X-ray repair cross complementing Group 1 (XRCC1) protein plays an important role in base excision repair. Single nucleotide polymorphisms (SNPs) in XRCC1 gene may affect DNA repairing ability, genetic susceptibility, and prognosis to oral and pharyngeal squamous cell cancer (OPSCC). This study was carried out to evaluate the association of three XRCC1 SNPs with the risk and prognosis of OPSCC. A total of 509 OPSCC cases and 678 cancer-free controls were recruited to detect the genotypes of XRCC1 by PCR-RFLP. Then, 447 case patients with surgical treatment and safety margins were included in the survival analysis. No association was observed for XRCC1 194 and the risk of OPSCC. As compared with the wild Arg/Arg genotype, the combined genotypes of 280 Arg/His and His/His were with decreased risk (AOR=0.73, 95% CI, 0.52-1.03, p = 0.069) of OPSCC and with a significantly decreased risk (AOR=0.67, 95% CI, 0.47-0.97, p = 0.035) of oral cavity. As compared with the Arg/Arg genotype of XRCC1 399, the Gln/Gln genotype was with the increased risk of OPSCC (AOR=2.06, 95%CI: 1.21-3.51, p = 0.008) and oral cavity cancer (AOR=1.89, 95%CI: 1.08-3.33, p = 0.026). We defined the ¡§putative high risk haplotypes¡¨ as ¡§Arg-Arg-Gln and Trp-Arg-Gln¡¨. The AOR were 1.29 (95% CI, 1.04-1.60, p = 0.020) for the ¡§putative high risk haplotypes¡¨ as compared with other haplotypes for OPSCC. Then, two putative high risk haplotypes were combined into ¡§putative high risk diplotypes¡¨. The AOR for the ¡§high risk diplotypes¡¨ were 1.98 (95% CI, 1.18-3.33, p = 0.010) as compared with other diplotypes for OPSCC. No association between XRCC1 polymorphisms and clinicopathological outcomes, except XRCC1 280. Those carriers of XRCC1 280His allele (combined Arg/His and His/His genotypes) were associated with late onset (≥50 yrs) of oral cavity cancers. No association between genetic variants in XRCC1 and disease-specific survival except XRCC1 399. Patients with 399 Arg/Gln and Gln/Gln genotypes showed a significant better survival as compared to Arg/Arg genotype carriers (AHR 0.41 95% CI: 0.18-0.93), especially for those patients younger than 50 years (p = 0.012), in pathological stage III or IV (p = 0.044), and without postoperative radiotherapy (p = 0.012). In summary, XRCC1 280 Arg/His and His/His genotypes were associated with decreased risk of oral cavity cancer. 399 Gln/Gln genotype was associated with increased risk of OPSCC and oral cavity cancer. The putative ¡§high risk haplotypes and diplotypes¡¨ were with increased risk of OPSCC. However, 399 Arg/Gln and Gln/Gln genotypes were prognostic factors, especially for those with young age, aggressive tumor stage, and without postoperative radiotherapy for oro and hypopharynx patients. These findings suggest that XRCC1 polymorphisms may play a role in the development and prognosis of OPSCC.

