Accurate staging and outcome prediction is a major problem in clinical management of oral cancer patients, hampering high precision treatment and adjuvant therapy planning. Here, we have built and validated multivariable models that integrate gene signatures with clinical and pathological variables to improve staging and survival prediction of patients with oral squamous cell carcinoma (OSCC). Gene expression profiles from 249 human papillomavirus (HPV)-negative OSCCs were explored to identify a 22-gene lymph node metastasis signature (LNMsig) and a 40-gene overall survival signature (OSsig). To facilitate future clinical implementation and increase performance, these signatures were transferred to quantitative polymerase chain reaction (qPCR) assays and validated in an independent cohort of 125 HPV-negative tumors. When applied in the clinically relevant subgroup of early-stage (cT1-2N0) OSCC, the LNMsig could prevent overtreatment in two-third of the patients. Additionally, the integration of RT-qPCR gene signatures with clinical and pathological variables provided accurate prognostic models for oral cancer, strongly outperforming TNM. Finally, the OSsig gene signature identified a subpopulation of patients, currently considered at low-risk for disease-related survival, who showed an unexpected poor prognosis. These well-validated models will assist in personalizing primary treatment with respect to neck dissection and adjuvant therapies.

DNA copy number alterations (CNAs) are frequent in cancer, and recently developed CNA signatures revealed their value in molecular tumor stratification for patient prognosis and platinum resistance prediction in ovarian cancer. Head and neck squamous cell carcinoma (HNSCC) is also characterized by high CNAs. In this study, we determined CNA in 173 human papilloma virus-negative HNSCC from a Dutch multicenter cohort by low-coverage whole genome sequencing and tested the prognostic value of seven cancer-derived CNA signatures for these cisplatin- and radiotherapy-treated patients. We find that a high CNA signature 1 (s1) score is associated with low values for all other signatures and better patient outcomes in the Dutch cohorts and The Cancer Genome Atlas HNSCC data set. High s5 and s7 scores are associated with increased distant metastasis rates and high s6 scores with poor overall survival. High cumulative cisplatin doses result in improved outcomes in chemoradiotherapy-treated HNSCC patients. Here we find that tumors high in s1 or low in s6 are most responsive to a change in cisplatin dose. High s5 values, however, significantly increase the risk for metastasis in patients with low cumulative cisplatin doses. Together this suggests that the processes causing these CNA signatures affect cisplatin response in HNSCC. In conclusion, CNA signatures derived from a different cancer type were prognostic and associated with cisplatin response in HNSCC, suggesting they represent underlying molecular processes that define patient outcome.

Purpose: Tumor markers that are related to hypoxia, proliferation, DNA damage repair and stem cell-ness, have a prognostic value in advanced stage HNSCC patients when assessed individually. Here we aimed to evaluate and validate this in a multifactorial context and assess interrelation and the combined role of these biological factors in determining chemo-radiotherapy response in HPV-negative advanced HNSCC. Methods: RNA sequencing data of pre-treatment biopsy material from 197 HPV-negative advanced stage HNSCC patients treated with definitive chemoradiotherapy was analyzed. Biological parameter scores were assigned to patient samples using previously generated and described gene expression signatures. Locoregional control rates were used to assess the role of these biological parameters in radiation response and compared to distant metastasis data. Biological factors were ranked according to their clinical impact using bootstrapping methods and multivariate Cox regression analyses that included clinical variables. Multivariate Cox regression analyses comprising all biological variables were used to define their relative role among all factors when combined. Results: Only few biomarker scores correlate with each other, underscoring their independence. The different biological factors do not correlate or cluster, except for the two stem cell markers CD44 and SLC3A2 (r = 0.4, p < 0.001) and acute hypoxia prediction scores which correlated with T-cell infiltration score, CD8+ T cell abundance and proliferation scores (r = 0.52, 0.56, and 0.6, respectively with p < 0.001). Locoregional control association analyses revealed that chronic (Hazard Ratio (HR) = 3.9) and acute hypoxia (HR = 1.9), followed by stem cell-ness (CD44/SLC3A2; HR = 2.2/2.3), were the strongest and most robust determinants of radiation response. Furthermore, multivariable analysis, considering other biological and clinical factors, reveal a significant role for EGFR expression (HR = 2.9, p < 0.05) and T-cell infiltration (CD8+T-cells: HR = 2.2, p < 0.05; CD8+T-cells/Treg: HR = 2.6, p < 0.01) signatures in locoregional control of chemoradiotherapy-treated HNSCC. Conclusion: Tumor acute and chronic hypoxia, stem cell-ness, and CD8+ T-cell parameters are relevant and largely independent biological factors that together contribute to locoregional control. The combined analyses illustrate the additive value of multifactorial analyses and support a role for EGFR expression analysis and immune cell markers in addition to previously validated biomarkers. This external validation underscores the relevance of biological factors in determining chemoradiotherapy outcome in HNSCC.

