Koos Boeve

23 General introduction and scope of this thesis Tumour cells and tumour DNA are often released in plasma and referred to as respectively circulating tumour cells (CTC) and circulating tumour DNA (ctDNA) [84]. Plasma collected via minimal-invasive blood collection method is referred to as liquid biopsy, and has great promise as a source for predictive testing and monitoring of treatment response [85]. The precise mechanism behind the release of ctDNA in plasma is not totally clarified. Probably that apoptotic neoplastic corpuscles are released into the bloodstream whether or not after phagocytation by white blood cells and necrotic tumour cell debris including DNA [85]. Another hypothesis is that viable tumour cells migrate into lymphatic or blood vessels and become necrotic after missing the opportunity to form a metastasis [85]. In addition to plasma, other body fluids can service as a source for CTCs and ctDNA including urine, cerebrospinal fluid and saliva [84]. Tumour DNA and tumour cells in saliva are more likely from apoptotic and necrotic cells which detach from the tumour surface in the oral cavity. The relative easy way to collect plasma (minimal-invasive) and especially saliva (non- invasive) makes it very promising sources for diagnosing (local) recurrences. ctDNA in saliva and plasma might also be used for monitoring of therapy response in patients with advanced disease and treated by radio-, chemo- or targeted therapy if complete resection by surgery is not possible, such as could be the case in for example oropharyngeal tumours [85]. Several types of biomarkers have been studied: DNA, RNA, methylation and protein based markers [85,86]. Some of these studies reported sensitivities and negative predictive values of more than 80% in detecting OSCC associated biomarkers in saliva [87] and serum [85,88]. Moreover, the marker concentration levels in serum were associated with overall survival and were independent prognostic markers [88]. Despite these promising results, none of the liquid biopsies is implemented clinically nowadays for OSCC. Biomarkers for diagnosing OSCC using liquid biopsies are facing challenges before being used clinically. Besides the release of ctDNA or CTCs into the bloodstreamor saliva, also often large amount of DNA derived from healthy cells and in saliva also from other organisms (bacteria, viruses, archaea and fungi) are present in these fluids [89]. In general, the amount of ctDNA is mostly a very low fraction of the total amount of DNA extracted from plasma or saliva referred to as cell free DNA from plasma (cfDNA) [85]. Therefore, a ctDNA marker needs to be very specific for OSCC in order to detect ctDNA in a background of total cfDNA. For this purpose, OSCC-specific methylation markers might also be useful in saliva to detect progression of disease or monitor treatment using a non (saliva) or very minimal-invasive (blood) approach to collect appropriate material. Recently, a review [85] included ten studies which reported using methylation markers in saliva from OSCC patients showing sensitivities from 34% to 93% and specificities from 72% to 93% for detecting OSCC in saliva. Although these promising results, validation with large and independent data and all tumour stages is not available. Also no other markers to identify ctDNA in saliva with appropriate accuracies up to 90% and clear validations are reported [85].