ktd

Kocatepe Tıp Dergisi

3061-9904

Afyonkarahisar Sağlık Bilimleri Üniversitesi

10.18229/kocatepetip.1628346

Cancer Diagnosis

Kanser Tanısı

MOLECULAR DIAGNOSIS IN CLINICAL BIOCHEMISTRY: LIQUID BIOPSY

KLİNİK BİYOKİMYADA MOLEKÜLER TANI: SIVI BİYOPSİ

https://orcid.org/0000-0002-7511-2547

Ellidağ

Hamit Yaşar

ANTALYA EĞİTİM VE ARAŞTIRMA HASTANESİ

04 16 2026

27 2 276 286 01 28 2025 05 27 2025

1999

Kocatepe Tıp Dergisi

Whether unicellular or multicellular, the fundamental drive of life is to survive and persist. This principle also applies to cancer cells. Humanity's relentless battle against cancer continues, and a crucial strategy in this fight is to determine the enemy’s position and strength. Currently, tissue biopsy remains the gold standard for tumor diagnosis. However, liquid biopsy has emerged as a promising alternative, focusing on detecting tumor-derived circulating free DNA (cfDNA), circulating free RNA (cfRNA), microvesicles (exosomes, extracellular vesicles: EVs), circulating tumor cells (CTCs), and tumor-educated platelets (TEPs) in bodily fluids such as blood, saliva, and urine. In cancer diagnosis, liquid biopsy plays a crucial role in early detection, monitoring treatment response, and tracking genetic changes in tumors. Advances in next-generation sequencing (NGS), quantitative polymerase chain reaction (qPCR), and digital droplet PCR (ddPCR) have significantly improved the sensitivity and specificity of data obtained through liquid biopsy. Beyond cancer, liquid biopsy is also being utilized in the diagnosis of neurodegenerative diseases, cardiovascular diseases, infections, and genetic disorders. In cardiovascular diseases, circulating microRNA profiles provide valuable insights into disease progression. However, several challenges must be addressed before liquid biopsy can be fully integrated into clinical practice. These challenges include standardizing methodologies, enhancing the sensitivity of biomarkers, and completing clinical validation processes. Additionally, advanced bioinformatics analysis techniques are needed to interpret the data obtained from liquid biopsy and translate it into clinical applications. In the future, liquid biopsy has the potential to become a standard diagnostic tool for managing various diseases, particularly cancer. The incorporation of artificial intelligence and machine learning-based data analysis methods is expected to further refine the accuracy and reliability of liquid biopsy results. This review will discuss the fundamental principles, advantages, limitations, and clinical applications of liquid biopsy, a rapidly evolving and highly relevant diagnostic approach.

İster tek hücreli, ister çok hücreli olsun, canlılığın en temel dürtüsü varlığını sürdürmek ve hayatta kalmaktır. Bu durum kanser hücreleri içinde geçerlidir. İnsanoğlunun kanserle amansız savaşı devam etmektedir. Bu savaş sırasında düşmanın konumunu ve gücünü tespit etmek temel stratejidir. Bu amaçla günümüzde doku biyopsisi tümör teşhisi için altın standart olmaya devam etmektedir. Bununla birlikte, sıvı biyopsi, tümör hücrelerinden salgılanan dolaşımdaki serbest DNA (cell-free tumor DNA : cftDNA), RNA (circulating cell-free RNA : cfRNA), mikrovezikül (ekzosomlar, ekstrasellüler veziküller :EVs), dolaşımdaki tümör hücreleri (circulating tumor cells (CTCs) ve tümör etkileşimli trombositlerin (tumor-educated platelets (TEPs) tespiti için başta kan, tükrük, idrar gibi vücut salgılarına odaklanan yöntemlerdir. Kanser tanısında sıvı biyopsi, hastalığın erken evrede belirlenmesi, tedavi yanıtının izlenmesi ve tümör genetiğindeki değişikliklerin takip edilmesi açısından büyük önem taşır. Yeni nesil dizileme (NGS), kantitatif polimeraz zincir reaksiyonu (qPCR) ve dijital damlacık PCR (ddPCR) gibi ileri teknolojiler sayesinde sıvı biyopsiden elde edilen verilerin duyarlılığı ve özgüllüğü artırılmaktadır. Kanser dışında sıvı biyopsi, nörodejeneratif hastalıklar, kardiyovasküler hastalıklar, enfeksiyonlar ve genetik bozuklukların teşhisinde de kullanılmaktadır. Kardiyovasküler hastalıklarda ise dolaşımdaki mikroRNA profilleri, hastalığın progresyonu hakkında önemli bilgiler sunmaktadır. Bununla birlikte, sıvı biyopsinin klinik uygulamalara tam anlamıyla entegre edilebilmesi için bazı zorluklar mevcuttur. Bu zorluklar arasında yöntemlerin standardizasyonunun sağlanması, biyobelirteçlerin duyarlılığının artırılması ve klinik doğrulama süreçlerinin tamamlanması bulunmaktadır. Sıvı biyopsiden elde edilen verilerin yorumlanması ve klinik pratiğe aktarılması için ileri biyoinformatik analiz yöntemlerinin geliştirilmesi gerekmektedir. Gelecekte sıvı biyopsi, kanser başta olmak üzere birçok hastalığın yönetiminde standart bir tanı yöntemi haline gelebilir. Özellikle yapay zeka ve makine öğrenmesi tabanlı veri analiz yöntemleri ile sıvı biyopsiden elde edilen bilgilerin daha hassas şekilde değerlendirilmesi mümkün olacaktır. Bu derlemede, şu an oldukça güncel olan sıvı biyopsi yöntemlerinin temel prensipleri, avantajları, sınırlamaları ve klinik uygulama alanları ele alınacaktır.

