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Liquid biopsy
- 1. Dr Kusuma KN MD,DNB
- 2. CONTENTS Introduction Definition What comes under liquid biopsy Indication Advantages Limitations Summary References
- 3. INTRODUCTION With highmorbidity and mortality, cancer has become a big health threat problem around the world . Two main techniques of tumor diagnosis Medical imaging ultrasonic testing, X-ray , CT, MRI, PETCT and endoscope Pathology tissue biopsies, serological indicators (eg. CEA and PSA) test and molecular pathology test (eg. FISH, RT-PCR and NGS)
- 4. LIMITATIONS Cancer isa heterogeneous disease Molecular properties differ within a tumour Primary tumour biopsy may not reflect current disease condition Therapy causes changes in tumour cells Biopsy invasive test High cost for procedure May not be feasible to do in all patients Impractical for periodic monitoring for progression/ recurrence
- 5. New diagnostic testwhich address these limitation simple, non-invasive Allows early disease detection Allows real time evaluation of metastasis actual treatment response Assessment of tumour heterogeneity and monitoring of tumour dynamics Can be cheaper and faster than classical biopsy testing
- 6. LIQUID BIOPSY Non-invasivetests that detect tumor biomarkers that are shed into the body fluid from the tumor. Samples included Blood Saliva Urine CSF
- 8. WHAT COMES UNDERLIQUID BIOPSY Circulating tumour cells Circulating tumour DNA Exosomes New addition : Tumour educated platelets
- 9. CIRCULATING TUMOR CELLS(CTCS) • Carries phenotypic and genotypic information of tumor 1869 Ashworth : first found and named CTC 2002 and 2003 Thiery and Steeg : found the formation mechanism of CTC. 2007 CTC has been recommended as new tumor biomarker by American Society of Clinical Oncology (ASCO)
- 10. SAMPLE COLLECTION Cellsave preservative tube made of negatively charged materials contain stabilizing reagents preventing blood cell breakdown for a period of about 5 days
- 11. TECHNOLOGIES TO DETECTAND CAPTURE CTCS: Biological property / EpCAM-affinity based: • CellSearch® system • AdnaTest Breast CancerDetect • CTC-Chip • MACS® (magnetic activated cell sorting system) • Mag Sweeper Physical properties- based: • ISET (isolation by size of epithelial tumor cells) • ScreenCell® • ApoStream™ • Density gradient centrifugation Other methods: • FAST (fiber-optic array scanning technology) • EPISPOT (epithelial immunospot) • FACS (flow cytometry) • PRO Onc Assay
- 15. ADVANTAGES CTC Count:significantly correlate with prognosis Allow both phenotypic and genotypic analysis Potential relation with the tumor progression and metastasis Various technologies for CTC enrichment or isolation High specificity Allow culturing and analysis in vitro
- 16. DISADVANTAGES Low concentration 1-10cells per 10 ml peripheral blood fragility of cells False negative and false positive results Cannot decrease the diagnosis bias from tumor heterogeneity
- 17. Complex heterogeneity morphologyof CTCs derived from different tumor tissues through EMT is significantly different. even within only one cancer, the morphology and amount of CTCs derived from different molecular subtypes of solid tumors or distant sites are distinct eg. prostate primary and bone metastatic cancers the percentage of patients whose CTCs can be detected is also different between cancers colorectal, ovarian and breast cancer : 50-70% non-small cell lung cancer is only 30%.
- 18. inter- and intra-patientheterogeneity Misses some small subclones of tumor cells like tissue biopsy. lead to diagnosis bias
- 19. CIRCULATING DNA’S Releasesby both healthy and tumour cells Released by solid tumor Have cancer- associated genetic mutations: Half life is less than 2 hrs Percentage of ctDNA is very low (0.01%-1%) Cell free DNA: cfDNA Circulating tumor DNA: ctDNA
- 21. High sensitivity Allow analysis of DNA sequence and methylation, including PCR and NGS Can decrease the diagnosis bias from tumor heterogeneity Timely and dynamically monitor tumor progression ctDNA is unstable requires fast processing low specificity because of cfDNA from normal tissue False positive and false negative results Advantages limitations
- 23. EXOSOMES Small (50–100nmin diameter) lipid bilayer membrane vesicles of endocytic origin. play a central role in cell-to-cell communication
- 25. Role intumour Initiation Progression Metastasis drug-resistance Based on these results, researchers have developed some exosomes targeted drugs and tried to use exosomes for drug delivery
- 26. Enrichment methods: similar to those of CTC. Analysis regular biological technologies, such as electron microscope, RT-PCR, western blot, FISH, flow cytometry and NGS
- 27. Allow analysisof DNA, RNA and proteins from solid tumor Potential relation with the tumor drug- resistance and metastasis Lacking effective enrichment method Advantages Limitation
- 28. New addition inliquid biopsy
- 29. TUMOUR EDUCATED PLATELATES Besides tumor cells and their products, normal cells present in the tumor microenvironment are also released into the blood stream. harbor important information about the tumor platelets have been studied extensively and gave promising results
- 30. interaction betweenblood platelets and tumor cells not only affects the expression of relevant genes in tumor cells, but also alters the RNA profile of blood platelets mRNA sequencing of TEPs can be done
- 32. INDICATIONS To monitor residual disease Treatment response disease progression and tumor evolution (i.e. development of tumor resistance). Helps to explore other options of treatment when the patient is resistant to current therapies. Provide an alternative method for biopsy when tissue is difficult to obtain or not available primary site of metastatic disease is unknown. Limited quantity of tissue and traditional molecular genotyping is requested. • Provide prognostic information.
- 33. ADVANTAGES Noninvasively takerepeated tumor samples Fewer side effects Rapid testing speed Decreasing the diagnosis bias from tumor heterogeneity Dynamically reflect the tumor progression
- 34. LIMITATIONS lack ofstandardization of techniques. sensitivity and specificity and current detection technologies need to be improved sample size in most of current researches is small
- 35. low concentrationof CTC, ctDNA and exosomes difficult to detect gene mutations, amplifications and fusions in parallel NGS can address these limitation However, cannot be used in general High cost requirement for high-quality DNA (not characteristic of cfDNA) extensive data analysis requiring a dedicated bioinformatician
- 36.
- 38.
- 40. DNA LiquidBiopsy Metastasis Test EGFR KRAS BRAF genes DNA Liquid Biopsy Lung Panel Metastasis Test BRAF, EGFR, ERBB2, KRAS, and PIK3CA genes DNA Liquid Biopsy Colon Cancer Metastasis Test KRAS, NRAS, EGFR, and PIK3CA genes
Editor's Notes
- #14 DAPI (4′,6-diamidino-2-phenylindole) is a blue-fluorescent DNA stain that exhibits ~20-fold enhancement of fluorescence upon binding to AT regions of dsDNA. It is excited by the violet (405 nm) laser line and is commonly used as a nuclear counterstain in fluorescence microscopy, flow cytometry, and chromosome staining.
- #20 average concentration in cancer patients’ blood is 180ng/mL