Promising epigenetic biomarkers for improved detection of head and neck cancer by quantitative real time multiplex PCR in saliva samples

Objectives Head and neck squamous cell carcinoma (HNSCC) is mainly diagnosed at advanced tumour stage after the onset of symptoms. We developed DNA methylation markers for early detection of HNSCC in DNA from primary tumour tissue. Our current feasibility study aims to prove that detection of these markers is also possible in non-invasive specimen.

Methods We used fresh-frozen tissue samples (20x HNSCC; 20x controls) for validation of markers Z1 – Z5 (reference ACTB) performing methylation-specific multiplex QPCR (cobas z 480 analyzer, Roche). Isolated genomic DNA is bisulfite-converted before use in QPCR. In a feasibility study we now collect both, tissue and saliva samples (goal: 200 patients), and perform multiplex QPCR for the validated markers. Specimen collection is performed at the Department of Otorhinolaryngology, Jena University Hospital.

Conclusions In the validation sample set consisting of HNSCC and control tissues, Z1 – Z5 yielded 100 % clinical sensitivity and 95 % specificity (1/5 markers positive). Single marker sensitivity ranged from 30 % to 70 %, at minimum specificity of 95 %. All tissue samples collected from the first six HNSCC patients included in the feasibility study showed positive Results for the markers Z1, Z3-Z5. Z1 showed matching Results between all tissue and saliva samples. Z5 showed positive detection in 6 tissue and 5 saliva samples. Z2 and Z4 had weak detection rates in saliva samples so far. Results from the control group are not yet available.

Conclusion Preliminary Results from validation and recent patient samples support our hypothesis that HNSCC markers may be robustly detectable in both, tissue and saliva. Using saliva for cancer-specific diagnostics based on epigenetic markers may be useful in secondary and tertiary prevention

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Publication History

Article published online:
10 June 2020