Liquid biopsy and PCR-free ultrasensitive detection systems in oncology (Review)

Finotti,Alessia; Allegretti,Matteo; Gasparello,Jessica; Giacomini,Patrizio; Spandidos,Demetrios  A.; Spoto,Giuseppe; Gambari,Roberto

doi:10.3892/ijo.2018.4516

Liquid biopsy and PCR-free ultrasensitive detection systems in oncology (Review)

Authors:
- Alessia Finotti
- Matteo Allegretti
- Jessica Gasparello
- Patrizio Giacomini
- Demetrios A. Spandidos
- Giuseppe Spoto
- Roberto Gambari
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Affiliations: Department of Life Sciences and Biotechnology, Ferrara University, 44121 Ferrara, Italy, Oncogenomics and Epigenetic Unit, IRCCS Regina Elena National Cancer Institute, 00144 Rome, Italy, Laboratory of Clinical Virology, Medical School, University of Crete, 71003 Heraklion, Crete, Greece, Department of Chemistry, Catania University, 95125 Catania, Italy
Published online on: August 6, 2018 https://doi.org/10.3892/ijo.2018.4516
Pages: 1395-1434
Copyright: © Finotti et al. This is an open access article distributed under the terms of Creative Commons Attribution License.

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Abstract

In oncology, liquid biopsy is used in the detection of next-generation analytes, such as tumor cells, cell-free nucleic acids and exosomes in peripheral blood and other body fluids from cancer patients. It is considered one of the most advanced non-invasive diagnostic systems to enable clinically relevant actions and implement precision medicine. Medical actions include, but are not limited to, early diagnosis, staging, prognosis, anticipation (lead time) and the prediction of therapy responses, as well as follow-up. Historically, the applications of liquid biopsy in cancer have focused on circulating tumor cells (CTCs). More recently, this analysis has been extended to circulating free DNA (cfDNA) and microRNAs (miRNAs or miRs) associated with cancer, with potential applications for development into multi-marker diagnostic, prognostic and therapeutic signatures. Liquid biopsies avoid some key limitations of conventional tumor tissue biopsies, including invasive tumor sampling, under-representation of tumor heterogeneity and poor description of clonal evolution during metastatic dissemination, strongly reducing the need for multiple sampling. On the other hand, this approach suffers from important drawbacks, i.e., the fragmentation of cfDNA, the instability of RNA, the low concentrations of certain analytes in body fluids and the confounding presence of normal, as well as aberrant DNAs and RNAs. For these reasons, the analysis of cfDNA has been mostly focused on mutations arising in, and pathognomonicity of, tumor DNA, while the analysis of cfRNA has been mostly focused on miRNA patterns strongly associated with neoplastic transformation/progression. This review lists some major applicative areas, briefly addresses how technology is bypassing liquid biopsy limitations, and places a particular emphasis on novel, PCR-free platforms. The ongoing collaborative efforts of major international consortia are reviewed. In addition to basic and applied research, we will consider technological transfer, including patents, patent applications and available information on clinical trials aimed at verifying the potential of liquid biopsy in cancer.

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1	Heitzer E, Auer M, Ulz P, Geigl JB and Speicher MR: Circulating tumor cells and DNA as liquid biopsies. Genome Med. 5:732013. View Article : Google Scholar : PubMed/NCBI
2	Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM, et al: Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 6:224ra242014. View Article : Google Scholar : PubMed/NCBI
3	Domínguez-Vigil IG, Moreno-Martínez AK, Wang JY, Roehrl MHA and Barrera-Saldaña HA: The dawn of the liquid biopsy in the fight against cancer. Oncotarget. 9:2912–2922. 2017.
4	Breitbach S, Tug S, Helmig S, Zahn D, Kubiak T, Michal M, Gori T, Ehlert T, Beiter T and Simon P: Direct quantification of cell-free, circulating DNA from unpurified plasma. PloS One. 9:e878382014. View Article : Google Scholar : PubMed/NCBI
5	Heitzer E, Ulz P and Geigl JB: Circulating tumor DNA as a liquid biopsy for cancer. Clin Chem. 61:112–123. 2015. View Article : Google Scholar
6	Yang M, Forbes ME, Bitting RL, O'Neill SS, Chou PC, Topaloglu U, Miller LD, Hawkins GA, Grant SC, DeYoung BR, et al: Incorporating blood-based liquid biopsy information into cancer staging: Time for a TNMB system. Ann Oncol. 29:311–323. 2018. View Article : Google Scholar
7	Kleppe M and Levine RL: Tumor heterogeneity confounds and illuminates: Assessing the implications. Nat Med. 20:342–344. 2014. View Article : Google Scholar : PubMed/NCBI
8	Diehl F, Schmidt K, Choti MA, Romans K, Goodman S, Li M, Thornton K, Agrawal N, Sokoll L, Szabo SA, et al: Circulating mutant DNA to assess tumor dynamics. Nat Med. 14:985–990. 2008. View Article : Google Scholar : PubMed/NCBI
9	Leon SA, Shapiro B, Sklaroff DM and Yaros MJ: Free DNA in the serum of cancer patients and the effect of therapy. Cancer Res. 37:646–650. 1977.PubMed/NCBI
10	Burz C, Pop VV, Buiga R, Daniel S, Samasca G, Aldea C and Lupan I: Circulating tumor cells in clinical research and monitoring patients with colorectal cancer. Oncotarget. 9:24561–24571. 2018. View Article : Google Scholar : PubMed/NCBI
11	Sozzi G, Conte D, Mariani L, Lo Vullo S, Roz L, Lombardo C, Pierotti MA and Tavecchio L: Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Res. 61:4675–4678. 2001.PubMed/NCBI
12	Spindler KL, Pallisgaard N, Vogelius I and Jakobsen A: Quantitative cell-free DNA, KRAS, and BRAF mutations in plasma from patients with metastatic colorectal cancer during treatment with cetuximab and irinotecan. Clin Cancer Res. 18:1177–1185. 2012. View Article : Google Scholar : PubMed/NCBI
13	Perkins G, Yap TA, Pope L, Cassidy AM, Dukes JP, Riisnaes R, Massard C, Cassier PA, Miranda S, Clark J, et al: Multi-purpose utility of circulating plasma DNA testing in patients with advanced cancers. PloS One. 7:e470202012. View Article : Google Scholar : PubMed/NCBI
14	Ignatiadis M, Lee M and Jeffrey SS: Circulating tumor cells and circulating tumor DNA: Challenges and opportunities on the path to clinical utility. Clin Cancer Res. 21:4786–4800. 2015. View Article : Google Scholar : PubMed/NCBI
15	Krebs MG, Hou JM, Ward TH, Blackhall FH and Dive C: Circulating tumour cells: Their utility in cancer management and predicting outcomes. Ther Adv Med Oncol. 2:351–365. 2010. View Article : Google Scholar
16	Millner LM, Linder MW and Valdes R Jr: Circulating tumor cells: a review of present methods and the need to identify heterogeneous phenotypes. Ann Clin Lab Sci. 43:295–304. 2013.PubMed/NCBI
17	Kuipers EJ and Spaander MC: Personalized screening for colorectal cancer. Nat Rev Gastroenterol Hepatol. 15:391–392. 2018. View Article : Google Scholar : PubMed/NCBI
18	Kloten V, Rüchel N, Brüchle NO, Gasthaus J, Freudenmacher N, Steib F, Mijnes J, Eschenbruch J, Binnebösel M, Knüchel R, et al: Liquid biopsy in colon cancer: Comparison of different circulating DNA extraction systems following absolute quantification of KRAS mutations using Intplex allele-specific PCR. Oncotarget. 8:86253–86263. 2017. View Article : Google Scholar : PubMed/NCBI
19	Thomsen CEB, Appelt AL, Andersen RF, Lindebjerg J, Jensen LH and Jakobsen A: The prognostic value of simultaneous tumor and serum RAS/RAF mutations in localized colon cancer. Cancer Med. 6:928–936. 2017. View Article : Google Scholar : PubMed/NCBI
20	Spindler KL, Pallisgaard N, Andersen RF, Brandslund I and Jakobsen A: Circulating free DNA as biomarker and source for mutation detection in metastatic colorectal cancer. PLoS One. 10:e01082472015. View Article : Google Scholar : PubMed/NCBI
21	Hardingham JE, Grover P, Winter M, Hewett PJ, Price TJ and Thierry B: Detection and clinical significance of circulating tumor cells in colorectal cancer-20 years of progress. Mol Med. 21:S25–S31. 2015. View Article : Google Scholar
22	Veldore VH, Choughule A, Routhu T, Mandloi N, Noronha V, Joshi A, Dutt A, Gupta R, Vedam R and Prabhash K: Validation of liquid biopsy: Plasma cell-free DNA testing in clinical management of advanced non-small cell lung cancer. Lung Cancer (Auckl). 9:1–11. 2018.
