Circulating Tumor Cells: Advances in Liquid Biopsy Technologies (Current Cancer Research)

Preface
	References
Acknowledgements
Contents
About the Editors
Part I: Liquid Biopsy Technologies
	CTC-Based Liquid Biopsies and Diagnostic Leukapheresis
		1 Introduction
		2 The Rationale to Use High Blood Volumes in CTC-Based Liquid Biopsies
		3 Leukapheresis as Starting Point Enabling CTC Analysis in Large Blood Volumes
		4 Establishment of DLA to Enable High Blood Volume CTC Analysis
		5 Validation of DLA and Its Application in Different Cancer Entities
		6 DLA in Breast Cancer
		7 DLA in Prostate Cancer
		8 DLA in Non-Small Cell Lung Cancer
		9 DLA in Pancreatic Cancer
		10 Conclusions, Challenges, and Outlook
		References
	Minor Allele Enrichment in Liquid Biopsies Using Nuclease-Assisted Elimination of Wild-Type DNA
		1 Introduction
		2 Materials and Methods
			Genomic DNA, cfDNA, and Bisulfite DNA Treatment
			NaME-PrO Treatment and PCR Amplification
				NaME-Pro
				MS-NaME
				MSI-NaME
			NaME-PrO Followed by Droplet Digital PCR (ddPCR)
			Capillary Electrophoresis for Microsatellite Analysis
		3 Results
			NaME-PrO-Based Mutation Enrichment Enhances Mutation Detection via ddPCR
			Multiplex NaME-PrO Mutation Enrichment
			Two-Round NaME-PrO Treatment Further Increases Mutation Enrichment in cfDNA
			MS-NaME for Enrichment of Methylated and/or Unmethylated DNA Targets
			MSI-NaME for Microsatellite Indel Enrichment on cfDNA
		4 Discussion
		References
	Exploiting Exosomes for Cancer Diagnosis and Treatment
		1 Introduction
		2 Exosomes as Cancer Biomarkers
		3 Exosomes as Therapeutic Vehicles for Cancer Treatment
		4 Concluding Remarks
		References
	Translational Opportunities of Extracellular Vesicles in Biomedicine
		1 Introduction
		2 Different EV Types and Their Formation
			Microvesicles (MVs)
			Apoptotic Bodies (ABs)
			Exosomes
		3 Isolation Methods
			Ultracentrifugation (UC)
			Density Gradient Centrifugation (DGC)
			Size-Based Isolation Techniques
			Immunoaffinity Capture
			Coprecipitation
			Microfluidics
		4 EV Analysis Platforms
			Physical Characterization
			Protein Detection
			Detection of EVs Nucleic Acids
			Detection of EV Lipids
		5 EVs as Cancer Biomarkers
			CNS Tumors
			Ovarian Cancer
			Pancreatic Cancer (PC)
			Colorectal Cancer (CRC)
			Lung Cancer
			Prostate Cancer (ProCA)
			Breast Cancer (BC)
			Melanoma
		6 Perspectives
			EV Subtyping
			Comparison with Other Circulating Markers
		References
	Capture and Concentration of Circulating Cancer-Associated Extracellular Vesicles
		1 Overview: Extracellular Vesicles (EVs) and Their Enrichment Technologies
			Introduction to EVs and Their Role in Cancer
			Introduction to Affinity-Based EV Isolation Technologies and Detection of Cancer-Derived EVs
		2 Ligand Selection and Functionalization
		3 Substrates and Isolation Formats
			Slides and Multi-well Plates: Microarrays
			Particle-Based Isolation
			Microfluidic Devices
		4 Isolation Performance Evaluation
		5 Conclusion
		References
	Circulating Tumor Cells (CTC) and Tumor-Derived Extracellular Vesicles (tdEV)
