REVIEW Libri Oncol. 2017;45(1):23–30 POSSIBILITIES OF LIQUID BIOPSY IN CLINICAL PRACTICE JOSIPA PERIŠA, PETRA BULIĆ, ZVJEZDANA ŠPACIR PRSKALO, MIHAELA GAĆE and LJILJANA MAYER Department of Medical Biochemistry in Oncology, University Hospital for Tumors, Sestre milosrdnice University Hospital Center, Zagreb, Croatia Summary Liquid biopsy represents innovation in the fi eld of oncology diagnosis. It refers to non-invasive blood collection method with isolation of circulating tumor cells, cell-free DNA (with circulating tumor cell free DNA fraction), circulating exosomes or micro RNA. Although requires specifi c methods for isolation and analysis of targets of interest, liquid biopsy could be of benefi t in preoperative prediction od overall survival of patients with carcinoma, for monitoring of disease and progression, along with surveillance of therapy response and early recurrence of cancer. The review describes methods for liquid biopsy analysis, together with concerns and achievements in implementing liquid biopsy in clinical practice. KEY WORDS: liquid biopsy, circulating tumor DNA - ctDNA, circulating tumor cells – CTCs, circulating microRNA – cmiRNA, exosomes, clinical practice MOGUĆNOSTI TEKUĆE BIOPSIJE U KLINIČKOJ PRAKSI Sažetak Tekuća biopsija predstavlja inovaciju na području onkološke dijagnostike. Neinvazivna je metoda kojom se iz uzorka krvi izoliraju cirkulirajuće tumorske stanice, slobodna cirkulirajuća DNA (uz frakciju tumorske DNA), cirkulirajući exosomi ili mikroRNA. Iako zahtjeva specifi čne metode izolacije i analize pojedinih elemenata od interesa, tekuća biopsija se može primijeniti u predoperativnoj predikciji preživljenja pacijenata s karcinomom, za praćenje tijeka i progresije bolesti, kao i za praćenje odgovora na terapiju ili ranog povratka bolesti. Članak se bavi opisom do danas poznatih metoda analize kompo- nenata tekuće biopsije, kao i problematikom i postignućima u uvođenju tekuće biopsije u kliničku praksu. KLJUČNE RIJEČI: tekuća biopsija, cirkulirajuća tumorska DNA - ctDNA, cirkulirajuće tumorske stanice – CTCs, cirkulirajuća mikroRNA - cmiRNA, egzosomi, klinička praksa Cancer incidence worldwide counted 14,1 residual tumor diagnosis. Consequently, interest million new cases in 2012, and the number is pre- in tumor molecular diagnostics is rapidly increas- dicted to overgrow 23 million cases each year by ing. Up to these days tissue biopsy has been the the 2030 (1). Early-stage tumor diagnosis repre- gold standard for cancer diagnosis, but its limita- sents leading problem in diagnosis of cancer with- tions and potential risks of complications lead to in asymptomatic population, considered „healthy“. comprehensive search for less invasive techniques. Great number of tumor biomarkers are being ex- The discovery of cell free DNA (cfDNA) by Man- amined nowadays, but not many have reached del and Metais in 1948 (2) and circulating tumor clinical practice. Sensitivity and specifi city of these cells (CTCs) in bloodstream revealed the new era biomarkers remain suboptimal for early-stage, or of molecular diagnostics of tumor diseases. Liquid 23 Lib Oncol. 2017;45(1):23–30 biopsy refers to less invasive technique which pro- early-stage cancer disease and can detect residual vides CTCs, cfDNA, cell free tumor DNA (ctD- lesions following therapy. High sensitivity and NA), circulating microRNA (cmiRNA) and exo- specifi city of ctDNA analytical methods can help somes from patient’s plasma. Development of mo- diagnose and monitoring of invasive cancer in lecular technologies such as PCR (polymerase early stage, together with recurrence of the cancer. chain reaction) or NGS (next generation sequenc- ing) made analysis of ctDNA possible, in despite of low concentration in plasma. Characteristics of liquid biopsy compounds CTCs-circulating tumor cells Tissue biopsy and liquid biopsy- compared Origin of CTCs can be primary tumor or sec- ondary metastatic sites. Detached cancer cells Tissue biopsy provides enough material for have a role in metastasis through blood circula- pathohistological and molecular analysis giving tion. CTCs found in blood have epithelial /mesen- information on pathohistological and molecular chymal tumor origin and express epithelial mark- changes within tumor cells. Although it allows ers on their surface (6) for example EpCAM malicious and benign tumor diff erentiation and (Epithelial Cell Adhesion Molecules) or CDH2 classifi cation, it also has shortcomings. Fine or (Cadherin-2, N-cadherin) – a molecule included in core needle biopsy has risks of infl ammation, tu- mor metastasis induced by biopsy (3), (4), bleed- cell-to-cell adhesion and transendothelial metas- ing, or other complications, also it is painful for tasis of cancer cells (7). Epithelial tumor cells go patient. Further