-induced changes in secondary structure to ensure a physiological lifetime compatible with signaling by plant cryptochrome.

Treatment of patients with neck lymph node metastasis of squamous cell carcinoma (SCC) from unknown primary tumor (NSCCUP) is challenging due to the risk of missing occult tumors or inducing toxicity to unaffected sites. Human papillomavirus (HPV) is a promising biomarker given its causal link to oropharyngeal SCC and superior survival of patients with HPV-driven oropharyngeal SCC and NSCCUP. Identification of HPV-driven NSCCUP could focus diagnostic work-up and treatment on the oropharynx. For the first time, we assessed HPV antibodies and their prognostic value in NSCCUP patients. Antibodies against E6 and E7 (HPV16/18/31/33/35), E1 and E2 (HPV16/18) were assessed in 46 NSCCUP patients in sera collected at diagnosis, and in follow-up sera from five patients. In 28 patients, HPV tumor status was determined using molecular markers (HPV DNA, mRNA and cellular p16INK4a ). Thirteen (28%) NSCCUP patients were HPV-seropositive for HPV16, 18, 31, or 33. Of eleven patients with HPV-driven NSCCUP, ten were HPV-seropositive, while all 17 patients with non-HPV-driven NSCCUP were HPV-seronegative, resulting in 91% sensitivity (95% CI: 59-100%) and 100% specificity (95% CI: 80-100%). HPV antibody levels decreased after curative treatment. Recurrence was associated with increasing levels in an individual case. HPV-seropositive patients had a better overall and progression-free survival with hazard ratios of 0.09 (95% CI: 0.01-0.42) and 0.03 (95% CI: 0.002-0.18), respectively. For the first time, seropositivity to HPV proteins is described in NSCCUP patients, and high sensitivity and specificity for HPV-driven NSCCUP are demonstrated. HPV seropositivity appears to be a reliable diagnostic and prognostic biomarker for patients with HPV-driven NSCCUP.

To determine the sensitivity and specificity of HPV16 serology as diagnostic marker for HPV16-driven oropharyngeal squamous cell carcinoma (OPSCC), 214 HNSCC patients from Germany and Italy with fresh-frozen tumor tissues and sera collected before treatment were included in this study. Hundred and twenty cancer cases were from the oropharynx and 94 were from head and neck cancer regions outside the oropharynx (45 oral cavity, 12 hypopharynx and 35 larynx). Serum antibodies to early (E1, E2, E6 and E7) and late (L1) HPV16 proteins were analyzed by multiplex serology and were compared to tumor HPV RNA status as the gold standard. A tumor was defined as HPV-driven in the presence of HPV16 DNA and HPV16 transformation-specific RNA transcript patterns (E6*I, E1∧ E4 and E1C). Of 120 OPSCC, 66 (55%) were HPV16-driven. HPV16 E6 seropositivity was the best predictor of HPV16-driven OPSCC (diagnostic accuracy 97% [95%CI 92-99%], Cohen's kappa 0.93 [95%CI 0.8-1.0]). Of the 66 HPV-driven OPSCC, 63 were HPV16 E6 seropositive, compared to only one (1.8%) among the 54 non-HPV-driven OPSCC, resulting in a sensitivity of 96% (95%CI 88-98) and a specificity of 98% (95%CI 90-100). Of 94 HNSCC outside the oropharynx, six (6%) were HPV16-driven. In these patients, HPV16 E6 seropositivity had lower sensitivity (50%, 95%CI 19-81), but was highly specific (100%, 95%CI 96-100). In conclusion, HPV16 E6 seropositivity appears to be a highly reliable diagnostic marker for HPV16-driven OPSCC with very high sensitivity and specificity, but might be less sensitive for HPV16-driven HNSCC outside the oropharynx.