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Introduction: Human papillomavirus (HPV) is changing the prevalence, survival and treatment paradigms in oropharyngeal squamous cell carcinoma (OPSCC). Improved survival of patients with HPV positive compared to HPV negative OPSCC has led to trials of treatment de-escalation. Current HPV detection methods are imprecise, therefore standardised assessment of transcriptionally active HPV in OPSCC is required. Furthermore, the differences in immune characteristics and/or the hypoxia response/effects could explain observed differences in prognosis between HPV positive and negative OPSCC. Rigorous HPV detection and subsequent biomarker evaluation should provide additional information required before introduction of treatment de-escalation in broad patient groupings. Methods: The study cohort was 218 patients with OPSCC who received radiotherapy with curative intent. HPV status was determined on pre-treatment, formalin-fixed paraffin-embedded blocks using: 1) polymerase chain reaction (PCR); 2) in-situ hybridisation (ISH) and 3) immuno-histochemistry (IHC). QuantiGene multiplex assay was designed to detect mRNA of reference sequences of the common high-risk HPV types (16, 18, 33, 35, 45, 52 and 58). HPV detection methods were compared with mRNA quantification. Multimarker IHC of immune cell markers using chromogenic and fluorescent staining was performed, analysed and compared with single marker IHC using automated multispectral image analysis. A validated multiplex IHC method was used for a) chromogenic (CD3, CD4, CD8, and FoxP3) and b) fluorescent (CD8, CD68 and PD1/PD-L1) evaluation in tumour and stroma compartments. Single marker IHC was used to investigate tumour hypoxia markers (HIF-1α and CA-IX) in HPV positive and negative OPSCC. Results: p16 IHC and ISH were the most sensitive and specific, respectively, for classifying HPV status. The combination of the three tests had the highest positive/negative predictive values compared with QuantiGene mRNA detection. Multiplex validation showed that, for serial sections up to 6 μm apart, there were highly significant correlations (P<0.0001) between single and multiplex counts for both chromogenic and fluorescent IHC. Overall there was less variation in cell counts with fluorescent staining when compared to chromogenic staining. Multiplex IHC of TILs in HPV positive and negative OPSCC showed higher infiltration in both tumour and stromal areas of CD3+CD4+ and CD3+CD8+ T cells but not CD4+FoxP3 Tregs in HPV positive compared with HPV negative OPSCC. Only CD3+CD8+ stromal and not tumour area infiltration was associated with increased survival (P=0.02). PD-L1 expression was higher in HPV negative OPSCC and this was related to macrophage (CD68) expression of PD-L1. In HPV negative tumours infiltration with CD68+PD-L1 was associated with a good prognosis. HPV negative patients had higher expression of HIF-1α but not CA-IX. High expression of both markers was associated with a poor prognosis irrespective of HPV status. Conclusions: There are other prognostic factors operating in the larger subdivision of HPV positive and negative OPSCC. Precise HPV detection and inclusion of other prognostic factors is required before treatment de-escalation is used. Expression of immune inhibitory factors (PD1/PD-L1) alone without contextualisation with immune cell density is insufficient for patient prognostication and potential selection for therapy using immune checkpoint inhibitors. Hypoxia modification of radiotherapy should be explored in both HPV positive and negative OPSCC.

Detection of p16 through immunohistochemistry (IHC) is the standard for determining the HPV status of the tumor according the TNM eighth edition released in 2017 and has become crucial for determining the HPV status of oropharyngeal squamous cell carcinomas (OPSCC) with direct impact on staging and prognostication. In recent years, detection of HPV DNA in mouthwashes has been proposed as a noninvasive alternative, both for OPSCCs and for other head and neck squamous cell carcinomas (HNSCCs). However, the prospect of using the mouthwashes to monitor the response to therapy is unclear. To evaluate the effect of curative therapy on the detection of HPV DNA, we performed a prospective study comparing the detection frequency of high-risk HPV DNA (HR-HPV-DNA) in pre- and post-therapy mouthwashes. We collected 137 mouthwashes from 88 pathologically confirmed HNSCC patients for DNA isolation and HPV genotyping with the Inno- LiPA assay. We show that HPV DNA in pretherapeutic mouthwashes can detect HPV-driven HNSCCs with a sensitivity of 50.0% and specificity of 85.4%, alongside a high negative predictive value of 79.5% and an accuracy of 74.5%. Furthermore, we observed a notable decrease in the detection frequency of HR-HPV-DNA after successful treatment (pre-therapy 50.0% (9/18) versus post-therapy 9.7% (3/28)). However, the comparatively low sensitivity regarding detection of HPV-driven OPSCC argues against its use in clinical routine.