		1 Introduction
		2 CTC and tdEV as Real-Time Liquid Biopsies
		3 The CellSearch CTC Definition
		4 Expansion of the CellSearch CTC Definition
		5 The Challenge to Identify CTC and tdEV in Blood
		6 Immunomagnetic Enrichment
		7 Size- and Density-Based Isolation
		8 Why Do We Want to Use CTC and tdEV and What Do We Want to Learn from Them?
		9 Scanning Electron Microscopy (SEM) to Reveal EV Formation
		10 Characterization of Single Tumor-Derived Extracellular Vesicles
		11 Outlook on CTC and tdEV
		References
Part II: Technology Development
	Mathematical Oncology to Integrate Multimodal Clinical and Liquid Biopsy Data for the Prediction of Survival
		1 Introduction
		2 Integration of Multicenter Demographic and Clinical Data
			Non-standardization and Sporadic Incompleteness in Multicenter Demographic and Clinical Data
			Imputation in Mixed Data Types
		3 Sparsity, Scale, and Dimensionality of Liquid Biopsy Data in Comprehensive Profiling
			Comprehensive CTC Profiling with HDSCA
			Morphometrics: Immunofluorescent Detection, Identification, and Enumeration of CTCs
			Genomics: Copy Number Variation Analysis in Single Cells and Cell-Free DNA
			Proteomics: Multiplex Imaging Mass Cytometry for Subcellular Features of Single Cells
		4 Integration and Augmentation of Multimodal and Multiscale Liquid Biopsy
			The Need for Methods for Multimodal Data Integration
			The Need to Augment Data to Address Sparsity in Liquid Biopsy
			Data Augmentation Using Generative Models and Information Geometry
		5 Advances in Machine and Deep Learning Methodologies for the Prediction of Survival
			Applications of Classical Machine Learning to Survival Prediction
			Advances in Deep Learning Models for Survival Prediction
			DeepSurv as a Case Example: Architecture and Hyperparameters
		6 Survival Prediction on Multicenter Demographic and Clinical Data in Two Breast Cancer Cohorts
			Patient Data, Integration, and Imputation of MSK and MDA Datasets
			Survival Prediction with a CPH, a Random Survival Forest, and DeepSurv
			DeepSurv Outperforms RSF and CPH in Individual and Merged Datasets
		7 Survival Prediction on Integrated Demographic, Clinical, and Liquid Biopsy Data in a Prostate Cancer Cohort
			Patient Data, Imputation, and Augmentation
			Survival Prediction with a CPH, an RSF, and DeepSurv
			Integrated Survival Prediction Models Outperform Those Built on Clinical Data Only
		8 Conclusion and Future Outlook
		References
	Microfluidics, CTC Capture, Analysis and Expansion
		1 Liquid Biopsy
		2 CTC Capture and Characterization
		3 Global Genomic Profiling of CTC
		4 Mutation Analyses of CTC
		5 CTC Profiling Beyond Mutations
		6 Cancer Stem Cells (CSCs)
		7 CSC in Dissemination
		8 Epithelial-to-Mesenchymal Transition (EMT)
		9 The CTC Microenvironment
		10 CTC Clusters
		11 CTC Propagation
		12 Microphysiological Systems
		13 MPS Design Parameters
		14 MPS Fabrication Methods
		15 Extravasation Models
		16 Modeling Breast Cancer Extravasation into Bone
		17 Future Outlook
		18 Conclusion
		References
	Development and Validation of Molecular Assays for Liquid Biopsy Applications
		1 Introduction
		2 Main Technologies for CTC Enrichment and Isolation
			Epitope-Dependent
			Epitope-Agnostic
			Affinity and Label-Free Microfluidics
			Diagnostic Leukapheresis (DLA)
		3 Molecular Assays for CTC Detection and Molecular Characterization
			Design, Development, and Analytical Validation of Gene Expression Assays for CTC Analysis
				RT-qPCR
				Multiplex RT-qPCR
				RT-dPCR
			Design, Development, and Analytical Validation of Molecular Assays for Mutation Detection in CTCs
			Design, Development, and Analytical Validation of Molecular Assays for DNA Methylation Markers in CTCs
				Methylation-Specific PCR (MSP)
				Whole Methylome Analysis of CTCs
		4 Single CTC Isolation and Analysis Systems