Background Human papillomavirus type 16 (HPV16)-E6 antibodies are detectable in peripheral blood before diagnosis in the majority of HPV16-driven oropharyngeal squamous cell carcinoma (OPSCC), but the timing of seroconversion is unknown. Patients and methods We formed the HPV Cancer Cohort Consortium which comprises nine population cohorts from Europe, North America and Australia. In total, 743 incident OPSCC cases and 5814 controls provided at least one pre-diagnostic blood sample, including 111 cases with multiple samples. Median time between first blood collection and OPSCC diagnosis was 11.4 years (IQR = 6–11 years, range = 0–40 years). Antibodies against HPV16-E6 were measured by multiplex serology (GST fusion protein based Luminex assay). Results HPV16-E6 seropositivity was present in 0.4% of controls (22/5814; 95% CI 0.2% to 0.6%) and 26.2% (195/743; 95% CI 23.1% to 29.6%) of OPSCC cases. HPV16-E6 seropositivity increased the odds of OPSCC 98.2-fold (95% CI 62.1–155.4) in whites and 17.2-fold (95% CI 1.7–170.5) in blacks. Seropositivity in cases was more frequent in recent calendar periods, ranging from 21.9% pre-1996 to 68.4% in 2005 onwards, in those with blood collection near diagnosis (lead time 30 years (N = 24), 20–30 years (N = 148), 10–20 years (N = 228), and

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth in Saccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Previously, we have shown that deletion of the MCK1 gene leads to sensitivity to sodium dodecyl sulfate; thus, we implemented a suppressor gene screening revealing that the overexpression of TAT2 tryptophan permease rescues cell growth in sodium dodecyl sulfate-treated Δmck1 cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show that trp1-1 cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil when cells are grown on rich media. While also critical in the response to tea tree oil, TRP1 does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as Calcofluor White, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discovered that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, we conclude that internal concentration of tryptophan and tyrosine makes cells more resistant to detergent such as sodium dodecyl sulfate.

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. �� In Copyright http://rightsstatements.org/vocab/InC/1.0/, What's new? Head and neck squamous cell carcinoma remains a deadly disease but new immunotherapeutic approaches are underexplored. Here the authors tested for antibody responses against human antigens to characterize the expression of such antigens in tumors positive or negative for human papillomavirus (HPV). Antibody responses were significantly different in prevalence and pattern based on HPV���status in a large patient cohort. The authors urge independent confirmation of their results but point out that multiplex serology of tumor antigens could be a promising strategy to identify immunotherapeutic targets based on HPV status., publishedVersion

Photoactive Yellow Protein (PYP) is a bacterial blue light receptor that enters a photocycle after excitation. The intermediate states are formed on time scales ranging from femtoseconds up to hundreds of milliseconds, after which the signaling state with a lifetime of about 1 s is reached. To investigate structural changes and dynamics, we incorporated the SCN IR label at distinct positions of the photoreceptor via cysteine mutation and cyanylation. FT-IR measurements of the SCN label at different sites of the well-established dark state structure of PYP characterized the spectral response of the label to differences in the environment. Under constant blue light irradiation, we observed the formation of the signaling state with significant changes of wavenumber and lineshape of the SCN bands. Thereby we deduced light-induced structural changes in the local environment of the labels. These results were supported by molecular dynamics simulations on PYP providing the solvent accessible surface area (SASA) at the different positions. To follow protein dynamics via the SCN label during the photocycle, we performed step-scan FT-IR measurements with a time resolution of 10 μs. Global analysis yielded similar time constants of τ1 = 70 μs, τ2 = 640 μs, and τ3 > 20 ms for the wild type and τ1 = 36 μs, τ2 = 530 μs, and τ3 > 20 ms for the SCN-labeled mutant PYP-A44C*, a mutant which provided a sufficiently large SCN difference signal to measure step-scan FT-IR spectra. In comparison to the protein (amide, E46) and chromophore bands the dynamics of the SCN label show a different behavior. This result indicates that the local kinetics sensed by the label are different from the global protein kinetics.

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.

