Liquid biopsy

In recent years, well established diagnostic method for the detection of circulating tumor cells (CTCs) or circulating cell-free tumor DNA (ctDNA) or cell-free tumor RNA (ctRNA) in body fluids such as blood and urine (Crowley E et al. 2013). The Liquid biopsy is used, among other things, to determine the response of a tumour to e.g. chemotherapy.

At present, molecular diagnostics is mostly performed from tissue biopsies obtained by needle biopsy or a (often complex) surgical procedure. Molecular diagnostics of DNA mutations are performed directly in fresh tissue samples or often in formalin-fixed and paraffin-embedded tissue samples (FFPE). Due to its methodology, this method is limited for many tumor entities and often does not allow a temporal cross-sectional analysis.

The Liquid biopsy from easily accessible body fluids (blood, urine) for the direct detection of circulating tumor cells (CTCs) does not have this major methodological disadvantage, so that cross-sectional analyses and repetitive examinations are possible without problems. Most of the available detection methods are based on the antibody-based imaging of epithelial markers, i.e. adhesion molecules and/or different types of antibodies. cytokines.

Circulating tumor cells (CTCs)

Among the tumor entities for which this method is already used are malignancies of the bladder, breast, liver, lung, ovaries, pancreas, prostate, colon and Merkel cell carcinoma (Bardelli A et al. 2017). In patients with metastatic breast, colorectal or prostate carcinoma, a significant correlation between CTC count and prognosis was demonstrated. This has led to the approval of the "CellSearch CTC system" (Menarini Silicon Biosystems, San Diego, USA) for these 3 entities. The extent to which this method will gain significance for malignant melanoma is the subject of further research (Gaiser MR et al. 2018).

For malignant melanoma the occurrence of CTCs and their association with tumor burden and therapeutic response has already been proven (Rodic S et al. 2014). Markers for tyrosinase, MAGE-A3,MART-1(Melan-A), PAX3 (paired box3) GalNac-T (polypdeptide-N-acetylgalactosaminyltransferase), MCAM (CD146) are used.

The different strategies used in the various Malignancies require different stratification of patients for appropriate treatment or for monitoring long-term relapses and patients without evidence of tumor progression (NED patients = no evidence of progression).

Cell-free ctDNA

Blood-based cell-free DNA (cell-free DNA or cfDNA) and cell-free RNA (cell-free RNA or cfRNA) are collectively referred to as "CNAPS", circulating nucleic acids in plasma and serum (CNAPS = circulating nucleic acids in plasma and serum). The main source of cfDNA in the body is passive release from cells, through mechanisms such as apoptosis and necrosis, as well as active secretion from living cells. In malignancies, the resulting circulating cell-free tumour DNA (ctDNA) is characterised by genetic defects, such as mutations (for malignant melanoma BRAF, NRAS; for other malignancies mutations in EGFR, PIK3CA, KRAS, BRAF, TP53, HER2, GNA11, KIT, MAP2K1, NRB) chromosomal aberrations, abnormal epigenetic patterns such as methylated DNA. The three most common detection methods are digital PCR (quantification of a target DNA in a sample), BEAMing and allele specific ligation PCR (LPCR) (Gaiser MR et al. 2018).

The genetic defects in the ctDNA are in high agreement with those in tumour tissue and are fundamentally different from the circulating DNA from healthy tissue. In blood, the ctDNA seems to be more unstable than the physiological DNA. The half-life of cell-free tumour DNA is 144 min (Diehl F et al. 2008). It is filtered out of the bloodstream by the spleen and especially by the liver and kidneys.

Other components of the blood, which can also be used as potential biomarkers for tumor diagnosis, include miRNA and extracellular circulating vesicles (EV) such as the 50-100nm exosomes.

The analysis of cell-free fetal DNA has become an established method for non-invasive prenatal diagnostics (NiPt). Examination of fetal DNA in the mother's blood can diagnose point mutations and aneuploidy as early as 7 weeks after conception. Circulating fetal DNA is not excreted through the kidney.

The clinical benefit of ctDNA analysis in oncology includes:

• Assessment of molecular heterogeneity
• Identification of specific genomic alterations for personalized treatment (Gaiser MR et al. 2018)
• Monitoring the tumor load
• Early detection of drug resistance of melanoma (Mirzaei H et al. 2016)
• Clarification of resistance mechanisms
• Blood-based replacement method for classical tissue biopsy

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