Serbest DNA Serbest RNA Ekstraselüler vezikül Serbest tümör hücreleri.

Cell fee DNA Cell fee RNA Extracellular vesicles Circulating tumor cells.

yok

1. Crosby D, Bhatia S, Brindle KM, et al. Early detection of cancer. Science. 2022;375(6586):eaay9040.

2. Vaidyanathan R, Soon RH, Zhang P, Jiang K, Lim CT. Cancer diagnosis: from tumor to liquid biopsy and beyond. Lab Chip. 2018;19(1):11–34.

3. Li W, Liu JB, Hou LK, et al. Liquid biopsy in lung cancer: significance in diagnostics, prediction, and treatment monitoring. Mol Cancer. 2022;21(1):25.

4. Casagrande GMS, Silva M de O, Reis RM, Leal LF. Liquid Biopsy for Lung Cancer: Up-to-Date and Perspectives for Screening Programs. Int J Mol Sci. 2023;24(3):2505.

5. Pantel K, Alix-Panabières C. Circulating tumour cells in cancer patients: challenges and perspectives. Trends Mol Med. 2010;16(9):398–406.

6. Fu Y, Zhang Y, Khoo BL. Liquid biopsy technologies for hematological diseases. Med Res Rev. 2021;41(1):246–74.

7. Lone SN, Nisar S, Masoodi T, et al. Liquid biopsy: a step closer to transform diagnosis, prognosis and future of cancer treatments. Mol Cancer. 2022;21(1):79.

8. Alix-Panabières C, Pantel K. Liquid Biopsy: From Discovery to Clinical Application. Cancer Discov. 2021;11(4):858–73.

9. Galvis MM, Romero CS, Bueno TO, Teng Y. Toward a New Era for the Management of Circulating Tumor Cells. Adv Exp Med Biol. 2021;1286:125–34.

10. Mandel P, Metais P. [Nuclear Acids In Human Blood Plasma]. C R Seances Soc Biol Fil. 1948;142(3–4):241–3.

11. Ma L, Guo H, Zhao Y, et al. Liquid biopsy in cancer current: status, challenges and future prospects. Signal Transduct Target Ther. 2024;9(1):336.

12. Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL. Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomark Prev Publ Am Assoc Cancer Res Cosponsored Am Soc Prev Oncol. 1994;3(1):67–71.

13. Alix-Panabières C, Pantel K. Liquid Biopsy: From Discovery to Clinical Application. Cancer Discov. 2021;11(4):858–73.

14. Salu P, Reindl KM. Advancements in Circulating Tumor Cell Research: Bridging Biology and Clinical Applications. Cancers. 2024;16(6):1213.

15. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004;351(8):781–91.