Sodium dodecyl sulfate is a detergent that disrupts cell membranes, activates cell wall integrity signaling and restricts cell growth inSaccharomyces cerevisiae. However, the underlying mechanism of how sodium dodecyl sulfate inhibits cell growth is not fully understood. Because deletion of theMCK1gene leads to sensitivity to sodium dodecyl sulfate, we implemented a suppressor gene screening revealing that theTAT2tryptophan permease rescues cell growth to sodium dodecyl sulfate-treatedΔmck1cells. Therefore, we questioned the involvement of tryptophan in the response to sodium dodecyl sulfate treatment. In this work, we show thatΔtrp1cells have a disadvantage in the response to sodium dodecyl sulfate compared to auxotrophy for adenine, histidine, leucine or uracil. While also critical in the response to tea tree oil,TRP1does not avert growth inhibition due to other cell wall/membrane perturbations that activate cell wall integrity signaling such as calcofluor white, Congo Red or heat stress. This implicates a distinction from the cell wall integrity pathway and suggests specificity to membrane stress as opposed to cell wall stress. We discover that tyrosine biosynthesis is also essential upon sodium dodecyl sulfate perturbation whereas phenylalanine biosynthesis appears dispensable. Finally, we observe enhanced tryptophan import within minutes upon exposure to sodium dodecyl sulfate indicating that these cells are not starved for tryptophan. In summary, our results expose a functional link between internal tryptophan levels and tryptophan biosynthesis in the response to plasma membrane damage.

DNA replication has to be tightly regulated to ensure genome integrity such that DNA replication takes place only once per cell cycle. The Cdc6 sequential phosphorylation by GSK-3 and Cdk1 creates a binding site for Cdc4 ubiquitin ligase to promote Cdc6 degradation., To ensure genome integrity, DNA replication takes place only once per cell cycle and is tightly controlled by cyclin-dependent kinase (Cdk1). Cdc6p is part of the prereplicative complex, which is essential for DNA replication. Cdc6 is phosphorylated by cyclin-Cdk1 to promote its degradation after origin firing to prevent DNA rereplication. We previously showed that a yeast GSK-3 homologue, Mck1 kinase, promotes Cdc6 degradation in a SCFCdc4-dependent manner, therefore preventing rereplication. Here we present evidence that Mck1 directly phosphorylates a GSK-3 consensus site in the C-terminus of Cdc6. The Mck1-dependent Cdc6 phosphorylation required priming by cyclin/Cdk1 at an adjacent CDK consensus site. The sequential phosphorylation by Mck1 and Clb2/Cdk1 generated a Cdc4 E3 ubiquitin ligase–binding motif to promote Cdc6 degradation during mitosis. We further revealed that Cdc6 degradation triggered by Mck1 kinase was enhanced upon DNA damage caused by the alkylating agent methyl methanesulfonate and that the resulting degradation was mediated through Cdc4. Thus, Mck1 kinase ensures proper DNA replication, prevents DNA damage, and maintains genome integrity by inhibiting Cdc6.

Treatment of patients with neck lymph node metastasis of squamous cell carcinoma (SCC) from unknown primary tumor (NSCCUP) is challenging due to the risk of missing occult tumors or inducing toxicity to unaffected sites. Human papillomavirus (HPV) is a promising biomarker given its causal link to oropharyngeal SCC and superior survival of patients with HPV-driven oropharyngeal SCC and NSCCUP. Identification of HPV-driven NSCCUP could focus diagnostic work-up and treatment on the oropharynx. For the first time, we assessed HPV antibodies and their prognostic value in NSCCUP patients. Antibodies against E6 and E7 (HPV16/18/31/33/35), E1 and E2 (HPV16/18) were assessed in 46 NSCCUP patients in sera collected at diagnosis, and in follow-up sera from five patients. In 28 patients, HPV tumor status was determined using molecular markers (HPV DNA, mRNA and cellular p16

Patients with neck squamous cell carcinomas of unknown primary tumour (NSCCUP) present with lymph node metastasis without evidence for a primary tumour. Most patients undergo an aggressive multimodal treatment, which induces severe, potentially unnecessary toxicity. Primary tumours of NSCCUP can be hidden in the oropharynx. Human papillomavirus (HPV) is causally involved in a subgroup of oropharyngeal squamous cell carcinomas (OPSCC) associated with early lymph node metastasis and good prognosis. Detection of markers for HPV transformation in NSCCUP could allow focussing on the oropharynx in primary tumour search and could be of value for choice and extent of treatment. In a retrospective multicentre study (Germany, Italy and Spain), we analysed metastatic lymph nodes from 180 NSCCUP patients for the presence of HPV DNA, HPV E6*I mRNA and cellular p16(INK4a) overexpression, a surrogate marker for HPV-induced transformation. HPV status, defined as positivity for viral mRNA with at least one additional marker, was correlated with clinical parameters and survival outcome. A substantial proportion (16%) of NSCCUP were HPV-driven, mainly by HPV16 (89%). HPV prevalence increased with-year of diagnosis from 9% during 1998-2004 to 23% during 2005-2014 (p = 0.007). HPV-driven NSCCUP had significantly better overall and progression-free survival rates (p < 0.008). Based on this survival benefit, it is contended that HPV RNA status should be included in NSCCUP diagnosis and in therapeutic decision-making. Deintensification of radiation in patients with HPV-driven NSCCUP, while concurrently concentrating on the oropharynx appears to be a promising therapeutic strategy, the efficacy of which should be assessed in prospective trials. To our knowledge, this is the largest study on HPV in NSCCUP. (C) 2017 Elsevier Ltd. All rights reserved.