Head and neck squamous cell carcinomas (HNSCCs) coincide with poor survival rates. The lack of driver oncogenes complicates the development of targeted treatments for HNSCC. Here, we follow-up on two previous genome-wide RNA and microRNA interference screens in HNSCC to cross-examine tumor-specific lethality by targeting ATM, ATR, CHEK1, or CHEK2. Our results uncover CHEK1 as the most promising target for HNSCC. CHEK1 expression is essential across a panel of HNSCC cell lines but redundant for growth and survival of untransformed oral keratinocytes and fibroblasts. LY2603618 (Rabusertib), which specifically targets Chk1 kinase, kills HNSCC cells effectively and specifically. Our findings show that HNSCC cells depend on Chk1-mediated signaling to progress through S-phase successfully. Chk1 inhibition coincides with stalled DNA replication, replication fork collapses, and accumulation of DNA damage. We further show that Chk1 inhibition leads to bimodal HNSCC cell killing. In the most sensitive cell lines, apoptosis is induced in S-phase, whereas more resistant cell lines manage to bypass replication-associated apoptosis, but accumulate chromosomal breaks that become lethal in subsequent mitosis. Interestingly, CDK1 expression correlates with treatment outcome. Moreover, sensitivity to Chk1 inhibition requires functional CDK1 and CDK4/6 to drive cell cycle progression, arguing against combining Chk1 inhibitors with CDK inhibitors. In contrast, Wee1 inhibitor Adavosertib progresses the cell cycle and thereby increases lethality to Chk1 inhibition in HNSCC cell lines. We conclude that Chk1 has become a key molecule in HNSCC cell cycle regulation and a very promising therapeutic target. Chk1 inhibition leads to S-phase apoptosis or death in mitosis. We provide a potential efficacy biomarker and combination therapy to follow-up in clinical setting.

The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, we mapped the genetic dependencies of human cell lines defective of cohesion regulators DDX11 and ESCO2. The obtained synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identify the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravates cohesion defects in ESCO2 mutant cells, leading to mitotic cell death. PAXIP1 promotes global chromatin association of cohesin, independent of DNA replication, a function that cannot be explained by indirect effects of PAXIP1 on transcription or DNA repair. Cohesin regulation by PAXIP1 requires its binding partner PAGR1 and a conserved FDF motif in PAGR1. PAXIP1 co-localizes with cohesin on multiple genomic loci, including active gene promoters and enhancers. Possibly, this newly identified role of PAXIP1-PAGR1 in regulating cohesin occupancy on chromatin is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.

Oral leukoplakia is the most common oral potentially malignant disorder. Malignant transformation of oral leukoplakia occurs at an annual rate of 1%-7%. WHO-defined classic epithelial dysplasia is an important predictor of malignant transformation of oral leukoplakia, but we have previously shown in a proof of concept study that prediction improves by incorporation of an architectural pattern of dysplasia, also coined as differentiated dysplasia. We aimed to analyze this finding in a larger cohort of patients.