23	Anfossi S, Babayan A, Pantel K and Calin GA: Clinical utility of circulating non-coding RNAs - an update. Nat Rev Clin Oncol. May 21–2018.Epub ahead of print. View Article : Google Scholar : PubMed/NCBI
24	Izzotti A, Carozzo S, Pulliero A, Zhabayeva D, Ravetti JL and Bersimbaev R: Extracellular MicroRNA in liquid biopsy: Applicability in cancer diagnosis and prevention. Am J Cancer Res. 6:1461–1493. 2016.PubMed/NCBI
25	Coombs CC, Zehir A, Devlin SM, Kishtagari A, Syed A, Jonsson P, Hyman DM, Solit DB, Robson ME, Baselga J, et al: Therapy-related clonal hematopoiesis in patients with non-hematologic cancers is common and associated with adverse clinical outcomes. Cell Stem Cell. 21:374–382e4. 2017. View Article : Google Scholar : PubMed/NCBI
26	Taly V, Pekin D, Benhaim L, Kotsopoulos SK, Le Corre D, Li X, Atochin I, Link DR, Griffiths AD, Pallier K, et al: Multiplex picodroplet digital PCR to detect KRAS mutations in circulating DNA from the plasma of colorectal cancer patients. Clin Chem. 59:1722–1731. 2013. View Article : Google Scholar : PubMed/NCBI
27	Wang JY, Hsieh JS, Chang MY, Huang TJ, Chen FM, Cheng TL, Alexandersen K, Huang YS, Tzou WS and Lin SR: Molecular detection of APC, K-ras, and p53 mutations in the serum of colorectal cancer patients as circulating biomarkers. World J Surg. 28:721–726. 2004. View Article : Google Scholar : PubMed/NCBI
28	Mohan S, Heitzer E, Ulz P, Lafer I, Lax S, Auer M, Pichler M, Gerger A, Eisner F, Hoefler G, et al: Changes in colorectal carcinoma genomes under anti-EGFR therapy identified by whole-genome plasma DNA sequencing. PLoS Genet. 10:e10042712014. View Article : Google Scholar : PubMed/NCBI
29	Feng WN, Gu WQ, Zhao N, Pan YM, Luo W, Zhang H, Liang JM, Yang J and Deng YM: Comparison of the SuperARMS and Droplet Digital PCR for detecting EGFR mutation in ctDNA from NSCLC patients. Transl Oncol. 11:542–545. 2018. View Article : Google Scholar : PubMed/NCBI
30	Newman AM, Bratman SV, To J, Wynne JF, Eclov NC, Modlin LA, Liu CL, Neal JW, Wakelee HA, Merritt RE, et al: An ultrasensitive method for quantitating circulating tumor DNA with broad patient coverage. Nat Med. 20:548–554. 2014. View Article : Google Scholar : PubMed/NCBI
31	Fujiwara K, Fujimoto N, Tabata M, Nishii K, Matsuo K, Hotta K, Kozuki T, Aoe M, Kiura K, Ueoka H, et al: Identification of epigenetic aberrant promoter methylation in serum DNA is useful for early detection of lung cancer. Clin Cancer Res. 11:1219–1225. 2005.PubMed/NCBI
32	Szpechcinski A, Chorostowska-Wynimko J, Struniawski R, Kupis W, Rudzinski P, Langfort R, Puscinska E, Bielen P, Sliwinski P and Orlowski T: Cell-free DNA levels in plasma of patients with non-small-cell lung cancer and inflammatory lung disease. Br J Cancer. 113:476–483. 2015. View Article : Google Scholar : PubMed/NCBI
33	Pisanic TR 2nd, Athamanolap P, Poh W, Chen C, Hulbert A, Brock MV, Herman JG and Wang TH: DREAMing: A simple and ultrasensitive method for assessing intratumor epigenetic heterogeneity directly from liquid biopsies. Nucleic Acids Res. 43:e1542015. View Article : Google Scholar : PubMed/NCBI
34	Dawson SJ, Tsui DW, Murtaza M, Biggs H, Rueda OM, Chin SF, Dunning MJ, Gale D, Forshew T, Mahler-Araujo B, et al: Analysis of circulating tumor DNA to monitor metastatic breast cancer. N Engl J Med. 368:1199–209. 2013. View Article : Google Scholar : PubMed/NCBI
35	Murtaza M, Dawson SJ, Tsui DW, Gale D, Forshew T, Piskorz AM, Parkinson C, Chin SF, Kingsbury Z, Wong AS, et al: Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature. 497:108–112. 2013. View Article : Google Scholar : PubMed/NCBI
36	Yanagawa T, Kagara N, Miyake T, Tanei T, Naoi Y, Shimoda M, Shimazu K, Kim SJ and Noguchi S: Detection of ESR1 mutations in plasma and tumors from metastatic breast cancer patients using next-generation sequencing. Breast Cancer Res Treat. 163:231–240. 2017. View Article : Google Scholar : PubMed/NCBI
37	Mastoraki S, Strati A, Tzanikou E, Chimonidou M, Politaki E, Voutsina A, Psyrri A, Georgoulias V and Lianidou E: ESR1 Methylation: A liquid biopsy-based epigenetic assay for the follow-up of patients with metastatic breast cancer receiving endocrine treatment. Clin Cancer Res. 24:1500–1510. 2018. View Article : Google Scholar
38	Gray ES, Rizos H, Reid AL, Boyd SC, Pereira MR, Lo J, Tembe V, Freeman J, Lee JH, Scolyer RA, et al: Circulating tumor DNA to monitor treatment response and detect acquired resistance in patients with metastatic melanoma. Oncotarget. 6:42008–42018. 2015. View Article : Google Scholar : PubMed/NCBI
39	Schreuer M, Meersseman G, Van Den Herrewegen S, Jansen Y, Chevolet I, Bott A, Wilgenhof S, Seremet T, Jacobs B, Buyl R, et al: Quantitative assessment of BRAF V600 mutant circulating cell-free tumor DNA as a tool for therapeutic monitoring in metastatic melanoma patients treated with BRAF/MEK inhibitors. J Transl Med. 14:952016. View Article : Google Scholar : PubMed/NCBI
40	Madic J, Piperno-Neumann S, Servois V, Rampanou A, Milder M, Trouiller B, Gentien D, Saada S, Assayag F, Thuleau A, et al: Pyrophosphorolysis-activated polymerization detects circulating tumor DNA in metastatic uveal melanoma. Clin Cancer Res. 18:3934–3941. 2012. View Article : Google Scholar : PubMed/NCBI
41	Pereira E, Camacho-Vanegas O, Anand S, Sebra R, Catalina Camacho S, Garnar-Wortzel L, Nair N, Moshier E, Wooten M, Uzilov A, et al: Personalized Circulating Tumor DNA Biomarkers dynamically predict treatment response and survival in gynecologic cancers. PLoS One. 10:e01457542015. View Article : Google Scholar : PubMed/NCBI
42	Forshew T, Murtaza M, Parkinson C, Gale D, Tsui DW, Kaper F, Dawson SJ, Piskorz AM, Jimenez-Linan M, Bentley D, et al: Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA. Sci Transl Med. 4:136ra682012. View Article : Google Scholar : PubMed/NCBI
43	No JH, Kim K, Park KH and Kim YB: Cell-free DNA level as a prognostic biomarker for epithelial ovarian cancer. Anticancer Res. 32:3467–3471. 2012.PubMed/NCBI
44	Giannopoulou L, Chebouti I, Pavlakis K, Kasimir-Bauer S and Lianidou ES: RASSF1A promoter methylation in high-grade serous ovarian cancer: A direct comparison study in primary tumors, adjacent morphologically tumor cell-free tissues and paired circulating tumor DNA. Oncotarget. 8:21429–21443. 2017. View Article : Google Scholar : PubMed/NCBI
