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.

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 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.

Oral squamous cell carcinomas (OSCCs) develop in genetically altered epithelium in the mucosal lining, also coined as fields, which are mostly not visible but occasionally present as white oral leukoplakia (OL) lesions. We developed a noninvasive genetic assay using next-generation sequencing (NGS) on brushed cells to detect the presence of genetically altered fields, including those that are not macroscopically visible. The assay demonstrated high accuracy in OL patients when brush samples were compared with biopsies as gold standard. In a cohort of Fanconi anemia patients, detection of mutations in prospectively collected oral brushes predicted oral cancer also when visible abnormalities were absent. We further provide insight in the molecular landscape of OL with frequent changes of TP53, FAT1 and NOTCH1. NGS analysis of noninvasively collected samples offers a highly accurate method to detect genetically altered fields in the oral cavity, and predicts development of OSCC in high-risk individuals. Noninvasive genetic screening can be employed to screen high-risk populations for cancer and precancer, map the extension of OL lesions beyond what is visible, map the oral cavity for precancerous changes even when visible abnormalities are absent, test accuracy of promising imaging modalities, monitor interventions and determine genetic progression as well as the natural history of the disease in the human patient.

The prognostic impact of human papillomavirus (HPV) in oropharyngeal cancer is generally acknowledged, and HPV-status is assessed routinely in clinical practice. Paradoxically, while the oral cavity seems the predilection site for productive HPV-infections, figures on HPV-attribution in oral cavity squamous cell carcinoma (OCSCC) differ widely, and prognostic impact is uncertain. Major obstacles are the lack of reproducible assays to detect HPV in nonoropharyngeal cancers, the relatively small cohorts studied and consequently the shortfall of convincing data. In our study, we used a validated, nucleic acid-based workflow to assess HPV-prevalence in a consecutive cohort of 1016 OCSCCs, and investigated its prognostic impact. In parallel, we analyzed p16-immunohistochemistry (p16-IHC) as surrogate marker for transforming HPV-infection and independent prognosticator. All OCSCC-patients diagnosed between 2008 and 2014 at two Dutch university medical centers were included (N = 1069). Formalin-fixed, paraffin-embedded (FFPE)-samples of 1016 OCSCCs could be retrieved. Punch biopsies were taken from the tumor area in the FFPE-blocks and tested for HPV. P16-IHC was performed on 580 OCSCCs, including all HPV-positive tumors. From 940 samples (92.5%), nucleic acids were of sufficient quality for HPV-testing. In total, 21 (2.2%) OCSCCs were HPV DNA-positive. All HPV DNA-positive tumors were E6 mRNA-positive and considered as true HPV-positive. There was no difference in survival between HPV-positive and HPV-negative OCSCCs. In total, 46 of 580 (7.9%) OCSCCs were p16-immunopositive, including all HPV-positive tumors. Survival was comparable in p16-positive and p16-negative OCSCCs. To conclude, HPV-prevalence is very low in OCSCC and neither HPV-status nor p16-status affects outcome. Based on these data, determining HPV-status in OCSCC seems irrelevant for clinical management.

Cost-effective and time-efficient detection of oncogenic mutations supports improved presymptomatic cancer diagnostics and post-treatment disease monitoring. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a is an RNA-guided endonuclease that, upon protospacer adjacent motif (PAM)-dependent recognition of target DNA in cis, exhibits indiscriminate ssDNase activity in trans, which can be harnessed for diagnostics. TP53, one of the most frequently mutated tumor suppressor genes in cancer, displays recurring point mutations at so-called "hotspots." In this study, we optimized Cas12a-based assay conditions for in vitro detection of six TP53 hotspot mutations at the codon for p.R273, located outside the Cas12a seed region, and evaluated the specificities of four commercial Cas12a variants. We found that nonengineered LbCas12a significantly outperformed the other tested nucleases specifically in distinguishing mutant p.R273 codons in synthetic DNA, mock cell-free DNA, and tissue biopsies, despite the suboptimal PAM-distal positioning of the corresponding mutations. Future clinical Cas12a-based applications may include point-of-care tumor analysis, cost-effective mutation screening, and improved monitoring of individual cancer patients.

The response rate to immune checkpoint inhibitors targeting programmed cell death 1 (PD-1) receptor is 13%-18% for patients with recurrent or metastatic head and neck squamous cell carcinoma (HNSCC). Detailed understanding of the tumor immune microenvironment (TIME) is crucial in order to explain and improve this response rate. HNSCCs arise at various anatomical locations including the oral cavity, hypopharynx, larynx and oropharynx. Studies directly comparing immune infiltration between anatomical sites are scarce. Since the distinct locations could drive deviating microenvironments, we questioned whether the immune composition varies across these HNSCC sites.