Cellular wound healing or the repair of plasma membrane/cell wall damage (plasma membrane damage) occurs frequently in nature. Although various cellular perturbations, such as DNA damage, spindle misalignment, and impaired daughter cell formation, are monitored by cell cycle checkpoint mechanisms in budding yeast, whether plasma membrane damage is monitored by any of these checkpoints remains to be addressed. Here, we define the mechanism by which cells sense membrane damage and inhibit DNA replication. We found that the inhibition of DNA replication upon plasma membrane damage requires GSK3/Mck1-dependent degradation of Cdc6, a component of the prereplicative complex. Furthermore, the CDK inhibitor Sic1 is stabilized in response to plasma membrane damage, leading to cell integrity maintenance in parallel with the Mck1-Cdc6 pathway. Cells defective in both Cdc6 degradation and Sic1 stabilization failed to grow in the presence of plasma membrane damage. Taking these data together, we propose that plasma membrane damage triggers G1 arrest via Cdc6 degradation and Sic1 stabilization to promote the cellular wound healing process.

In budding yeast, B-type cyclin (Clb)-dependent kinase activity is essential for S phase and mitosis. In newborn G1 cells, Clb kinase accumulation is blocked, in part because of the Sic1 stoichiometric inhibitor. Previous results strongly suggested that G1 cyclin-dependent Sic1 phosphorylation, and its consequent degradation, is essential for S phase. However, cells containing a precise endogenous gene replacement of SIC1 with SIC1-0P (all nine phosphorylation sites mutated) were fully viable. Unphosphorylatable Sic1 was abundant and nuclear throughout the cell cycle and effectively inhibited Clb kinase in vitro. SIC1-0P cells had a lengthened G1 and increased G1 cyclin transcriptional activation and variable delays in the budded part of the cell cycle. SIC1-0P was lethal when combined with deletion of CLB2, CLB3, or CLB5, the major B-type cyclins. Sic1 phosphorylation provides a sharp link between G1 cyclin activation and Clb kinase activation, but failure of Sic1 phosphorylation and proteolysis imposes a variable cell cycle delay and extreme sensitivity to B-type cyclin dosage, rather than a lethal cell cycle block.

Photoplethysmography (PPG) is a non-invasive, inexpensive and unobtrusive method to achieve heart rate monitoring during physical exercises. Motion artifacts during exercise challenge the heart rate estimation from wrist-type PPG signals. This paper presents a methodology to overcome these limitation by incorporating acceleration information. The proposed algorithm consisted of four stages: (1) A wavelet based denoising, (2) an acceleration based denoising, (3) a frequency based approach to estimate the heart rate followed by (4) a postprocessing step. Experiments with different movement types such as running and rehabilitation exercises were used for algorithm design and development. Evaluation of our heart rate estimation showed that a mean absolute error 1.96 bpm (beats per minute) with standard deviation of 2.86 bpm and a correlation of 0.98 was achieved with our method. These findings suggest that the proposed methodology is robust to motion artifacts and is therefore applicable for heart rate monitoring during sports and rehabilitation.