45	Chan KC, Jiang P, Zheng YW, Liao GJ, Sun H, Wong J, Siu SS, Chan WC, Chan SL, Chan AT, et al: Cancer genome scanning in plasma: Detection of tumor-associated copy number aberrations, single-nucleotide variants, and tumoral heterogeneity by massively parallel sequencing. Clin Chem. 59:211–224. 2013. View Article : Google Scholar
46	Zhang P, Wen X, Gu F, Deng X, Li J, Dong J, Jiao J and Tian Y: Methylation profiling of serum DNA from hepatocellular carcinoma patients using an Infinium Human Methylation 450 BeadChip. Hepatol Int. 7:893–900. 2013. View Article : Google Scholar : PubMed/NCBI
47	Ren N, Qin LX, Tu H, Liu YK, Zhang BH and Tang ZY: The prognostic value of circulating plasma DNA level and its allelic imbalance on chromosome 8p in patients with hepatocellular carcinoma. J Cancer Res Clin Oncol. 132:399–407. 2006. View Article : Google Scholar : PubMed/NCBI
48	Huang A, Zhang X, Zhou SL, Cao Y, Huang XW, Fan J, Yang XR and Zhou J: Detecting circulating tumor DNA in hepatocellular carcinoma patients using Droplet Digital PCR is feasible and reflects intratumoral heterogeneity. J Cancer. 7:1907–1914. 2016. View Article : Google Scholar : PubMed/NCBI
49	Heitzer E, Ulz P, Belic J, Gutschi S, Quehenberger F, Fischereder K, Benezeder T, Auer M, Pischler C, Mannweiler S, et al: Tumor-associated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing. Genome Med. 5:302013. View Article : Google Scholar : PubMed/NCBI
50	Annala M, Vandekerkhove G, Khalaf D, Taavitsainen S, Beja K, Warner EW, Sunderland K, Kollmannsberger C, Eigl BJ, Finch D, et al: Circulating tumor DNA genomics correlate with resistance to Abiraterone and Enzalutamide in prostate cancer. Cancer Discov. 8:444–457. 2018. View Article : Google Scholar : PubMed/NCBI
51	Delgado PO, Alves BC, Gehrke Fde S, Kuniyoshi RK, Wroclavski ML, Del Giglio A and Fonseca FL: Characterization of cell-free circulating DNA in plasma in patients with prostate cancer. Tumour Biol. 34:983–986. 2013. View Article : Google Scholar
52	Buelens S, Claeys T, Dhondt B, Poelaert F, Vynck M, Yigit N, Thas O, Ost P, Vandesompele J, Lumen N, et al: Prognostic and therapeutic implications of circulating androgen receptor gene copy number in prostate cancer patients using Droplet Digital polymerase chain reaction. Clin Genitourin Cancer. 16:197–205. 2017. View Article : Google Scholar
53	Pu XX, Huang GL, Guo HQ, Guo CC, Li H, Ye S, Ling S, Jiang L, Tian Y and Lin TY: Circulating miR-221 directly amplified from plasma is a potential diagnostic and prognostic marker of colorectal cancer and is correlated with p53 expression. J Gastroenterol Hepatol. 25:1674–1680. 2010. View Article : Google Scholar : PubMed/NCBI
54	Liu GH, Zhou ZG, Chen R, Wang MJ, Zhou B, Li Y and Sun XF: Serum miR-21 and miR-92a as biomarkers in the diagnosis and prognosis of colorectal cancer. Tumour Biol. 34:2175–2181. 2013. View Article : Google Scholar : PubMed/NCBI
55	Cheng H, Zhang L, Cogdell DE, Zheng H, Schetter AJ, Nykter M, Harris CC, Chen K, Hamilton SR and Zhang W: Circulating plasma miR-141 is a novel biomarker for metastatic colon cancer and predicts poor prognosis. PLoS One. 6:e177452011. View Article : Google Scholar : PubMed/NCBI
56	Lv ZC, Fan YS, Chen HB and Zhao DW: Investigation of microRNA-155 as a serum diagnostic and prognostic biomarker for colorectal cancer. Tumour Biol. 36:1619–1625. 2015. View Article : Google Scholar
57	Krawczyk P, Powrózek T, Olesiński T, Dmitruk A, Dziwota J, Kowalski D and Milanowski J: Evaluation of miR-506 and miR-4316 expression in early and non-invasive diagnosis of colorectal cancer. Int J Colorectal Dis. 32:1057–1060. 2017. View Article : Google Scholar : PubMed/NCBI
58	Huang Z, Huang D, Ni S, Peng Z, Sheng W and Du X: Plasma microRNAs are promising novel biomarkers for early detection of colorectal cancer. Int J Cancer. 127:118–126. 2010. View Article : Google Scholar
59	Liu C, Eng C, Shen J, Lu Y, Takata Y, Mehdizadeh A, Chang GJ, Rodriguez-Bigas MA, Li Y, Chang P, et al: Serum exosomal miR-4772-3p is a predictor of tumor recurrence in stage II and III colon cancer. Oncotarget. 7:76250–76260. 2016.PubMed/NCBI
60	Ogata-Kawata H, Izumiya M, Kurioka D, Honma Y, Yamada Y, Furuta K, Gunji T, Ohta H, Okamoto H, Sonoda H, et al: Circulating exosomal microRNAs as biomarkers of colon cancer. PLoS One. 9:e929212014. View Article : Google Scholar : PubMed/NCBI
61	Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, Poon TC, Ng SS and Sung JJ: Differential expression of microRNAs in plasma of patients with colorectal cancer: A potential marker for colorectal cancer screening. Gut. 58:1375–1381. 2009. View Article : Google Scholar : PubMed/NCBI
62	Zheng G, Du L, Yang X, Zhang X, Wang L, Yang Y, Li J and Wang C: Serum microRNA panel as biomarkers for early diagnosis of colorectal adenocarcinoma. Br J Cancer. 111:1985–1992. 2014. View Article : Google Scholar : PubMed/NCBI
63	Dou H, Wang Y, Su G and Zhao S: Decreased plasma let-7c and miR-152 as noninvasive biomarker for non-small-cell lung cancer. Int J Clin Exp Med. 8:9291–9298. 2015.PubMed/NCBI
64	Yu H, Jiang L, Sun C, Li Guo L, Lin M, Huang J and Zhu L: Decreased circulating miR-375: A potential biomarker for patients with non-small-cell lung cancer. Gene. 534:60–65. 2014. View Article : Google Scholar : PubMed/NCBI
65	Li N, Ma J, Guarnera MA, Fang H, Cai L and Jiang F: Digital PCR quantification of miRNAs in sputum for diagnosis of lung cancer. J Cancer Res Clin Oncol. 140:145–150. 2014. View Article : Google Scholar :
66	Geng Q, Fan T, Zhang B, Wang W, Xu Y and Hu H: Five microRNAs in plasma as novel biomarkers for screening of early-stage non-small cell lung cancer. Respir Res. 15:1492014. View Article : Google Scholar : PubMed/NCBI
67	Hu Z, Chen X, Zhao Y, Tian T, Jin G, Shu Y, Chen Y, Xu L, Zen K, Zhang C, et al: Serum microRNA signatures identified in a genome-wide serum microRNA expression profiling predict survival of non-small-cell lung cancer. J Clin Oncol. 28:1721–1726. 2010. View Article : Google Scholar : PubMed/NCBI