16. Diehl F, Schmidt K, Choti MA, , et al. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14(9):985–90.

17. Smirnov DA, Zweitzig DR, Foulk BW, et al. Global gene expression profiling of circulating tumor cells. Cancer Res. 2005;65(12):4993–7.

18. Cristofanilli M, Budd GT, Ellis MJ, et al. Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med. 2004;351(8):781–91.

19. Soda N, Rehm BHA, Sonar P, et al. Advanced liquid biopsy technologies for circulating biomarker detection. J Mater Chem B. 2019;7(43):6670–704.

20. Pantel K, Alix-Panabières C. Liquid biopsy and minimal residual disease - latest advances and implications for cure. Nat Rev Clin Oncol. 2019;16(7):409–24.

21. Russo GI, Musso N, Romano A, et al. The Role of Dielectrophoresis for Cancer Diagnosis and Prognosis. Cancers. 2021;14(1):198.

22. Lozar T, Jesenko T, Kloboves Prevodnik V, et al. Preclinical and Clinical Evaluation of Magnetic-Activated Cell Separation Technology for CTC Isolation in Breast Cancer. Front Oncol. 2020;10:554554.

23. Petrik J, Verbanac D, Fabijanec M, et al. Circulating Tumor Cells in Colorectal Cancer: Detection Systems and Clinical Utility. Int J Mol Sci. 2022;23(21):13582.

24. Chen Z, Li C, Zhou Y, et al. Liquid biopsies for cancer: From bench to clinic. MedComm. 2023;4(4):e329.

25. Malapelle U, Buono M, Pisapia P, et al. Circulating tumor DNA in cancer: Predictive molecular pathology meets mathematics. Crit Rev Oncol Hematol. 2021;163:103394.

26. Stejskal P, Goodarzi H, Srovnal J, et al. Circulating tumor nucleic acids: biology, release mechanisms, and clinical relevance. Mol Cancer. 2023;22(1):15.

27. Underhill HR, Kitzman JO, Hellwig S, et al. Fragment Length of Circulating Tumor DNA. PLoS Genet. 2016;12(7):e1006162.

28. Ponti G, Manfredini M, Tomasi A. Non-blood sources of cell-free DNA for cancer molecular profiling in clinical pathology and oncology. Crit Rev Oncol Hematol. 2019;141:36–42.

29. Stewart CM, Kothari PD, Mouliere F, et al. The value of cell-free DNA for molecular pathology. J Pathol. 2018;244(5):616–27.

30. Stroun M, Anker P, Maurice P, et al. Neoplastic characteristics of the DNA found in the plasma of cancer patients. Oncology. 1989;46(5):318–22.

31. Holm M, Andersson E, Osterlund E, et al. Detection of KRAS mutations in liquid biopsies from metastatic colorectal cancer patients using droplet digital PCR, Idylla, and next generation sequencing. PloS One. 2020;15(11):e0239819.

32. Diehl F, Schmidt K, Choti MA, et al. Circulating mutant DNA to assess tumor dynamics. Nat Med. 2008;14(9):985–90.

33. Pandoh PK, Corbett RD, McDonald H, et al. A high-throughput protocol for isolating cell-free circulating tumor DNA from peripheral blood. BioTechniques. 2019;66(2):85–92.

34. Kang Q, Henry NL, Paoletti C, Jiang H, et al. Comparative analysis of circulating tumor DNA stability In K3EDTA, Streck, and CellSave blood collection tubes. Clin Biochem. 2016;49(18):1354–60.

35. Meddeb R, Pisareva E, Thierry AR. Guidelines for the Preanalytical Conditions for Analyzing Circulating Cell- Free DNA. Clin Chem. 2019;65(5):623–33.

36. Hannigan B, Ye W, Mehrotra M, et al. Liquid biopsy assay for lung carcinoma using centrifuged supernatants from fine-needle aspiration specimens. Ann Oncol Off J Eur Soc Med Oncol. 2019;30(6):963–9.

37. Olmedillas-López S, Olivera-Salazar R, García-Arranz M, García-Olmo D. Current and Emerging Applications of Droplet Digital PCR in Oncology: An Updated Review. Mol Diagn Ther. 2022;26(1):61–87.

38. Ståhlberg A, Krzyzanowski PM, Egyud M, et al. Simple multiplexed PCR-based barcoding of DNA for ultrasensitive mutation detection by next-generation sequencing. Nat Protoc. 2017;12(4):664–82.