Introduction Human papillomavirus (HPV) infection is a risk factor for various cancer types, including head and neck squamous cell carcinoma (HNSCC). HPV-positive HNSCC show better survival compared to HPV-negative HNSCC despite their frequent early metastatic spread to cervical lymph nodes. Patients initially presenting only with a cervical lymph node metastasis (CLNM) during work-up are frequently diagnosed with HNSCC. In a few CLNM cases no primary tumor is detected. Aggressive multimodal treatment of these cancers of unknown primary site (CUP) often leads to substantial side effects and loss of quality of life. Recent studies provide evidence that the HPV status may have a prognostic value in CUP and may serve as a predictor for oropharyngeal localization of the primary tumor. Material and methods In a multinational retrospective study (Heidelberg, Barcelona, Treviso) we want to determine the prevalence of HPV-driven SCC in CLNM with unknown or identified primary tumor. Presence of 51 mucosal HPV types will be analyzed in fresh-frozen and/or formalin-fixed paraffin-embedded tissue from lymph node metastases and, if available, the corresponding primary tumor. To identify truly HPV-driven cases additional markers including viral load, E6*I oncogene mRNA and expression levels of the cellular proteins p16INK4a, p53 and pRb will be determined. In addition, if fresh-frozen tissue and serum are available viral transformation-associated RNA patterns and anti-HPV antibodies, respectively, will be analyzed. Results Results of ongoing HPV status analyses will be presented. Conclusion These data will be correlated with clinical parameters to assess, whether active HPV involvement is associated with a better prognosis and may help to direct the search for the primary tumor towards the oropharynx. Furthermore, we want to study pairs of HPV-driven oropharyngeal tumors and the corresponding cervical lymph node metastases by comparing the HPV status and integration patterns. The study is open for participation of additional clinical collaborators.

Regulation of cyclin abundance is central to eukaryotic cell cycle control. Strong overexpression of mitotic cyclins is known to lock the system in mitosis, but the quantitative behavior of the control system as this threshold is approached has only been characterized in the in vitro Xenopus extract system. Here, we quantitate the threshold for mitotic block in budding yeast caused by constitutive overexpression of the mitotic cyclin Clb2. Near this threshold, the system displays marked loss of robustness, in that loss or even heterozygosity for some regulators becomes deleterious or lethal, even though complete loss of these regulators is tolerated at normal cyclin expression levels. Recently, we presented a quantitative kinetic model of the budding yeast cell cycle. Here, we use this model to generate biochemical predictions for Clb2 levels, asynchronous as well as through the cell cycle, as the Clb2 overexpression threshold is approached. The model predictions compare well with biochemical data, even though no data of this type were available during model generation. The loss of robustness of the Clb2 overexpressing system is also predicted by the model. These results provide strong confirmation of the model's predictive ability.

Although Toll-like receptors (TLRs) are critical mediators of the immune response to pathogens, the influence of polymorphisms in this gene family on human susceptibility to infection is poorly understood. We demonstrated recently that TLR5 recognizes flagellin, a potent inflammatory stimulus present in the flagellar structure of many bacteria. Here, we show that a common stop codon polymorphism in the ligand-binding domain of TLR5 (TLR5392STOP) is unable to mediate flagellin signaling, acts in a dominant fashion, and is associated with susceptibility to pneumonia caused by Legionella pneumophila, a flagellated bacterium. We also show that flagellin is a principal stimulant of proinflammatory cytokine production in lung epithelial cells. Together, these observations suggest that TLR5392STOP increases human susceptibility to infection through an unusual dominant mechanism that compromises TLR5's essential role as a regulator of the lung epithelial innate immune response.