68	Yu L, Todd NW, Xing L, Xie Y, Zhang H, Liu Z, Fang H, Zhang J, Katz RL and Jiang F: Early detection of lung adenocarcinoma in sputum by a panel of microRNA markers. Int J Cancer. 127:2870–2878. 2010. View Article : Google Scholar
69	Liu XG, Zhu WY, Huang YY, Ma LN, Zhou SQ, Wang YK, Zeng F, Zhou JH and Zhang YK: High expression of serum miR-21 and tumor miR-200c associated with poor prognosis in patients with lung cancer. Med Oncol. 29:618–626. 2012. View Article : Google Scholar
70	Leng Q, Lin Y and Jiang F, Lee CJ, Zhan M, Fang H, Wang Y and Jiang F: A plasma miRNA signature for lung cancer early detection. Oncotarget. 8:111902–111911. 2017. View Article : Google Scholar
71	Asaga S, Kuo C, Nguyen T, Terpenning M, Giuliano AE and Hoon DS: Direct serum assay for microRNA-21 concentrations in early and advanced breast cancer. Clin Chem. 57:84–91. 2011. View Article : Google Scholar
72	Zhu W, Qin W, Atasoy U and Sauter ER: Circulating microRNAs in breast cancer and healthy subjects. BMC Res Notes. 2:892009. View Article : Google Scholar : PubMed/NCBI
73	Heneghan HM, Miller N, Lowery AJ, Sweeney KJ, Newell J and Kerin MJ: Circulating microRNAs as novel minimally invasive biomarkers for breast cancer. Ann Surg. 251:499–505. 2010. View Article : Google Scholar : PubMed/NCBI
74	Stückrath I, Rack B, Janni W, Jäger B, Pantel K and Schwarzenbach H: Aberrant plasma levels of circulating miR-16, miR-107, miR-130a and miR-146a are associated with lymph node metastasis and receptor status of breast cancer patients. Oncotarget. 6:13387–13401. 2015. View Article : Google Scholar : PubMed/NCBI
75	Mangolini A, Ferracin M, Zanzi MV, Saccenti E, Ebnaof SO, Poma VV, Sanz JM, Passaro A, Pedriali M, Frassoldati A, et al: Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR. Biomark Res. 3:122015. View Article : Google Scholar : PubMed/NCBI
76	Kleivi Sahlberg K, Bottai G, Naume B, Burwinkel B, Calin GA, Børresen-Dale AL and Santarpia L: A serum microRNA signature predicts tumor relapse and survival in triple-negative breast cancer patients. Clin Cancer Res. 21:1207–1214. 2015. View Article : Google Scholar
77	Margue C, Reinsbach S, Philippidou D, Beaume N, Walters C, Schneider JG, Nashan D, Behrmann I and Kreis S: Comparison of a healthy miRNome with melanoma patient miRNomes: are microRNAs suitable serum biomarkers for cancer. Oncotarget. 6:12110–121127. 2015. View Article : Google Scholar : PubMed/NCBI
78	Fleming NH, Zhong J, da Silva IP, Vega-Saenz de Miera E, Brady B, Han SW, Hanniford D, Wang J, Shapiro RL, Hernando E, et al: Serum-based miRNAs in the prediction and detection of recurrence in melanoma patients. Cancer. 121:51–59. 2015. View Article : Google Scholar
79	Ono S, Oyama T, Lam S, Chong K, Foshag LJ and Hoon DS: A direct plasma assay of circulating microRNA-210 of hypoxia can identify early systemic metastasis recurrence in melanoma patients. Oncotarget. 6:7053–7064. 2015. View Article : Google Scholar : PubMed/NCBI
80	Stark MS, Klein K, Weide B, Haydu LE, Pflugfelder A, Tang YH, Palmer JM, Whiteman DC, Scolyer RA, Mann GJ, et al: The prognostic and predictive value of melanoma-related microRNAs using tissue and serum: A microRNA expression analysis. EBioMedicine. 2:671–680. 2015. View Article : Google Scholar : PubMed/NCBI
81	Friedman EB, Shang S, de Miera EV, Fog JU, Teilum MW, Ma MW, Berman RS, Shapiro RL, Pavlick AC, Hernando E, et al: Serum microRNAs as biomarkers for recurrence in melanoma. J Transl Med. 10:1552012. View Article : Google Scholar : PubMed/NCBI
82	Kanemaru H, Fukushima S, Yamashita J, Honda N, Oyama R, Kakimoto A, Masuguchi S, Ishihara T, Inoue Y, Jinnin M, et al: The circulating microRNA-221 level in patients with malignant melanoma as a new tumor marker. J Dermatol Sci. 61:187–193. 2011. View Article : Google Scholar : PubMed/NCBI
83	Fogli S, Polini B, Carpi S, Pardini B, Naccarati A, Dubbini N, Lanza M, Breschi MC, Romanini A and Nieri P: Identification of plasma microRNAs as new potential biomarkers with high diagnostic power in human cutaneous melanoma. Tumour Biol. 39:10104283177016462017. View Article : Google Scholar : PubMed/NCBI
84	Yokoi A, Yoshioka Y, Hirakawa A, Yamamoto Y, Ishikawa M, Ikeda S, Kato T, Niimi K, Kajiyama H, Kikkawa F, et al: A combination of circulating miRNAs for the early detection of ovarian cancer. Oncotarget. 8:89811–89823. 2017. View Article : Google Scholar : PubMed/NCBI
85	Todeschini P, Salviato E, Paracchini L, Ferracin M, Petrillo M, Zanotti L, Tognon G, Gambino A, Calura E, Caratti G, et al: Circulating miRNA landscape identifies miR-1246 as promising diagnostic biomarker in high-grade serous ovarian carcinoma: A validation across two independent cohorts. Cancer Lett. 388:320–327. 2017. View Article : Google Scholar
86	Zuberi M, Mir R, Das J, Ahmad I, Javid J, Yadav P, Masroor M, Ahmad S, Ray PC and Saxena A: Expression of serum miR-200a, miR-200b, and miR-200c as candidate biomarkers in epithelial ovarian cancer and their association with clinicopathological features. Clin Transl Oncol. 17:779–787. 2015. View Article : Google Scholar : PubMed/NCBI
87	Liang H, Jiang Z, Xie G and Lu Y: Serum microRNA-145 as a novel biomarker in human ovarian cancer. Tumour Biol. 36:5305–5313. 2015. View Article : Google Scholar : PubMed/NCBI
88	Gao YC and Wu J: MicroRNA-200c and microRNA-141 as potential diagnostic and prognostic biomarkers for ovarian cancer. Tumour Biol. 36:4843–4850. 2015. View Article : Google Scholar : PubMed/NCBI
89	Zhou J, Gong G, Tan H, Dai F, Zhu X, Chen Y, Wang J, Liu Y, Chen P, Wu X, et al: Urinary microRNA-30a-5p is a potential biomarker for ovarian serous adenocarcinoma. Oncol Rep. 33:2915–2923. 2015. View Article : Google Scholar : PubMed/NCBI
90	Shah JS, Gard GB, Yang J, Maidens J, Valmadre S, Soon PS and Marsh DJ: Combining serum microRNA and CA-125 as prognostic indicators of preoperative surgical outcome in women with high-grade serous ovarian cancer. Gynecol Oncol. 148:181–188. 2018. View Article : Google Scholar
91	Gui J, Tian Y, Wen X, Zhang W, Zhang P, Gao J, Run W, Tian L, Jia X and Gao Y: Serum microRNA characterization identifies miR-885-5p as a potential marker for detecting liver pathologies. Clin Sci (Lond). 120:183–193. 2011. View Article : Google Scholar
