Recent studies have reported that p16 protein overexpression qualifies as a surrogate marker identifying an oncogenic human papillomavirus (HPV) infection in oropharyngeal squamous cell carcinoma (OPSCC). However, there is still a percentage of OPSCCs that are positive for p16 immunohistochemistry (p16 IHC) but lack HPV DNA. The objective of this study was to characterize this group at the molecular level by performing sensitive HPV DNA-and RNA-based PCR methods and genetic profiling. All patients diagnosed with an OPSCC in the period 2000-2006 in two Dutch university medical centers were included (n 5 841). The presence of HPV in a tumor sample was tested by p16 IHC followed by an HPV DNA GP51/61 PCR. p16 IHC scored positive in 195 samples, of which 161 were HPV DNA-positive and 34 (17%) HPV DNA-negative. In the latter group, a SPF 10 -LiPA25 assay, an HPV16 type-specific E7 PCR and an E6 mRNA RT-PCR were performed. Next, ten of these cases were further analyzed for loss of heterozygosity (LOH) of 15 microsatellite markers at chromosome arms 3p, 9p and 17p. Of the 34 p16-positive but PCR-negative OPSCCs, two samples tested positive by SPF 10 assay, HPV16 E7 PCR and HPV16 E6 mRNA RT-PCR. Three samples tested positive by SPF 10 assay but negative by the HPV16-specific assays. Nine of ten cases that were tested for LOH showed a genetic pattern comparable to that of HPV-negative tumors. This study categorizes p16-positive but HPV DNA-negative OPSCCs as HPV-negative tumors based on genetic profiling. This study highlights the importance of performing HPV testing in addition to p16 IHC for proper identification of HPV-associated OPSCCs.Head and neck squamous cell carcinoma (HNSCC) arises in the oral cavity, oropharynx, larynx or hypopharynx and is the sixth most common cancer worldwide, accounting for 4% of all tumors.1 Besides tobacco and alcohol consumption, high-risk human papillomavirus (HPV) is also etiologically linked to the development of HNSCCs, particularly those carcinomas that arise in the oropharyngeal region. It is widely acknowledged that HPV-attributable oropharyngeal squamous cell carcinomas (OPSCCs) have a unique biology and also have improved treatment responses and patient outcomes.2,3 Knowledge of HPV status does provide prognostic information, and more importantly, it may guide specific treatment decisions. At this moment, clinical trials are being conducted to investigate de-escalation of therapy for patients with an HPV-positive OPSCC. The hazards to patient safety of inaccurately assigning HPV-negative tumors to a HPVpositive category are clear. Therefore, a reliable HPV detection method is of major importance.The most commonly applied HPV detection methods are based on PCR amplification of HPV DNA. The problem with most of these assays is their extremely high sensitivity, which may easily identify transient infections or contaminations yielding false-positive results rather than identifying biologically relevant oncogenic infections. 4 Another approach is the use of surrogate biomarkers of t...

Patients with advanced head and neck squamous cell carcinomas (HNSCCs) are often treated with concomitant chemotherapy and radiotherapy, but only 50% is cured. A possible explanation for treatment failure is therapy resistance of the cancer stem cells (CSCs). The application of compounds specifically targeting these CSCs, in addition to routinely used therapeutics, would likely improve clinical outcome. We demonstrate that the previously described monoclonal antibody K984 recognizes the CD98 cell surface protein, which is specifically expressed by cells forming the squamous basal cell layer, the region where the squamous stem cells reside. Moreover, CD98 is highly resistant to the proteolytic enzymes required for CSC enrichment procedures. We show that CD98(high) cells, in contrast to CD98(low) cells, are able to generate tumors in immunodeficient mice, indicating that CD98(high) cells have stem cell characteristics. Furthermore, the CD98(high) subpopulation expresses high levels of cell cycle control and DNA repair genes, while the CD98(low) fraction shows expression patterns that represent the more differentiated cells forming the bulk of the tumor. CD98 is a promising CSC enrichment marker in HNSCC. Our data support the CSC concept in head and neck cancer and the potential relevance of these cells for treatment outcome.

HPV-positive OPSCCs harbour fewer cells expressing the CSC enrichment markers CD44 and CD98. Furthermore, OS and PFS were significantly worse for patients with HPV-positive OPSCC with a high percentage of CD98-positive cells.

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Summary Chromosomal copy number aberrations can be efficiently detected and quantified using low-coverage whole-genome sequencing, but analysis is hampered by the lack of knowledge on absolute DNA copy numbers and tumor purity. Here, we describe an analytical tool for Absolute Copy number Estimation, ACE, which scales relative copy number signals from chromosomal segments to optimally fit absolute copy numbers, without the need for additional genetic information, such as SNP data. In doing so, ACE derives an estimate of tumor purity as well. ACE facilitates analysis of large numbers of samples, while maintaining the flexibility to customize models and generate output of single samples. Availability and implementation ACE is freely available via www.bioconductor.org and at www.github.com/tgac-vumc/ACE. Supplementary information Supplementary data are available at Bioinformatics online.

Head and neck squamous cell carcinomas (HNSCC) develop in fields of genetically altered cells. These fields are often dysplastic and a subset can be recognized as (erythro)leukoplakia, but most are macroscopically invisible. There is a lack of adequate treatment options to eradicate these fields, whereas they underlie the development of primary tumors as well as part of the local relapses. Unfortunately, there are almost no representative cellular models available to identify suitable treatment options. To this end, clinical biopsy specimens ( = 98) were cultured from normal appearing mucosa of the surgical margins of patients with primary HNSCCs ( = 32) to generate precancer cell culture models. This collection was extended with six previously established precancer cell cultures. Genetic analysis was performed on cultures with an extended life span (≥20 population doublings), the previously established cultures, and some randomly selected cultures. In total, cancer-associated changes were detected in 18 out of 34 (53%) cultures analyzed, which appeared to be independent of life span. A variety of genetic changes were identified, including somatic mutations as well as chromosomal copy-number aberrations (CNA). Loss of /p16 and mutations in /p53 were most prominent. Remarkably, in some of these precancer cell cultures only chromosomal CNAs were detected, and none of the frequently occurring driver mutations. The precancer cell cultures, characterized herein, form a representative collection of field models that can be exploited to identify and validate new therapeutic strategies to prevent primary HNSCCs and local relapses.

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 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 HPVinfections, 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 FFPEblocks 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 HPVstatus nor p16-status affects outcome. Based on these data, determining HPV-status in OCSCC seems irrelevant for clinical management.Abbreviations: (m)RNA, (messenger) ribonucleic acid; Ct-value, cycle threshold value; DFS, disease-free survival; DNA, deoxyribonucleic acid; FFPE, formalin-fixed, paraffin-embedded; H&E, hemotoxylin and eosin; HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; OCSCC, oral cavity squamous cell carcinoma; OPSCC, oropharyngeal squamous cell carcinoma; OS, overall survival; p16-IHC, p16-immunohistochemistry; PCR, polymerase chain reaction.