			Single CTC Isolation Systems
			Single CTC Analysis at the DNA Level
			Single CTC Transcriptomics
		5 Applications of CTC Analysis Molecular Assays in the Clinical Setting
			Breast Cancer
				Early BrCa
				Metastatic Breast Cancer
			Prostate Cancer
			Non-small Cell Lung Cancer
		6 Quality Control and Standardization of CTC Analysis
		7 Conclusions: Future Perspectives
		References
	State of the Art in the Propagation of Circulating Tumor Cells
		1 Introduction
		2 Propagating CTCs In Vitro on a Tissue Culture Plate
			The First CTC Cultures: 2013–2015
			Understanding the Identity of CTCs: 2015–2020
			Functionally Significant CTCs: 2020–Present
			The Future of CTC Cultures
		3 CTC-Derived Xenografts
		4 Unlocking the Potential of CTCs
		References
Part III: Biology
	Transcriptomic Analysis of CTCs
		1 Introduction
		2 The CTC Phenotype
			CTCs in Blood and Other Biological Fluids
			CTC Diagnostic and Prognostic Utility
		3 The CTC Proliferative and Metastatic Phenotype
		4 The CTC Resting/Dormant Phenotype
			Bone Marrow-Resident CTCs
			The CTC Dormant State at Organs Distinct from Bone Marrow
		5 Future Perspectives
		6 Conclusions
		References
	Insight into Intratumoral Heterogeneity Through Single CTC Sequencing and CDX Analysis
		1 Introduction
		2 Tumor Heterogeneity Investigation Through Single CTC Sequencing in ALK-Positive NSCLC
			Clinical Issues in ALK-Positive NSCLC
			Workflows for Single CTC Isolation and Sequencing in ALK-Positive Patients
			Genetic Alterations in Single CTCs at Resistance to ALK-TKI
			Intratumor Heterogeneity Revealed by Single CTC at Resistance to ALK-TKI
		3 Genetic Characterization of a Unique Neuroendocrine Trans-differentiation Prostate Circulating Tumor Cell-Derived eXplant Model
			Establishment of the Prostate CDX
			Phenotypical and Genetic Characterization of the Prostate CDX Model
			A CDX Model of Phenotypic Plasticity and Therapeutic Resistance to AR-Directed Therapies
		References
	Molecular Characterization of Single Circulating Tumor Cells in Breast and Ovarian Cancer
		1 Clinical Characteristics and Therapeutic Options in Ovarian Cancer
			Introduction
			First-Line Treatment
			Recurrence
		2 Disseminated Tumor Cells in Ovarian Cancer
		3 CTCs in EOC: Heading Toward Single-Cell Analysis?
		4 Circulating Cell-Free (cf) and Tumor (ct) DNA in Ovarian Cancer
			Methods
			Methylation
			Mutations
			The Role of cfDNA/ctDNA in Prognosis, Therapy Monitoring, and Recurrence Prediction
		5 MicroRNAs
			The Most Frequently Detected miRs in OC
			MiRs in EVs
			MiRs in the Context of Prognosis, Metastasis, and Resistance
			MiRs and the TME
		6 Circulating Immune Marker
			PD-1, PDL1, PD-L2, and CTLA-4
			HLA-G
		7 Conclusion and Future Directions
		References
	Circulating Tumour Cell Isolation and Molecular Profiling; Potential Therapeutic Intervention
		1 Background
		2 Cellular and Molecular Features of CTCs
			Metastatic Potential of CTCs
			Unrevealing Tumour Heterogeneity Using CTCs
		3 CTC Isolation Methods
			Immunoaffinity-Based Separation Methods
			Physical Property-Based Separation Methods
		4 Single-Cell Isolation Approaches
			Micro-pipetting and Micromanipulation
			Laser Capture Microdissection
			Serial Dilution
			Dielectrophoresis
			Fluorescence- and Magnetic-Activated Cell Sorting
			Droplet Generation and Single-Cell Encapsulation
		5 Single-Cell Analysis of CTCs
			Genomics
			Transcriptomics
			Proteomics
			Metabolomics
		6 Clinical Application of Single-Cell Analysis of Single/Cluster CTCs in the Prediction of Targeted Therapy Response
		7 Conclusion and Outlook
		References
	Dissecting the Molecular Profiles of Circulating Tumor Cells in Models of Breast and Prostate Cancers
		1 Introduction
		2 Breast Cancer
			Breast Cancer: Molecular Characterisation