92	Yamamoto Y, Kosaka N, Tanaka M, Koizumi F, Kanai Y, Mizutani T, Murakami Y, Kuroda M, Miyajima A, Kato T, et al: MicroRNA-500 as a potential diagnostic marker for hepatocellular carcinoma. Biomarkers. 14:529–538. 2009. View Article : Google Scholar : PubMed/NCBI
93	Xu J, Wu C, Che X, Wang L, Yu D, Zhang T, Huang L, Li H, Tan W, Wang C, et al: Circulating microRNAs, miR-21, miR-122, and miR-223, in patients with hepatocellular carcinoma or chronic hepatitis. Mol Carcinog. 50:136–142. 2011. View Article : Google Scholar : PubMed/NCBI
94	Zhou J, Yu L, Gao X, Hu J, Wang J, Dai Z, Wang JF, Zhang Z, Lu S, Huang X, et al: Plasma microRNA panel to diagnose hepatitis B virus-related hepatocellular carcinoma. J Clin Oncol. 29:4781–4788. 2011. View Article : Google Scholar : PubMed/NCBI
95	Li LM, Hu ZB, Zhou ZX, Chen X, Liu FY, Zhang JF, Shen HB, Zhang CY and Zen K: Serum microRNA profiles serve as novel biomarkers for HBV infection and diagnosis of HBV-positive hepatocarcinoma. Cancer Res. 70:9798–9807. 2010. View Article : Google Scholar : PubMed/NCBI
96	Moshiri F, Salvi A, Gramantieri L, Sangiovanni A, Guerriero P, De Petro G, Bassi C, Lupini L, Sattari A, Cheung D, et al: Circulating miR-106b-3p, miR-101-3p and miR-1246 as diagnostic biomarkers of hepatocellular carcinoma. Oncotarget. 9:15350–15364. 2018. View Article : Google Scholar : PubMed/NCBI
97	Kachakova D, Mitkova A, Popov E, Popov I, Vlahova A, Dikov T, Christova S, Mitev V, Slavov C and Kaneva R: Combinations of serum prostate-specific antigen and plasma expression levels of let-7c, miR-30c, miR-141, and miR-375 as potential better diagnostic biomarkers for prostate cancer. DNA Cell Biol. 34:189–200. 2015. View Article : Google Scholar :
98	Bryant RJ, Pawlowski T, Catto JW, Marsden G, Vessella RL, Rhees B, Kuslich C, Visakorpi T and Hamdy FC: Changes in circulating microRNA levels associated with prostate cancer. Br J Cancer. 106:768–774. 2012. View Article : Google Scholar : PubMed/NCBI
99	Lin HM, Castillo L, Mahon KL, Chiam K, Lee BY, Nguyen Q, Boyer MJ, Stockler MR, Pavlakis N, Marx G, et al: Circulating microRNAs are associated with docetaxel chemotherapy outcome in castration-resistant prostate cancer. Br J Cancer. 110:2462–2471. 2014. View Article : Google Scholar : PubMed/NCBI
100	Kotb S, Mosharafa A, Essawi M, Hassan H, Meshref A and Morsy A: Circulating miRNAs 21 and 221 as biomarkers for early diagnosis of prostate cancer. Tumour Biol. 35:12613–12617. 2014. View Article : Google Scholar : PubMed/NCBI
101	Srivastava A, Goldberger H, Dimtchev A, Ramalinga M, Chijioke J, Marian C, Oermann EK, Uhm S, Kim JS, Chen LN, et al: MicroRNA profiling in prostate cancer-the diagnostic potential of urinary miR-205 and miR-214. PLoS One. 8:e769942013. View Article : Google Scholar
102	Singh PK, Preus L, Hu Q, Yan L, Long MD, Morrison CD, Nesline M, Johnson CS, Koochekpour S, Kohli M, et al: Serum microRNA expression patterns that predict early treatment failure in prostate cancer patients. Oncotarget. 5:824–840. 2014. View Article : Google Scholar : PubMed/NCBI
103	Farran B, Dyson G, Craig D, Dombkowski A, Beebe-Dimmer JL, Powell IJ, Podgorski I, Heilbrun L, Bolton S and Bock CH: A study of circulating microRNAs identifies a new potential biomarker panel to distinguish aggressive prostate cancer. Carcinogenesis. 39:556–561. 2018. View Article : Google Scholar : PubMed/NCBI
104	Cochetti G, Poli G, Guelfi G, Boni A, Egidi MG and Mearini E: Different levels of serum microRNAs in prostate cancer and benign prostatic hyperplasia: Evaluation of potential diagnostic and prognostic role. Onco Targets Ther. 9:7545–7553. 2016. View Article : Google Scholar : PubMed/NCBI
105	Brychta N, Krahn T and von Ahsen O: Detection of KRAS mutations in circulating tumor DNA by digital PCR in early stages of pancreatic cancer. Clin Chem. 62:1482–1491. 2016. View Article : Google Scholar : PubMed/NCBI
106	Kinugasa H, Nouso K, Miyahara K, Morimoto Y, Dohi C, Tsutsumi K, Kato H, Matsubara T, Okada H and Yamamoto K: Detection of K-ras gene mutation by liquid biopsy in patients with pancreatic cancer. Cancer. 121:2271–2280. 2015. View Article : Google Scholar : PubMed/NCBI
107	Couraud S, Zalcman G, Milleron B, Morin F and Souquet PJ: Lung cancer in never smokers-a review. Eur J Cancer. 48:1299–1311. 2012. View Article : Google Scholar : PubMed/NCBI
108	Perez-Carbonell L, Sinicrope FA, Alberts SR, Oberg AL, Balaguer F, Castells A, Boland CR and Goel A: miR-320e is a novel prognostic biomarker in colorectal cancer. Br J Cancer. 113:83–90. 2015. View Article : Google Scholar : PubMed/NCBI
109	Case M, Matheson E, Minto L, Hassan R, Harrison CJ, Bown N, Bailey S, Vormoor J, Hall AG and Irving JA: Mutation of genes affecting the RAS pathway is common in childhood acute lymphoblastic leukemia. Cancer Res. 68:6803–6809. 2008. View Article : Google Scholar : PubMed/NCBI
110	Lin CC, Huang WL, Wei F, Su WC and Wong DT: Emerging platforms using liquid biopsy to detect EGFR mutations in lung cancer. Expert Rev Mol Diagn. 15:1427–1440. 2015. View Article : Google Scholar : PubMed/NCBI
111	Warton K, Mahon KL and Samimi G: Methylated circulating tumor DNA in blood: Power in cancer prognosis and response. Endocr Relat Cancer. 23:R157–171. 2016. View Article : Google Scholar : PubMed/NCBI
112	Mitchell SM, Ho T, Brown GS, Baker RT, Thomas ML, McEvoy A, Xu ZZ, Ross JP, Lockett TJ, Young GP, et al: Evaluation of methylation biomarkers for detection of crculating tumor DNA and application to colorectal cancer. Genes (Basel). 7:E1252016. View Article : Google Scholar
113	Ghelani HS, Rachchh MA and Gokani RH: MicroRNAs as newer therapeutic targets: A big hope from a tiny player. J Pharmacol Pharmacother. 3:217–227. 2012. View Article : Google Scholar : PubMed/NCBI
114	Krol J, Loedige I and Filipowicz W: The widespread regulation of microRNA biogenesis, function and decay. Nat Rev Genet. 11:597–610. 2010. View Article : Google Scholar : PubMed/NCBI
115	Sun K and Lai EC: Adult-specific functions of animal microRNAs. Nat Rev Genet. 14:535–548. 2013. View Article : Google Scholar : PubMed/NCBI