E2F transcription factors control the oscillating expression pattern of multiple target genes during the cell cycle. Activator E2Fs, E2F1-3, induce an upswing of E2F targets, which is essential for the G1-to-S phase transition, whereas atypical E2Fs, E2F7 and E2F8, mediate a downswing of the same targets during late S, G2, and M phases. Expression of atypical E2Fs is induced by E2F1-3, but it is unknown how atypical E2Fs are inactivated in a timely manner. Here, we demonstrate that E2F7 and E2F8 are substrates of the anaphase-promoting complex/cyclosome (APC/C). Removal of CDH1, or mutating the CDH1-interacting KEN boxes, stabilized E2F7/8 from anaphase onwards and during G1. Expressing KEN mutant E2F7 during G1 impairs S phase entry and eventually results in cell death. Furthermore, we show that E2F8, but not E2F7, interacts also with APC/C(C) (dc20). Importantly, atypical E2Fs can activate APC/C(C) (dh1) by repressing its inhibitors cyclin A, cyclin E, and Emi1. In conclusion, we discovered a feedback loop between atypical E2Fs and APC/C(C) (dh1), which ensures balanced expression of cell cycle genes and normal cell cycle progression.

Prediction in high dimensional settings is difficult due to the large number of variables relative to the sample size. We demonstrate how auxiliary 'co-data' can be used to improve the performance of a Random Forest in such a setting.

Activation of cyclin B1-cyclin-dependent kinase 1 (Cdk1), triggered by a positive feedback loop at the end of G2, is the key event that initiates mitotic entry. In metaphase, anaphase-promoting complex/cyclosome-dependent destruction of cyclin B1 inactivates Cdk1 again, allowing mitotic exit and cell division. Several models describe Cdk1 activation kinetics in mitosis, but experimental data on how the activation proceeds in mitotic cells have largely been lacking. We use a novel approach to determine the temporal development of cyclin B1-Cdk1 activity in single cells. By quantifying both dephosphorylation of Cdk1 and phosphorylation of the Cdk1 target anaphase-promoting complex/cyclosome 3, we disclose how cyclin B1-Cdk1 continues to be activated after centrosome separation. Importantly, we discovered that cytoplasmic cyclin B1-Cdk1 activity can be maintained even when cyclin B1 translocates to the nucleus in prophase. These experimental data are fitted into a model describing cyclin B1-Cdk1 activation in human cells, revealing a striking resemblance to a bistable circuit. In line with the observed kinetics, cyclin B1-Cdk1 levels required to enter mitosis are lower than the amount of cyclin B1-Cdk1 needed for mitotic progression. We propose that gradually increasing cyclin B1-Cdk1 activity after centrosome separation is critical to coordinate mitotic progression.

Despite advances in treatments, 30% to 50% of stage III-IV head and neck squamous cell carcinoma (HNSCC) patients relapse within 2 years after treatment. The Big Data to Decide (BD2Decide) project aimed to build a database for prognostic prediction modeling.

To investigate the course of health-related quality of life (HRQOL) from diagnosis to 2 years follow-up among patients with oropharyngeal cancer (OPSCC), in relation to human papilloma virus (HPV) status.

This study aimed to evaluate the presence and prognostic value of tumor-infiltrating T cells in the tumor epithelium in advanced stage, HPV-negative head and neck squamous cell carcinoma (HNSCC) patients treated with primary chemoradiotherapy using digital pathology.

ERCC1-XPF is a multifunctional endonuclease involved in nucleotide excision repair (NER), interstrand cross-link (ICL) repair, and DNA double-strand break (DSB) repair. Only two patients with bi-allelic ERCC1 mutations have been reported, both of whom had features of Cockayne syndrome and died in infancy. Here, we describe two siblings with bi-allelic ERCC1 mutations in their teenage years. Genomic sequencing identified a deletion and a missense variant (R156W) within ERCC1 that disrupts a salt bridge below the XPA-binding pocket. Patient-derived fibroblasts and knock-in epithelial cells carrying the R156W substitution show dramatically reduced protein levels of ERCC1 and XPF. Moreover, mutant ERCC1 weakly interacts with NER and ICL repair proteins, resulting in diminished recruitment to DNA damage. Consequently, patient cells show strongly reduced NER activity and increased chromosome breakage induced by DNA cross-linkers, while DSB repair was relatively normal. We report a new case of ERCC1 deficiency that severely affects NER and considerably impacts ICL repair, which together result in a unique phenotype combining short stature, photosensitivity, and progressive liver and kidney dysfunction.