39. El Marabti E, Younis I. The Cancer Spliceome: Reprograming of Alternative Splicing in Cancer. Front Mol Biosci. 2018;5:80.

40. Cabús L, Lagarde J, Curado J, et al. Current challenges and best practices for cell-free long RNA biomarker discovery. Biomark Res. 2022;10(1):62.

41. Tzimagiorgis G, Michailidou EZ, Kritis A, et al. Recovering circulating extracellular or cell-free RNA from bodily fluids. Cancer Epidemiol. 2011;35(6):580–9.

42. Pös O, Biró O, Szemes T, Nagy B. Circulating cell-free nucleic acids: characteristics and applications. Eur J Hum Genet EJHG. 2018;26(7):937–45.

43. Drula R, Ott LF, Berindan-Neagoe I, Pantel K, Calin GA. MicroRNAs from Liquid Biopsy Derived Extracellular Vesicles: Recent Advances in Detection and Characterization Methods. Cancers. 2020;12(8):2009.

44. Heitzer E, Haque IS, Roberts CES, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet. 2019;20(2):71–88.

45. Kolenda T, Guglas K, Baranowski D, et al. cfRNAs as biomarkers in oncology - still experimental or applied tool for personalized medicine already? Rep Pract Oncol Radiother J Gt Cancer Cent Poznan Pol Soc Radiat Oncol. 2020;25(5):783–92.

46. Glinge C, Clauss S, Boddum K, J et al. Stability of Circulating Blood-Based MicroRNAs - Pre-Analytic Methodological Considerations. PloS One. 2017;12(2):e0167969.

47. Lin Y, Leng Q, Zhan M, Jiang F. A Plasma Long Noncoding RNA Signature for Early Detection of Lung Cancer. Transl Oncol. 2018;11(5):1225–31.

48. Umu SU, Langseth H, Bucher-Johannessen C, et al. A comprehensive profile of circulating RNAs in human serum. RNA Biol. 2018;15(2):242–50.

49. Li X, Mauro M, Williams Z. Comparison of plasma extracellular RNA isolation kits reveals kit-dependent biases. BioTechniques. 2015;59(1):13–7.

50. Verwilt J, Trypsteen W, Van Paemel R, et al. When DNA gets in the way: A cautionary note for DNA contamination in extracellular RNA-seq studies. Proc Natl Acad Sci U S A. 2020;117(32):18934–6.

51. Chen K, Hu Z, Xia Z, et al. The Overlooked Fact: Fundamental Need for Spike-In Control for Virtually All Genome-Wide Analyses. Mol Cell Biol. 2015;36(5):662–7.

52. Yuan T, Huang X, Woodcock M, et al. Plasma extracellular RNA profiles in healthy and cancer patients. Sci Rep. 2016;6:19413.

53. Ward Gahlawat A, Lenhardt J, Witte T, et al. Evaluation of Storage Tubes for Combined Analysis of Circulating Nucleic Acids in Liquid Biopsies. Int J Mol Sci. 2019;20(3):704.

54. van Niel G, Carter DRF, Clayton A, et al. Challenges and directions in studying cell-cell communication by extracellular vesicles. Nat Rev Mol Cell Biol. 2022;23(5):369–82.

55. Zhou Y, Zhang Y, Gong H, Luo S, Cui Y. The Role of Exosomes and Their Applications in Cancer. Int J Mol Sci. 2021;22(22):12204.

56. Han QF, Li WJ, Hu KS, et al. Exosome biogenesis: machinery, regulation, and therapeutic implications in cancer. Mol Cancer. 2022;21(1):207.

57. Wang J, Ni J, Beretov J, Thompson J, Graham P, Li Y. Exosomal microRNAs as liquid biopsy biomarkers in prostate cancer. Crit Rev Oncol Hematol. 2020;145:102860.

58. Wang J, Yang K, Yuan W, Gao Z. Determination of Serum Exosomal H19 as a Noninvasive Biomarker for Bladder Cancer Diagnosis and Prognosis. Med Sci Monit Int Med J Exp Clin Res. 2018;24:9307–16.

59. Keerthikumar S, Chisanga D, Ariyaratne D, et al. ExoCarta: A Web-Based Compendium of Exosomal Cargo. J Mol Biol. 2016;428(4):688–92.