116	Chekulaeva M and Filipowicz W: Mechanisms of miRNAmediated post-transcriptional regulation in animal cells. Curr Opin Cell Biol. 21:452–460. 2009. View Article : Google Scholar : PubMed/NCBI
117	Guo H, Ingolia NT, Weissman JS and Bartel DP: Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature. 466:835–840. 2010. View Article : Google Scholar : PubMed/NCBI
118	Cammaerts S, Strazisar M, De Rijk P and Del Favero J: Genetic variants in microRNA genes: Impact on microRNA expression, function, and disease. Front Genet. 6:1862015. View Article : Google Scholar : PubMed/NCBI
119	Friedländer MR, Lizano E, Houben AJS, Bezdan D, Báñez-Coronel M and Kudla G: Evidence for the biogenesis of more than 1,000 novel human microRNAs. Genome Biol. 15:R572014. View Article : Google Scholar : PubMed/NCBI
120	Cheng WC, Chung IF, Tsai CF, Huang TS, Chen CY and Wang SC: YM500v2: A small RNA sequencing (smRNA-seq) database for human cancer miRNome research. Nucleic Acids Res. 43:D862–D867. 2015. View Article : Google Scholar :
121	Londin E, Loher P, Telonis AG, Quann K, Clark P and Jing Y: Analysis of 13 cell types reveals evidence for the expression of numerous novel primate-and tissue-specific microRNAs. Proc Natl Acad Sci USA. 112:E1106–E1115. 2015. View Article : Google Scholar
122	Griffiths-Jones S, Grocock RJ, van Dongen S, Bateman A and Enright AJ: miRBase: MicroRNA sequences, targets and gene nomenclature. Nucleic Acids Res. 34:D140–D144. 2006. View Article : Google Scholar :
123	Gambari R, Fabbri E, Borgatti M, Lampronti I, Finotti A, Brognara E, Bianchi N, Manicardi A, Marchelli R and Corradini R: Targeting microRNAs involved in human diseases: A novel approach for modification of gene expression and drug development. Biochem Pharmacol. 82:1416–1429. 2011. View Article : Google Scholar : PubMed/NCBI
124	Piva R, Spandidos DA and Gambari R: From microRNA functions to microRNA therapeutics: Novel targets and novel drugs in breast cancer research and treatment. Int J Oncol. 43:985–994. 2013. View Article : Google Scholar : PubMed/NCBI
125	Gambari R, Brognara E, Spandidos DA and Fabbri E: Targeting oncomiRNAs and mimicking tumor suppressor miRNAs: Νew trends in the development of miRNA therapeutic strategies in oncology. Int J Oncol. 49:5–32. 2016. View Article : Google Scholar : PubMed/NCBI
126	Bertucci A, Prasetyanto EA, Septiadi D, Manicardi A, Brognara E, Gambari R, Corradini R and De Cola L: Combined Delivery of temozolomide and anti-miR221 PNA using mesoporous silica nanoparticles induces apoptosis in resistant glioma cells. Small. 11:5687–5695. 2015. View Article : Google Scholar : PubMed/NCBI
127	Gheinani AH, Vögeli M, Baumgartner U, Vassella E, Draege RA, Burkhard FC and Monastyrskaya K: Improved isolation strategies to increase the yield and purity of human urinary exosomes for biomarker discovery. Sci Rep. 8:39452018. View Article : Google Scholar : PubMed/NCBI
128	da Silveira JC, Andrade GM, Del Collado M, Sampaio RV, Sangalli JR, Silva LA, Pinaffi FVL, Jardim IB, Cesar MC, Nogueira MFG, et al: Supplementation with small-extracellular vesicles from ovarian follicular fluid during in vitro production modulates bovine embryo development. PLoS One. 12:e01794512017. View Article : Google Scholar : PubMed/NCBI
129	Halvorsen AR, Helland Å, Gromov P, Wielenga VT, Talman MM, Brunner N, Sandhu V, Børresen-Dale AL, Gromova I and Haakensen VD: Profiling of microRNAs in tumor interstitial fluid of breast tumors - a novel resource to identify biomarkers for prognostic classification and detection of cancer. Mol Oncol. 11:220–234. 2017. View Article : Google Scholar : PubMed/NCBI
130	Valentino A, Reclusa P, Sirera R, Giallombardo M, Camps C, Pauwels P, Crispi S and Rolfo C: Exosomal microRNAs in liquid biopsies: Future biomarkers for prostate cancer. Clin Transl Oncol. 19:651–657. 2017. View Article : Google Scholar : PubMed/NCBI
131	Wecker T, Hoffmeier K, Plötner A, Grüning BA, Horres R, Backofen R, Reinhard T and Schlunck G: MicroRNA profiling in aqueous humor of individual human eyes by next-generation sequencing. Invest Ophthalmol Vis Sci. 57:1706–1713. 2016. View Article : Google Scholar : PubMed/NCBI
132	Nishida-Aoki N and Ochiya T: Interactions between cancer cells and normal cells via miRNAs in extracellular vesicles. Cell Mol Life Sci. 72:1849–1861. 2015. View Article : Google Scholar : PubMed/NCBI
133	Schetter AJ, Okayama H and Harris CC: The role of microRNAs in colorectal cancer. Cancer J. 18:244–252. 2012. View Article : Google Scholar : PubMed/NCBI
134	Markowitz SD and Bertagnolli MM: Molecular origins of cancer: Molecular basis of colorectal cancer. N Engl J Med. 361:2449–2460. 2009. View Article : Google Scholar : PubMed/NCBI
135	Bellassai N and Spoto G: Biosensors for liquid biopsy: Circulating nucleic acids to diagnose and treat cancer. Anal Bioanal Chem. 408:7255–7264. 2016. View Article : Google Scholar : PubMed/NCBI
136	Das J, Ivanov I, Montermini L, Rak J, Sargent EH and Kelley SO: An electrochemical clamp assay for direct, rapid analysis of circulating nucleic acids in serum. Nat Chem. 7:569–575. 2015. View Article : Google Scholar : PubMed/NCBI
137	Pinheiro LB, Coleman VA, Hindson CM, Herrmann J, Hindson BJ, Bhat S and Emslie KR: Evaluation of a Droplet Digital polymerase chain reaction format for DNA copy number quantification. Anal Chem. 84:1003–1011. 2012. View Article : Google Scholar :
138	Podlesniy P and Trullas R: Biomarkers in cerebrospinal fluid: Analysis of cell-free circulating mitochondrial DNA by digital PCR. Methods Mol Biol. 1768:111–126. 2018. View Article : Google Scholar : PubMed/NCBI
139	Macagno N, Fina F, Penel N, Bouvier C, Nanni I, Duffaud F, Rouah R, Lacarelle B, Ouafik L, Bonvalot S, et al: Proof of concept: Prognostic value of the plasmatic concentration of circulating cell free DNA in desmoid tumors using ddPCR. Oncotarget. 9:18296–18308. 2018. View Article : Google Scholar : PubMed/NCBI
140	DiNardo CD, Routbort MJ, Bannon SA, Benton CB, Takahashi K, Kornblau SM, Luthra R, Kanagal-Shamanna R, Medeiros LJ, Garcia-Manero G, et al: Improving the detection of patients with inherited predispositions to hematologic malignancies using next-generation sequencing-based leukemia prognostication panels. Cancer. 124:2704–2713. 2018. View Article : Google Scholar : PubMed/NCBI