The aim of this prospective cohort study was to estimate the relationship between the course of HRQOL in the first 2 years after diagnosis and treatment of head and neck cancer (HNC) and personal, clinical, psychological, physical, social, lifestyle, HNC-related, and biological factors.

Fanconi anemia is a rare genome instability disorder with extreme susceptibility to squamous cell carcinoma of the head and neck and anogenital tract. In patients with this inherited disorder, the risk of head and neck cancer is 800-fold higher than in the general population, a finding which might suggest a viral etiology. Here, we analyzed the possible contribution of human polyomaviruses to FA-associated head and neck squamous cell carcinoma (HNSCC) by a pan-polyomavirus immunohistochemistry test which detects the T antigens of all known human polyomaviruses. We observed weak reactivity in 17% of the HNSCC samples suggesting that based on classical criteria, human polyomaviruses are not causally related to squamous cell carcinomas analyzed in this study.

To assess the reduction in the vaccine preventable burden of cancer in men if boys are vaccinated along with girls against oncogenic human papillomavirus (HPV).

The ubiquitin ligase anaphase-promoting complex/cyclosome (APC/C) is activated at prometaphase by mitotic phosphorylation and binding of its activator, Cdc20. This initiates cyclin A degradation, whereas cyclin B1 is stabilized by the spindle checkpoint. Upon checkpoint release, the RXXL destruction box (D box) was proposed to direct cyclin B1 to core APC/C or Cdc20. In this study, we report that endogenous cyclin B1-Cdk1 is recruited to checkpoint-inhibited, phosphorylated APC/C in prometaphase independently of Cdc20 or the cyclin B1 D box. Like cyclin A, cyclin B1 binds the APC/C by the Cdk cofactor Cks and the APC3 subunit. Prior binding to APC/C(Cdc20) makes cyclin B1 a better APC/C substrate in metaphase, driving mitotic exit and cytokinesis. We conclude that in prometaphase, the phosphorylated APC/C can recruit both cyclin A and cyclin B1 in a Cks-dependent manner. This suggests that the spindle checkpoint blocks D box recognition of APC/C-bound cyclin B1, whereas distinctive complexes between the N terminus of cyclin A and Cdc20 evade checkpoint control.

The combination of systemic cisplatin with local and regional radiotherapy as primary treatment of head and neck squamous cell carcinoma (HNSCC) leads to cure in approximately half of the patients. The addition of cisplatin has significant effects on outcome, but despite extensive research the mechanism underlying cisplatin response is still not well understood.

A major challenge in radiomics is assembling data from multiple centers. Sharing data between hospitals is restricted by legal and ethical regulations. Distributed learning is a technique, enabling training models on multicenter data without data leaving the hospitals ("privacy-preserving" distributed learning). This study tested feasibility of distributed learning of radiomics data for prediction of two year overall survival and HPV status in head and neck cancer (HNC) patients. Pretreatment CT images were collected from 1174 HNC patients in 6 different cohorts. 981 radiomic features were extracted using Z-Rad software implementation. Hierarchical clustering was performed to preselect features. Classification was done using logistic regression. In the validation dataset, the receiver operating characteristics (ROC) were compared between the models trained in the centralized and distributed manner. No difference in ROC was observed with respect to feature selection. The logistic regression coefficients were identical between the methods (absolute difference <10-7). In comparison of the full workflow (feature selection and classification), no significant difference in ROC was found between centralized and distributed models for both studied endpoints (DeLong p > 0.05). In conclusion, both feature selection and classification are feasible in a distributed manner using radiomics data, which opens new possibility for training more reliable radiomics models.

Oral squamous cell carcinoma (OSCC) is a main cause of oral cancer mortality and morbidity in central south Asia. To improve the clinical outcome of OSCC patients, detection markers are needed, which are preferably non-invasive and thus independent of a tissue biopsy.