BACKGROUND:Tumorous SEPT9 (septin 9, SEPTIN9) circulating cell-free DNA (ccfDNA) methylation in blood plasma is a powerful biomarker for diagnosis, molecular staging, prognosis, and recurrence monitoring in head and neck squamous cell carcinoma (HNSCC) patients. The present study aimed to evaluate the clinical performance of SEPT9 ccfDNA methylation to detect post-surgical minimal residual disease (MRD) in patients with localized or locally advanced HNSCC treated with curative intent.METHODS:We applied quasi-digital methylation-specific real-time PCR to quantify SEPT9 ccfDNA methylation levels 2 to 30 days post-surgically in plasma from n = 219 prospectively enrolled HNSCC patients. We tested the associations of SEPT9 ccfDNA methylation with clinicopathological parameters and used Kaplan-Meier and Cox proportional hazards analyses for univariate, pairwise bivariate, and multivariate analyses of disease-free survival.RESULTS:Of 219 patients, 26.5% (58/219) were post-surgically SEPT9 ccfDNA methylation positive. SEPT9 ccfDNA methylation positivity was significantly associated with tumor site, American Joint Committee on Cancer/Union for International Cancer Control (AJCC/UICC; 8th edition) tumor stage, nodal category and extracapsular extension, lymphatic and vascular invasion, and surgical margin. Bivariate Cox proportional hazards analysis proved post-surgical SEPT9 ccfDNA methylation positivity to be an independent prognostic factor tested together with AJCC/UICC tumor stage (SEPT9: hazard ratio [HR] = 2.43, 95% CI, 1.37-4.30, P = 0.002; AJCC/UICC stage: HR = 1.48, 95% CI, 1.11-1.98, P = 0.008).CONCLUSIONS:Post-surgical SEPT9 ccfDNA methylation may aid to identify high-risk HNSCC patients who could benefit from an intensified adjuvant treatment and surveillance.

Liquid biopsies allow for the non-invasive detection of tumorous circulating cell-free DNA (ccfDNA) in blood plasma of head and neck squamous cell carcinoma (HNSCC) patients. The current study tested the performance of post-surgical SEPT9 ccfDNA methylation as a molecular staging parameter in HNSCC as adjunct to established prognostic factors. A cohort of n = 219 prospectively enrolled HNSCC patients suffering from localized or locally advanced disease were tested for post-surgical SEPT9 ccfDNA methylation in their blood plasma via quantitative methylation specific real-time PCR. SEPT9 ccfDNA methylation positivity was then correlated with established prognostic factors, namely 8th edition of AJCC/UICC stage, surgical margin, lymphatic and vascular invasion. Post-surgical SEPT9 DNA methylation positivity was detected in 26.5 % (58/219) of all analyzed cases and is significantly associated with AJCC/UICC stage (P = 0.001), lymphatic invasion (P = 0.032), vascular invasion (P = 0.012), and a histopathologically verified positive surgical margin (P < 0.001) in localized and locally advanced HNSCC.Post-surgical SEPT9 ccfDNA methylation in blood is a promising molecular staging parameter in localized and locally advanced HNSCC patients. Future studies are necessary to evaluate the clinical relevance of post-surgical SEPT9 ccfDNA methylation positivity to personalize patients’ adjuvant treatment.

Flüssigbiopsien („Liquid Biopsies”) ermöglichen die nicht-invasive Bestimmung tumoröser zellfreier zirkulierender DNA (ccfDNA) im Blutplasma von Kopf-Hals-Karzinom (HNSCC) Patienten. Die aktuelle Studie untersucht die Leistungsfähigkeit der postoperativ gemessenen SEPT9 ccfDNA-Methylierung als molekularen Staging-Parameter für HNSCC-Patienten ergänzend zu etablierten prognostischen Parametern. Eine prospektiv zusammengestellte HNSCC-Patientenkohorte, bestehend aus n=219 Patienten mit lokalisierter oder lokal fortgeschrittener Tumorerkrankung, wurde postoperativ mittels methylierungsspezifischer real-time PCR auf die SEPT9 ccfDNA-Methylierung im Blutplasma untersucht. Anschließend wurde der statistische Zusammenhang zwischen der SEPT9 ccfDNA-Methylierung und den etablierten prognostischen Faktoren UICC-TNM Stadium (8. Auflage), dem Resektionsrand sowie der Lymphgefäß- und Veneninvasion analysiert. Postoperative Positivität der SEPT9 ccfDNA-Methylierung wurde in 26,5% (58/219) aller untersuchten Proben nachgewiesen. Die SEPT9 ccfDNA-Methylierungs-Positivität korrelierte signifikant mit dem UICC-TNM Stadium (P=0,001), der Lymphgefäß- (P=0,032) sowie Veneninvasion (P=0,012) und dem Resektionsrand (P<0,001). Die postoperative Bestimmung der SEPT9 ccfDNA-Methylierung im Blutplasma ist ein vielversprechender molekularer Staging-Parameter für Patienten mit lokalisierten oder lokal fortgeschrittenen Plattenepithelkarzinomen des Kopf-Hals-Bereiches. Zukünftige Studien sind erforderlich um die klinische Relevanz der postoperativ bestimmten SEPT9 ccfDNA-Methylierung für die Personalisierung der adjuvanten Therapie der Patienten zu untersuchen.

Background. Our study describes the neoexpression (Juno) and suppression (catsperD, dysferlin, Fer1L5 and otoferlin) of selected genes in oral squamous cell carcinomas (OSCCs). As the expression pattern of these genes allows a “yes” or “no” statement by exhibiting an inverse expression pattern in malignant versus benign tissues, they represent potential biomarkers for the characterization of oral malignancies, particularly OSCCs. Methods. Differential expression analyses of selected genes of interest were examined by quantitative PCR of oral cancer tissues compared to normal. Results. Five candidates out of initially nine genes were examined, demonstrating Juno as a putative new tumor marker selectively expressed in OSCCs. Interestingly, the expression of four other genes in benign tissues was completely repressed in tumor tissues with a specificity and sensitivity of 100%. No correlation was observed regarding patients’ sex, tumor staging and grading, and tumor site. Conclusion. The present study shows novel candidates that might be useful tools for oral cancer diagnosis. The neoexpression of Juno in cancerous tissues makes it a promising target molecule regarding its potential in diagnosis as well a therapeutic tool. Moreover, our observations suggest that also the repression of gene expression can be used for diagnosing—at least—OSCCs.