Cdc6p is an essential component of the pre-replicative complex (pre-RC), which binds to DNA replication origins to promote initiation of DNA replication. Only once per cell cycle does DNA replication take place. After initiation, the pre-RC components are disassembled in order to prevent re-replication. It has been shown that the N-terminal region of Cdc6p is targeted for degradation after phosphorylation by Cyclin Dependent Kinase (CDK). Here we show that Mck1p, a yeast homologue of GSK-3 kinase, is also required for Cdc6 degradation through a distinct mechanism. Cdc6 is an unstable protein and is accumulated in the nucleus only during G1 and early S-phase in wild-type cells. In mck1 deletion cells, CDC6p is stabilized and accumulates in the nucleus even in late S phase and mitosis. Overexpression of Mck1p induces rapid Cdc6p degradation in a manner dependent on Threonine-368, a GSK-3 phosphorylation consensus site, and SCFCDC4. We show evidence that Mck1p-dependent degradation of Cdc6 is required for prevention of DNA re-replication. Loss of Mck1 activity results in synthetic lethality with other pre-RC mutants previously implicated in re-replication control, and these double mutant strains over-replicate DNA within a single cell cycle. These results suggest that a GSK3 family protein plays an unexpected role in preventing DNA over-replication through Cdc6 degradation in Saccharomyces cerevisiae. We propose that both CDK and Mck1 kinases are required for Cdc6 degradation to ensure a tight control of DNA replication., Author Summary DNA replication is a fundamental cellular process that takes place in all living organisms. This cellular event has to be tightly regulated to ensure an accurate genome integrity such that DNA replication takes place only once per cell cycle. Here we show a mechanism by which DNA re-replication is controlled by Cyclin Dependent Kinase (CDK) and a yeast GSK-3 kinase (Mck1p) in S. cerevisiae. We found that Mck1p promoted Cdc6 protein degradation. Mck1p targets Cdc6p through a GSK-3 consensus site (T368), and Cdc6p protein degradation was also mediated through the same T368 site. The GSK-3 kinase has diverse cellular functions in higher eukaryotes including roles in tumorigenesis. This finding is particularly important, since this is the first evidence to show that a GSK-3 family kinase regulates DNA replication.

After i.p. infection of mice with the intracellular bacterium Mycobacterium bovis bacillus Calmette-Guérin, macrophages recovered from the peritoneal cavity display classical signs of immune activation. We have identified a member of the serine protease inhibitor (serpin) family which is highly induced in macrophages during bacillus Calmette-Guérin infection. Serpin 2a (spi2a) expression is also induced in macrophages in vivo during infection with Salmonella typhimurium and Listeria monocytogenes, and in vitro by a variety of bacteria and bacterial products. The cytokine IFN-γ also induces spi2a expression in macrophages, and this induction is synergistic with bacterial products. We also demonstrate here that a ubiquitin homolog, IFN-stimulated gene of 15-kDa (ISG15), is strongly induced during in vitro and in vivo activation of macrophages and that it conjugates to spi2a in activated macrophages. The ISG15-spi2a conjugates were identified by tandem mass spectrometry and contained spi2a conjugated to either one or two molecules of ISG15. Whereas spi2a was induced by either bacterial products or IFN-γ, ISG15 was induced only by bacterial products. Although many protein targets have been described for ubiquitin conjugation, spi2a is the first ISG15-modified protein to be reported. Macrophage activation is accompanied by the activation of a variety of proteases. It is of interest that a member of the serine protease inhibitor family is concomitantly induced and modified by a ubiquitin-like protein.

Toll-like receptors (TLRs) have been shown to participate in the recognition of pathogens by the innate immune system, but it is not clear how a restricted family of receptors has the capacity to recognize the wide spectrum of TLR stimuli known to exist. We report here that two members of the TLR family, TLR2 and TLR6, together coordinate macrophage activation by Gram-positive bacteria and the yeast cell-wall particle, zymosan. TLR6 and TLR2 both are recruited to the macrophage phagosome, where they recognize peptidoglycan, a Gram-positive pathogen component. By contrast, TLR2 recognizes another component, bacterial lipopeptide, without TLR6. The requirement for TLR cooperation is supported by the finding that TLR2 needs a partner to activate tumor necrosis factor-α production in macrophages. Dimerization of the cytoplasmic domain of TLR2 does not induce tumor necrosis factor-α production in macrophages, whereas similar dimerization of the TLR4 cytoplasmic domain does. We show that the cytoplasmic domain of TLR2 can form functional pairs with TLR6 or TLR1, and this interaction leads to cytokine induction. Thus, the cytoplasmic tails of TLRs are not functionally equivalent, with certain TLRs requiring assembly into heteromeric complexes, whereas others are active as homomeric complexes. Finally, we show that TLR6, TLR2, and TLR1 are recruited to macrophage phagosomes that contain IgG-coated erythrocytes that do not display microbial components. The data suggest that TLRs sample the contents of the phagosome independent of the nature of the contents, and can establish a combinatorial repertoire to discriminate among the large number of pathogen-associated molecular patterns found in nature.