60. Wu X, Zhou Z, Xu S, et al. Extracellular vesicle packaged LMP1-activated fibroblasts promote tumor progression via autophagy and stroma-tumor metabolism coupling. Cancer Lett. 2020;478:93–106.

61. Kalluri R, LeBleu VS. The biology, function, and biomedical applications of exosomes. Science. 2020;367(6478):eaau6977.

62. Li P, Kaslan M, Lee SH, Yao J, Gao Z. Progress in Exosome Isolation Techniques. Theranostics. 2017;7(3):789– 804.

63. Tauro BJ, Greening DW, Mathias RA, et al. Comparison of ultracentrifugation, density gradient separation, and immunoaffinity capture methods for isolating human colon cancer cell line LIM1863-derived exosomes. Methods San Diego Calif. 2012;56(2):293–304.

64. Tian Y, Gong M, Hu Y, et al. Quality and efficiency assessment of six extracellular vesicle isolation methods by nano-flow cytometry. J Extracell Vesicles. 2020;9(1):1697028.

65. Yakubovich EI, Polischouk AG, Evtushenko VI. Principles and Problems of Exosome Isolation from Biological Fluids. Biochem Mosc Suppl Ser Membr Cell Biol. 2022;16(2):115–26.

66. Baranyai T, Herczeg K, Onódi Z, et al. Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods. PloS One. 2015;10(12):e0145686.

67. Kang D, Oh S, Ahn SM, Lee BH, Moon MH. Proteomic analysis of exosomes from human neural stem cells by flow field-flow fractionation and nanoflow liquid chromatography-tandem mass spectrometry. J Proteome Res. 2008;7(8):3475–80.

68. Pan W, Miao Q, Yin W, et al. The role and clinical applications of exosomes in cancer drug resistance. Cancer Drug Resist. 2024;7:43.

69. Yang Z, Atiyas Y, Shen H, Siedlik MJ, Wu J, Beard K, et al. Ultrasensitive Single Extracellular Vesicle Detection Using High Throughput Droplet Digital Enzyme-Linked Immunosorbent Assay. Nano Lett. 2022;22(11):4315–24.

70. Thon JN, Italiano JE. Platelet formation. Semin Hematol. 2010;47(3):220–6.

71. Scurfield G, Radley JM. Aspects of platelet formation and release. Am J Hematol. 1981;10(3):285–96.

72. Lefrançais E, Ortiz-Muñoz G, Caudrillier A, et al. The lung is a site of platelet biogenesis and a reservoir for haematopoietic progenitors. Nature. 2017;544(7648):105–9.

73. Schubert S, Weyrich AS, Rowley JW. A tour through the transcriptional landscape of platelets. Blood. 2014;124(4):493–502.

74. Nagalla S, Shaw C, Kong X, et al. Platelet microRNA-mRNA coexpression profiles correlate with platelet reactivity. Blood. 2011;117(19):5189–97.

75. Alhasan AA, Izuogu OG, Al-Balool HH, et al. Circular RNA enrichment in platelets is a signature of transcriptome degradation. Blood. 2016;127(9):e1–11.

76. Schlesinger M. Role of platelets and platelet receptors in cancer metastasis. J Hematol OncolJ Hematol Oncol. 2018;11(1):125.

77. Plantureux L, Mège D, Crescence L, et al. The Interaction of Platelets with Colorectal Cancer Cells Inhibits Tumor Growth but Promotes Metastasis. Cancer Res. 2020;80(2):291–303.

78. Eslami-S Z, Cortés-Hernández LE, Glogovitis I, et al. In vitro cross-talk between metastasis-competent circulating tumor cells and platelets in colon cancer: a malicious association during the harsh journey in the blood. Front Cell Dev Biol. 2023;11:1209846.

79. Wang X, Zhao S, Wang Z, Gao T. Platelets involved tumor cell EMT during circulation: communications and interventions. Cell Commun Signal CCS. 2022;20(1):82.

80. Best MG, Sol N, Kooi I, et al. RNA-Seq of Tumor-Educated Platelets Enables Blood-Based Pan-Cancer, Multiclass, and Molecular Pathway Cancer Diagnostics. Cancer Cell. 2015;28(5):666–76.