Liquid Biopsy For Early Disease Detection
When treatment is disappointing, PC remains one of the most dismal diseases worldwide, with incidence nearly parallel to mortality. There has been much effort invested in identifying accurate tumor markers, ideally present in the timeframe between cancer onset and invasion, to allow diagnosing PC in early curable stages, to ultimately improve patient’s survival (67, 68). In this setting, the ideal biomarker should be easily detected with satisfactory sensitivity and specificity and should distinguish PC from other benign pancreatic lesions. In the context of early detection, the identification of preneo¬plastic conditions, such as PanINs, IPMNs and MCNs, is of great impor¬tance (69).
The concept of liquid biopsy refers to the analysis of biomarkers present in a sample of a body fluid collected through a simple and painless, minimally invasive technique. The body fluids mostly used for biomarker isolation are essentially blood, urine and saliva (70-72). A myriad of circulating molecules may be used as tumor markers, including cell-free DNA (cfDNA), cell-free RNA (cfRNA), circulating tumor cells (CTC), circulating tumor proteins, and extracellular vesicles, more specifically exosomes (66, 73). Blood is easily accessible and relatively stable, making serum an ideal specimen to explore potential biomarkers. However, biomarkers secreted into serum are extremely diluted and probably obscured by other more-abundant serum proteins (74).
Technological advances in the last decade have provided more opportunities to discover circulating biomarkers based on “omics” analyses, including methods focused on proteins, nucleic acids, CTCs, and exosomes. Numerous proteins of low abundance can be analyzed by mass spectrometry-based approaches and proteomic technologies (24). In recent years, based on the expression of transcriptional profiles and structural variations, different molecular subtypes of PC have been described thought genomic analyses (75-78). The early detection of mutant genes that identify PC and its subtypes is essential for an effective strategy for the management of the disease. In PDAC, there are four major driver genes (one oncogene and three tumour suppressor genes) implicated in tumorigenesis (5).
KRAS is the most frequently altered oncogene that encodes a GTPase which mediates downstream signaling from growth factor receptors; somatic mutations, clustered in specific hotspots (most in codon 12), are identified in more than 90% of PC (79). CDKN2A, by turn, is the most frequently altered tumour suppressor gene, with loss of function in more than 90% of tumors; it encodes an important cell-cycle regulator (79). TP53 is another tumour suppressor gene, with essential role in cellular response to stress, also exhibits frequent somatic mutations (79).
Lastly, SMAD4, a tumor suppressor gene mediating downstream signaling of the transforming growth factor β (TGFβ) receptor is inactivated in about half of the PC cases (79).Since Mandel and Metais, in 1948, (80) first described circulating free DNA in body fluids, an exponential interest in noninvasive technology for disease monitoring has been the focus of research in many centers worldwide. Recently, due to the possibility to pair genomic tests with tests on CTCs, circulating tumor nucleic acids (ctNAs) and tumor-derived exosomes, liquid biopsies have gained increased value for clinical application (71, 81, 82).
During PC initiation and progression, many different genetic modifications take place, including genetic diversification, amplifications and homozygous deletions, an increase in duplicate chromosomal number, recapitulation of clonal expansion, clonal selection, driver mutations and losses of heterozygosity (10, 83-90).Next-generation sequencing techniques provide deeper insight into somatic mutations and epigenetics analysis of the genome and broaden the characterization of circulating tumor DNA (ctDNA) and cell-free RNA. With the development of cell tracking techniques and flow cytometry, it is now possi¬ble to capture and analyze CTCs and exosomes (24).In this manner, the capabilities of liquid biopsy are enormous, allowing the characterization of tumor biomarkers in the same way tissue biopsy does, favoring improvement of the knowledge of tumor heterogeneity and, most importantly, contributing to early detection, monitoring of disease progression and response to treatment (71, 82, 91-96).
