Liquid biopsy: Tumor diagnosis and treatment monitoring in a blood test
Radmila Jankovic, PhD Institute for Oncology and Radiology of Serbia
Term “Liquid biopsy” stands for:
Circulating tumor cells (CTCs)
Cell-free DNA (cfDNA) Plasma Serum Other liquid material
Exosomes
Circulating tumor cells (CTCs)
etiology disseminated cancer cells cancer stem cells
rare cells in a dormant, nonproliferative state
unaffected by chemotherapy unrecognized by the host immune system difficult to isolate advanced cancer has less then 10 CTCs per 7.5ml of blood
Origin of CTCs
(Paterlini-Brechot et al. Cancer Letters 2007. 253:180.)
Main approaches for CTC detection
Immunomagnetic Capture composite
CD45
control
cytokeratin
AntiEpCAM Ferrofluid
Anti CD45-APC
EpCAM Nucleus DAPI
Nucleus DAPI
CD45
CK AntiCK-PE
Circulating Tumor Cell
CellSearch™ System
DAPI
Leukocyte
Individual Cell Sorting with DEPArray™ Recovery
Parking
1. Inject, trap and image all cells 2. Move all cells of interest into Parking chamber 3. Move separately to Recovery chamber and flush
Main chamber
Features Multiparametric image-based sorting Single cell resolution Small cell loads Gentle on cells No a priori thresholds choose best cells Sorting based on slow kinetics (10-100s minutes) possible
p. 7
In vivo isolation of circulating tumor cells from peripheral blood of cancer patients
Saucedo-Zeni N et al, Int J Oncol. 2012 Oct; 41(4): 1241–1250.
Microtube surface is coated with both E-selectin and antiepithelial antibodies (EpCAM or PSMA)
Halloysite Nanotube Protein G
E-Selectin-Fc Chimera Anti-EpCAM/PSMA mAb
Clinical relevance of CTCs in breast cancer Applications Prognosis
Monitor
Metastasis Drug Activity Cell culture Xenograft Model
Beyond the enumeration: Is the CTC count relevant for development & progression of metastases?
The independent prognostic effect of CTC count on progression-free survival and overall survival. CTC count also improves the prognostication of metastatic breast cancer when added to full clinicopathological predictive models, whereas serum tumour markers do not.
Beyond the enumeration: Is the CTC count relevant for therapy efficacy monitoring?
Study confirms the prognostic significance of CTCs in patients with MBC receiving first-line chemotherapy. For patients with persistently increased CTCs after 21 days of first-line chemotherapy, early switching to an alternate cytotoxic therapy was not effective in prolonging OS.
Negative study?
Beyond the enumeration: Molecular characterization of CTCs Nat Genet 2013 Dec;45(12):1415-6. doi: 10.1038/ng.2831. The search for ESR1 mutations in breast cancer. Oesterreich S, Davidson NE
Term “Liquid biopsy” stands for:
Circulating tumor cells (CTC)
Cell-free DNA (cfDNA) Plasma Serum Other liquid material
Exosomes
Origin of Circulating Tumour DNA (ctDNA) What is ctDNA?
ctDNA is tumour DNA that has been shed into the bloodstream ctDNA can be present in 0.01% - >90% of the total Cell Free DNA (cfDNA)
The amount of ctDNA is related to the tumour burden and varies between patients with different clinical presentations
Diaz and Bardelli, 2014 Journal of Clincial Oncology 32
Clinical relevance of ctDNA
Applications:
Tumor genotyping: Tissue versus liquid biopsy Monitoring tumor burden, therapeutic response and treatment Minimal residual disease monitoring and early detection
Approaches: PCR-based approaches Nested real-time PCR PARE ARMS-Scorpion PCR PAP-A amplification BEAMing Microfluidic digital PCR Droplet-based digital PCR NGS-based approaches Targeted deep sequencing Tam-Seq Safe-Seq CAPP-Seq Ion-AmpliSeqTM Whole exome sequencing Whole genome Sequencing 19
FFPE vs. ctDNA ctDNA Samples
FFPE Samples
Problems with quality of DNA due to fixation Mixture of normal and tumour DNA Some patients have no tumour sample available The sample represents the tumour at one fixed time point
ctDNA shed directly from tumour Extracted from the plasma component of whole blood Large fragment sizes possible Small quantities extracted ~ 30ng/ 5ml plasma Serial samples can be taken at various time points during the patient’s treatment
Isolation and processing of ctDNA
Blood sample taken
Sample spun down to isolate the plasma
Plasma is stored at -80ºc
ctDNA is extracted from the plasma using the QIAamp Circulating Nucleic Acid or some other commercial kit
Applications:: Next-generation sequencing Quantative PCR BEAMing Digital PCR
Major techical advantage compared to CTC
Possible problems?
ctDNA is unstable: requires fast processing
Getting only 30ng of ctDNA per 5ml plasma extraction
The technique used must be sensitive enough to pick up the low level variants
Diaz and Bardelli, 2014 Journal of Clincial Oncology 32
ctDNA: methods What is Droplet Digital PCR?
Expensive equipment?
ctDNA for monitoring metastasis
This proof-of-concept analysis showed that circulating tumor DNA is an informative, inherently specific, and highly sensitive biomarker of metastatic breast cancer
ctDNA for monitoring drug resistance
Exome-wide analysis of circulating tumour DNA could complement current invasive biopsy approaches to identify mutations associated with acquired drug resistance in advanced cancers. Serial analysis of cancer genomes in plasma constitutes a new paradigm for the study of clonal evolution in human cancers.
ctDNA as biomarkers of microscopic residual disease after surgery?
Study demonstrates accurate mutation detection in tumor tissues using ddPCR, and that ptDNA can be detected in blood before and after surgery in patients with early-stage breast cancer. ptDNA could identifies patients at risk for recurrence, which could guide chemotherapy decisions for individual patients.