biopsies during treatment are of- through epithelial to mesenchymal transition ten impossible to be done due to the risks listed. (EMT) - changing surface molecules and weaken- Another disadvantage of tissue biopsy is aff ord- ing intercellular adhesion. This process aff ects the ability as it is performed by experienced profes- success of the isolation of CTCs using antibodies sional and is usually guided by ultrasound, com- targeting epithelial surface molecules. Transition- puted tomography scan (CT scan) or mammogra- al cells gain the ability to survive inside blood- phy. CT guided fi ne needle aspiration biopsy cost stream and invade distance tissue. Adhesion of for lung cancer patients was presented in a study. CTCs and progression of metastatic cancer de- The cost (including procedure, adverse events and pend on optimal fl ow of blood - “mechanic hy- pathology tests) was $4130,09 on average, while pothesis” and local microenvironment of the ad- blood-based genome test (including blood draw hesion site – the “seed and soil” hypothesis (8). and pathology tests) summed up to $836,45. An- Great number of researches had shown correla- other tissue biopsy disadvantage of concern is tion evidence between blood fl ow patt ern and me- turnaround time (TAT); while DNA analysis re- tastasis of primary tumor cells. CTCs (mean diam- sults can be obtained within 71 hours from blood eter of 12 to 20 μm) are usually trapped inside collection, biopsy results are usually given within capillaries of lungs and liver (diameter 3-8 μm), or 1 or 2 weeks from sampling. Molecular analysis other capillary perfused organs (7). CTCs need to obtained from formalin-fi xed paraffi n-embedded escape anoikis (programmed cell death that oc- (FFPE) tissue sample has weaknesses; non-stan- curs when cells detach from the surrounding ex- dardized preparation methods and toxic fi xatives tracellular matrix) and immune system defence induce DNA degradation, thus DNA integrity prior to metastasis (9). Some of these mechanisms verifi cation is necessary prior to molecular analy- include clustering with other cells, such as plate- sis (5). As it grows, tumor mutates and tumor tis- lets -“cloaking eff ect” while other mechanisms are sue becomes molecularly heterogeneous – one yet to be described. Platelets and monocytes needle aspiration can hardly collect all types of (found in CTC clusters) and macrophages mediate tumor cells, and sometimes re-aspiration is need- adhesion and thrive the metastatic progress ed -this is where liquid biopsy is welcomed. False (10,11). Value of CTCs number clinically correlates negative pathohistological or cytological results of with invasive progression of cancer, tumor mass patients with early-stage cancer can be conse- and decreased overall survival in patients with quence of imprecise needle aspiration. On the con- diff erent cancer varieties. Using diff erent plat- trary, ctDNA can be found in some patients with forms circulating tumor cells can be suffi ciently 24 Lib Oncol. 2017;45(1):23–30 separated from other blood cells, despite the low to tumor cell-DNA biopsy, if the samples were count in which they are found (1 CTC per 106 – 108 taken properly. Garcia-Olmoand coworkers es- white blood cells or 0-10 CTC/mL of blood (12)). tablished the hypothesis of genometastasis in 1999 Single tumor cells can be isolated using EpCAM with remarkable discovery of cancer transforma- expressed on their surface, or can be separated by tion of cultured cells induced by addition of plas- size using microporous fi lter membranes, since ma taken from tumor bearing rats (17). The fol- CTCs are bigger in size in relation to blood cells. lowing studies demonstrated oncogenic transfor- Isolated cells can be characterized by immunos- mation of susceptible cultured cells caused by taining of cytokeratin or other specifi c molecules addition of plasma containing ctDNA obtained expressed in tumor cells. On the other hand, clus- from colorectal carcinoma (CRC) patients (18). ters of CTCs, which have higher metastatic poten- Trejo-Becerril et al. brought evidence to the hy- tial, can be separated physically using microfl u- pothesis of genometastasis with research showing idic bifurcating traps (13). Morphological charac- the ability of ctDNA to prompt horizontal tumor teristics defi ne best isolation methods to be used progression in an immunocompetent colon-carci- in particular research purposes. nogenesis rat model (19). Oncogenic transforma- tion of cells did not occur when serum and super- ctDNA – cell free tumor DNA natants were depleted of DNA, thus confi rming tumorigenic characteristics of ctDNA. The mecha- ctDNA can be isolated from plasma or serum nism of ctDNA uptake by the host cells remains of cancer patients, together with other cell-free