				CTCs and Development of Metastases
				CTC Heterogeneity
				CTCs and Hormone Receptors
				Digital Molecular CTC-Based Signatures
			Prostate Cancer
			Prostate Cancer: Molecular Characterisation
				CTCs and Early Disease Monitoring
				CTCs and Development of Metastases
				CTCs and Hormone Receptors
				Digital Molecular CTC-Based Signatures
		3 Epithelial-Mesenchymal Plasticity in Breast and Prostate Cancer CTCs
		4 Unmet Clinical Needs and Current Challenges in CTCs
		References
Part IV: Clinical Application of the Liquid Biopsy
	Creating an Annotated Biospecimen Resource for Liquid Biopsy Applications
		1 Introduction and Rationale
		2 Existing Resources
		3 Defining Scope and Charter
		4 Biospecimen Collection
		5 Biospecimen Processing
		6 Biospecimen Storage
		7 Biospecimen Annotation and Data Management
		8 Resource Sustainability
		9 Conclusions
		References
	Opportunities for Liquid Biopsies to Meet the Challenges of Precision Medicine
		1 Introduction
			Challenges for Precision Medicine in Oncology: Opportunities for Liquid Biopsy
			Uses of Liquid Biopsy in Developing Complex Biomarkers
		2 Concepts Critically Important for ‘Omics Biomarker Development for Liquid Biopsy and Precision Medicine
		3 Critical Considerations in Development and Use of Liquid Biopsy Assays in Precision Oncology—ctDNA as Example
		4 How ctDNA Liquid Biopsy Is Used in Precision Medicine
			Predictive Biomarkers
			Response Biomarkers
			Biomarkers of Emerging Resistance
			Early Detection Biomarkers
		5 Standards Are Needed
			Blood Profiling Atlas in Cancer (BloodPAC)
			Foundation for the National Institutes of Health (FNIH) ctDNA Quality Control Materials (QCM)
			SEQC2 Oncopanel
			Friends of Cancer Research ctMoniTR
			International Liquid Biopsy Standardization Alliance (ILSA)
			Regulatory Guidance
		6 Conclusion—Challenges and Research Approaches
		References
Part V: Prognosis and Therapeutic Monitoring
	CTCs in Early Breast Cancer
		1 Introduction
		2 Genetic Profile of CTCs in Breast Cancer
		3 Prevalence and Prognostic Relevance of CTCs in the Context of Neoadjuvant Treatment
			GeparQuattro
			GeparQuinto
			GeparSixto and GeparSepto
			AVASTEM
			Remagus 02
			NeoALTTO
			NEOZOL
			Dynamics of Circulating Tumor Cells in Early Breast Cancer Under Neoadjuvant Therapy
			Analysis of the Serial Circulating Tumor Cell Count During Neoadjuvant Chemotherapy in Breast Cancer Patients
		4 Prognostic Value of CTCs Before, During, and Immediately After Adjuvant Therapy
			Cytokeratin-19 mRNA-Positive Circulating Tumor Cells After Adjuvant Chemotherapy in Patients with Early Breast Cancer
			Success-A
			TREAT-CTC Study
			Monitoring the Response of Circulating Epithelial Tumor Cells to Adjuvant Chemotherapy in Breast Cancer Allows Detection of Patients at Risk of Early Relapse
			Influence of Adjuvant Radiotherapy on Circulating Epithelial Tumor Cells and Circulating Cancer Stem Cells in Primary Non-metastatic Breast Cancer
			Pooled Analysis of the Prognostic Relevance of Circulating Tumor Cells in Primary Breast Cancer
		5 CTC Evaluation as a Screening Method and During Recurrence-Free Follow-Up
		6 Conclusions and Therapeutic Implications
		References
	NGS Analysis of Plasma cfDNA and cfmiRNA Signatures in Melanoma Brain Metastasis Patients
		1 Introduction
		2 ctDNA Assessment of MBM Patients
		3 Detection of ctDNA Genomic Aberrations in MBM Patients
		4 SNV Profiles of Paired MBM Tumors and Blood ctDNA
		5 ctDNA SNV Profiles and Respective Patient MBM Tumor Burden
		6 ctDNA Longitudinal Bleed SNV Profiling in Monitoring Tumor Progression After Complete Lymph Node Dissection (CLND) to MBM Development
		7 cfmiRNA Ability to Compensate for Lack of ctDNA in the Brain
		8 Discussion
		References
	ctDNA and Lung Cancer
		1 History of Cell-Free DNA
		2 Cell-Free DNA Biology