141	Müllauer L: Next generation sequencing: Clinical applications in solid tumours. Memo. 10:244–247. 2017. View Article : Google Scholar
142	Giuffrida MC and Spoto G: Integration of isothermal amplification methods in microfluidic devices: Recent advances. Biosens Bioelectron. 90:174–186. 2017. View Article : Google Scholar
143	Giuffrida MC, Zanoli LM, D'Agata R, Finotti A, Gambari R and Spoto G: Isothermal circular-strand-displacement polymerization of DNA and microRNA in digital microfluidic devices. Anal Bioanal Chem. 407:1533–1543. 2015. View Article : Google Scholar : PubMed/NCBI
144	Garcia-Olmo DC, Gutierrez-Gonzalez L, Ruiz-Piqueras R, Picazo MG and Garcia-Olmo D: Detection of circulating tumor cells and of tumor DNA in plasma during tumor progression in rats. Cancer Lett. 217:115–123. 2005. View Article : Google Scholar
145	Rago C, Huso DL, Diehl F, Karim B, Liu G, Papadopoulos N, Samuels Y, Velculescu VE, Vogelstein B, Kinzler KW, et al: Serial assessment of human tumor burdens in mice by the analysis of circulating DNA. Cancer Res. 67:9364–9370. 2007. View Article : Google Scholar : PubMed/NCBI
146	Thierry AR, Mouliere F, Gongora C, Ollier J, Robert B, Ychou M, Del Rio M and Molina F: Origin and quantification of circulating DNA in mice with human colorectal cancer xenografts. Nucleic Acids Res. 38:6159–6175. 2010. View Article : Google Scholar : PubMed/NCBI
147	Mitchell PS, Parkin RK, Kroh EM, Fritz BR, Wyman SK, Pogosova-Agadjanyan EL, Peterson A, Noteboom J, O'Briant KC, Allen A, et al: Circulating microRNAs as stable blood-based markers for cancer detection. Proc Natl Acad Sci US A. 105:10513–10518. 2008. View Article : Google Scholar
148	Selth LA, Townley S, Gillis JL, Ochnik AM, Murti K, Macfarlane RJ, Chi KN, Marshall VR, Tilley WD and Butler LM: Discovery of circulating microRNAs associated with human prostate cancer using a mouse model of disease. Int J Cancer. 131:652–661. 2012. View Article : Google Scholar
149	Waters PS, McDermott AM, Wall D, Heneghan HM, Miller N, Newell J, Kerin MJ and Dwyer RM: Relationship between circulating and tissue microRNAs in a murine model of breast cancer. PLoS One. 7:e504592012. View Article : Google Scholar : PubMed/NCBI
150	Greystoke A, Ayub M, Rothwell DG, Morris D, Burt D, Hodgkinson CL, Morrow CJ, Smith N, Aung K, Valle J, et al: Development of a circulating miRNA assay to monitor tumor burden: From mouse to man. Mol Oncol. 10:282–291. 2016. View Article : Google Scholar :
151	Gasparello J, Allegretti M, Tremante E, Fabbri E, Amoreo CA, Romania P, Melucci E, Messana K, Borgatti M, Giacomini P, et al: Liquid biopsy in mice bearing colorectal carcinoma xenografts: Gateways regulating the levels of circulating tumor DNA (ctDNA) and miRNA (ctmiRNA). J Exp Clin Cancer Res. 37:1242018. View Article : Google Scholar : PubMed/NCBI
152	Hofman P: Liquid biopsy for early detection of lung cancer. Curr Opin Oncol. 29:73–78. 2017. View Article : Google Scholar
153	Pérez-Ramírez C, Cañadas-Garre M, Robles AI, Molina MÁ, Faus-Dáder MJ and Calleja-Hernández MÁ: Liquid biopsy in early stage lung cancer. Transl Lung Cancer Res. 5:517–524. 2016. View Article : Google Scholar : PubMed/NCBI
154	Bedin C, Enzo MV, Del Bianco P, Pucciarelli S, Nitti D and Agostini M: Diagnostic and prognostic role of cell-free DNA testing for colorectal cancer patients. Int J Cancer. 140:1888–1898. 2017. View Article : Google Scholar
155	Allenson K, Castillo J, San Lucas FA, Scelo G, Kim DU, Bernard V, Davis G, Kumar T, Katz M, Overman MJ, et al: High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients. Ann Oncol. 28:741–747. 2017.PubMed/NCBI
156	Shimomura A, Shiino S, Kawauchi J, Takizawa S, Sakamoto H, Matsuzaki J, Ono M, Takeshita F, Niida S, Shimizu C, et al: Novel combination of serum microRNA for detecting breast cancer in the early stage. Cancer Sci. 107:326–334. 2016. View Article : Google Scholar : PubMed/NCBI
157	Schröck A, Leisse A, de Vos L, Gevensleben H, Dröge F, Franzen A, Wachendörfer M, Schröck F, Ellinger J, Teschke M, et al: Free-circulating methylated DNA in blood for diagnosis, staging, prognosis, and monitoring of head and neck squamous cell carcinoma patients: An observational prospective cohort study. Clin Chem. 63:1288–1296. 2017. View Article : Google Scholar : PubMed/NCBI
158	Quandt D, Dieter Zucht H, Amann A, Wulf-Goldenberg A, Borrebaeck C, Cannarile M, Lambrechts D, Oberacher H, Garrett J, Nayak T, et al: Implementing liquid biopsies into clinical decision making for cancer immunotherapy. Oncotarget. 8:48507–48520. 2018.
159	Goodall J, Mateo J, Yuan W, Mossop H, Porta N, Miranda S, Perez-Lopez R, Dolling D, Robinson DR, Sandhu S, et al: Circulating cell-free DNA to quide prostate cancer treatment with PARP inhibition. Cancer Discov. 7:1006–1017. 2017. View Article : Google Scholar : PubMed/NCBI
160	He J, Tan W and Ma J: Circulating tumor cells and DNA for real-time EGFR detection and monitoring of non-small-cell lung cancer. Future Oncol. 13:787–797. 2017. View Article : Google Scholar : PubMed/NCBI
161	Craw P and Balachandran W: Isothermal nucleic acid amplification technologies for point-of-care diagnostics: A critical review. Lab Chip. 12:2469–2486. 2012. View Article : Google Scholar : PubMed/NCBI
162	Kim J and Easley CJ: Isothermal DNA amplification in bioanalysis: strategies and applications. Bioanalysis. 3:227–239. 2011. View Article : Google Scholar : PubMed/NCBI
163	Guo Q, Yang X, Wang K, Tan W, Li W, Tang H and Li H: Sensitive fluorescence detection of nucleic acids based on isothermal circular strand-displacement polymerization reaction. Nucleic Acids Res. 37:e202009. View Article : Google Scholar : PubMed/NCBI
164	D'Agata R, Breveglieri G, Zanoli LM, Borgatti M, Spoto G and Gambari R: Direct detection of point mutations in nonamplified human genomic DNA. Anal Chem. 83:8711–8717. 2011. View Article : Google Scholar : PubMed/NCBI
165	Albitar M: Hematopoietic cell phenotyping using circulating cell-free markers. US Patent 9,255,926 B2. Filed August 17, 2006, issued February 9, 2016.