			Sources of cfDNA
			Clues of Nucleosome Positioning in cfDNA Fragments
			Lipoprotein-Associated cfDNA
			Cell-Free Mitochondrial DNA
			Neutrophil and Eosinophil Extracellular Traps
			Extrachromosomal Circular DNA
			Extracellular Vesicles
			Nuclease Activity and Apoptotic cfDNA Fragments
		3 Circulating Tumor DNA in Lung Cancer
		4 Characteristics of Cell-Free DNA for Lung Cancer Diagnostics
		5 Laboratory Methods for Cell-Free DNA Analysis
			cfDNA Extraction
			Quantitative PCR
			Digital PCR
			EFIRM
			Next-Generation Sequencing
		6 Cell-Free DNA Clinical Usage in Lung Cancer
			Non-invasive Driver Mutation Detection for NSCLC
			Non-invasive Measurement of Tumor Mutation Burden
			Longitudinal Monitoring of Tumor Mutations to Guide Therapy
			Longitudinal Measurement of ctDNA Levels to Assess Immunotherapy Efficacy
			Minimal Residual Disease and Recurrence Monitoring
		7 Considerations and Future Outlook
		References
	Clinical Perspectives in the Use of Liquid Biopsy in Metastatic Breast Cancer
		1 From Prognosis to Clinical Decision Making
			CTCs for Clinical Staging: Stage IV Indolent and Stage IV Aggressive
			CTCs and Treatment Choice
		2 The Best of Two Worlds: Combining CTCs and ctDNA
		3 A Glimpse of the Future: New Perspectives
			Beyond Canonical CTCs
				CTCs and Epithelial-to-Mesenchymal Transition (EMT)
				Dual-Positive Cells
				Cancer-Associated Macrophage-Like Cells
			New CTC-Based Biomarkers in MBC
				Sequence-Based Characterization
				Epigenetics
				Gene Expression
				MicroRNA
		References
	Circulating Tumor Cells in Men Treated for Prostate Cancer
		1 Introduction
		2 Challenges in the Clinical Assessment of Prostate Cancer Risk
		3 CTC Detection Methods in Prostate Cancer Patients
		4 Characteristics of CTCs in Non-metastatic Prostate Cancer
		5 Pre- and Post-treatment Studies Using CellSearch and Alternative CTC Assays in Patients with High-Risk Localized Prostate Cancer
		6 Strategies to Overcome Limitations of CTC Analysis
		7 Pilot Studies of CTC Response to RT
		8 Future Directions
		References
	The Role of Liquid Biopsy in Brain Tumors
		1 Glioma and Glioma Diagnosis
		2 2021 WHO Classification of CNS Tumors
		3 Liquid Biopsy
			Cell-Free DNA
			Circulating Tumor Cells
			Extracellular Vesicles
				EV Cargo: DNA
				EV Cargo: Protein
				EV Cargo: Lipids
				EV Cargo: Metabolites
				EV Cargo: mRNA
				EV Cargo: miRNA
				EV Cargo: lncRNA
				EV Cargo: Circular RNA
		4 Diagnostic Opportunities Based on New WHO Classifications
		5 Challenges in EVs for Liquid Biopsy
		6 Future Directions for EVs in Liquid Biopsy and Beyond
		References
Part VI: The Next Frontier: Liquid Biopsy and Early Cancer Detection
	Early Cancer Detection: Challenges and Opportunities
		1 Liquid Biopsy – Opportunities for Streamlining Cancer Screening and Improving Adherence
		2 Analytical and Biological Considerations and Challenges of Liquid Biopsy
		3 Challenges in Clinical Implementation of MCED Liquid Biopsy Tests
		4 Mitigating the Challenges
			Standard Data Collection
			Robust Examination of Limit of Detection
			Rigorous Study Design
			Diagnostic Follow-Ups
		5 Future of MCED Test Screening
		References
	The Galleri Assay
		1 Background
		2 The Challenges of Developing a Cancer Screening Test, Scientific Background, and the Birth of GRAIL
		3 Galleri Test Overview
		4 Clinical Program
			The Circulating Cell-Free Genome Atlas Study
				First CCGA Substudy (CCGA1)
				The Second CCGA Substudy (CCGA2)
				The Third CCGA Substudy (CCGA3)
				Prognostic Significance of MCED Results
			The PATHFINDER Study
			STRIVE
			SUMMIT
			NHS-Galleri
			SYMPLIFY
			REFLECTION and Real-World Evidence
			PATHFINDER2
		5 Tumor Fraction Drives MCED Detection
		6 Clinical Utility
		7 Modeling the Impact of MCEDs
		8 Conclusions
		References
Index