166	Thierry A and Molina F: Analytical methods for cell free nucleic acids and applications. European Patent 2,426,217A1. Filed September 9, 2010; issued March 7, 2012.
167	Platica O: Method of mutation detection in blood cell-free DNA using primer extension (PE) and PCR. US Patent 9,062,350 B2. Filed March 11, 2012; issued June 23, 2015.
168	Hoon DSB and Taback B: DNA markers for management of cancer. US Patent 7,718,364 B2. Filed March 25, 2004; issued May 18, 2010.
169	Cortese Rand Petronis A: Method for analysis of DNA methylation profiles of cell-free circulating DNA in bodily fluids. European Patent 2,483,426 A4. Filed October 1, 2010; issued April 4, 2013.
170	Schutz E, Beck J and Urnovitz H: Colorectal cancer associated circulating nucleic acid biomarkers. US Patent 2014/0303008A1. Filed October 19, 2012; issued October 9, 2014.
171	Murtaza M and Contente-Cuomo T: Quality assessment of circulating cell-free DNA using multiplexed droplet digital PCR. WO Patent 2016/168844A1. Filed April 17, 2015; issued October 10, 2016.
172	Hoon DSB, Umetani N and Sunami E: Use of free circulating DNA for diagnosis, prognosis, and treatment of cancer. WO Patent 2006/128192 A2. Filed May 27, 2005; issued November 30, 2006.
173	Raymond CK, Lim LP and Armour CD: Methods for quantitative genetic analysis of cell free DNA. US Patent 2016/0053301 A1. Filed August 22, 2014; issued February 25, 2016.
174	Ambros V, Lee R and Fusco AP: Isolating Circulating microRNA (miRNA). US Patent 9,896,683 B2. Filed July 29, 2015; issued February 20, 2018.
175	Taylor DD and Gercel-Taylor C: Cancer-derived microvesicle-associated microrna as a diagnostic marker. US Patent 8,216,784 B2. Filed July 25, 2008; issued July 10, 2012.
176	Taylor DD and Gercel-Taylor C: Exosome-associated microRNA as a diagnostic marker. European Patent 2,806,273 B1. Filed August 12, 2013; issued December 5, 2013.
177	Croce CM, Calin GA and Volinia S: Methods for Diagnosing Pancreatic Cancer Using MicroRNAs. US Patent 2013/0324589 A1. Filed August 12, 2013; issued December 5, 2013.
178	Ditzel H and Kodahl AR: Circulating microRNA based cancer biomarkers. European Patent 3,011,058 A1. Filed December 24, 2014; issued April 27, 2016.
179	Croce CM: MicroRNA signatures in human ovarian cancer. European Patent 3,138,926 A3. Filed September 8, 2008; issued April 5, 2017.
180	Plasma microRNAs for the detection of early colorectal cancer. European Patent 2,944,700 B1. Filed October 10, 2012; issued October 18, 2017.
181	Zhang C, Zeng K, Zhang J, Ba Y, Chen X and Li H: Serum or plasma microRNA as biomarkers for non-small cell lung cancer. US Patent 9,388,470 B2. Filed December 14, 2009; issued July 12, 2016.
182	Croce CM, Calin GA and Volinia S: Methods for diagnosing breast cancer using MicroRNAs. US Patent. 8,603,744 B2. Filed February 28, 2012; issued December 10, 2013.
183	NCT02639832, A Pilot Surveillance Study to Monitor Natural Killer Cells and Circulating Tumor Cells in Women With Previously Treated Non-metastatic Triple Negative Breast Cancer and Women With Previously Treated Non-metastatic Breast Cancer With a Confirmed BRCA Mutation, 2015
184	NCT02626039, Characterization & Comparison of Drugable Mutations in Primary and Metastatic Tumors, CTCs and cfDNA in MBCpatients, 2015
185	NCT02186236, Detection of Oncogenic Tumor Mutations in the Urine and Blood of Lung and Colorectal Cancer Patients, 2014
186	NCT02788084, Development of a Tissue-Based & Cell Free DNA Next-Generation Sequencing Workflow, 2016
187	NCT02883517, Cell-free Circulating DNA in Primary Cutaneous Lymphomas, 2016
188	NCT02887612, ctDNA for Prediction of Relapse in Gastric Cancer, 2016
189	NCT02738593, Detection Cell Free DNA in Lung Cancer Patients, 2016
190	NCT02610218, Liquid Biopsy in Monitoring the Therapeutic Efficacy of Targeted Therapy in Advanced/Metastatic Gastric Cancer, 2015
191	NCT02872779, Correlation Between Circulating Tumour Markers Early Variations and Clinical Response in First Line Treatment of Metastatic Colorectal Cancer (COCA-MACS), 2016
192	NCT02443948, Circulating Cell-free Tumor DNA in the Plasma of Patients With Gastrointestinal Stromal Tumors (GIST), 2015
193	NCT02133222, Mutational Analyses in Consecutive Measurement Before and After Chemotherapy (AMMAM), 2014
194	NCT02934984, Circulating Cell-free Tumor DNA (ctDNA) in Pancreatic Cancer, 2016
195	NCT02784639, Comparison of KRAS/BRAF Mutational Status With Conventional Techniques and Plasma Samples Analysis, 2016
196	NCT02036216, Circulating Cell-free DNA as a Predictive Biomarker for Hepatocelluar Carcinoma, 2014
197	NCT02791217, Identification of Hematological Malignancies and Therapy Predication Using microRNAs as a Diagnostic Tool, 2016
198	NCT02928627, Clinical Significance of Hepatic and Circulating microRNAs miR-221 and miR-222 in Hepatocellular Carcinoma, 2016
199	NCT02964351, microRNA Profiles Identification in Adeno Carcinoma Prostate Cancer, 2016
200	NCT01541800, Circulating microRNAs as Disease Markers in Pediatric Cancers, 2012
201	NCT02065908, Circulating MicroRNA as Biomarker of Cardiotoxicity in Breast Cancer, 2014
202	NCT02812680, The Utility of Circulating Tumour Cells and Plasma microRNA in Esophageal Adenocarcinoma, 2016
203	NCT01612871, Circulating miRNAs as Biomarkers of Hormone Sensitivity in Breast Cancer (MIRHO), 2012
204	NCT01722851. Circulating miRNAs ICORG. 10(11): V22012.
205	D'Agata R, Corradini R, Ferretti C, Zanoli L, Gatti M, Marchelli R and Spoto G: Ultrasensitive detection of non-amplified genomic DNA by nanoparticle-enhanced surface-plasmon resonance imaging. Biosens Bioelectron. 25:2095–2100. 2010. View Article : Google Scholar : PubMed/NCBI
206	D'Agata R and Spoto G: Surface plasmon resonance imaging for nucleic acid detection. Anal Bioanal Chem. 405:573–584. 2013. View Article : Google Scholar