Innovations in Liquid Biopsies for Precision Cancer Diagnosis

Welcome
Innovations in Liquid Biopsies for Precision Cancer Diagnosis
Name: Vijaya Lakshmi Kanna Reddy
Qualification: MSc Diabetes Care
and Management
Student ID Here: 243/122023
10/18/2022
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@clinosolresearch 1

Index
• Introduction
• Liquid Biopsy Components
• Applications of Liquid Biopsy Biomarkers
• Advantages and Disadvantages of Liquid Biopsies
• Future Perspectives in Liquid Biopsy
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Introduction
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 A liquid biopsy is a medical test where biological fluids are sampled and analyzed.
 Comparing liquid biopsies to traditional blood tests and urinalyses: understanding the commonalities in sampling
bodily fluids like blood and urine for medical analysis.
 The broader scope of liquid biopsies: beyond blood and urine, encompassing other biological fluids such as
saliva, sputum, pleural fluid, and cerebrospinal fluid.
 Liquid biopsy-based biomarkers are increasingly vital in precision medicine, offering a less invasive and
potentially more sensitive alternative to traditional tissue biopsies and imaging.
 These biomarkers, including circulating tumour DNA (ctDNA), are used for detecting and monitoring cancer,
aiding in early disease detection, and tracking treatment progress, and patient care.

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 They leverage advanced technologies like next-generation sequencing to analyse various components such
as DNA, circulating free DNA [cfDNA] or circulating tumour DNA [ctDNA]), RNA, microRNA, and
proteins free in blood or bone marrow provide detailed insights into a patient’s tumour and its
microenvironment.
 This approach supports precision medicine by offering detailed genomic, proteomic, and immune status
information.
 The development and application of these biomarkers are being standardised by the FDA and international
initiatives, reflecting their growing importance in drug development.
 Diagnostic Potential: These tests serve similar purposes as traditional blood tests and urinalyses but are
gaining recognition for their broader diagnostic capabilities and resemblance to solid tissue biopsies in
clinical use.

Liquid Biopsy Components
• Liquid Biopsy (LB) involves analyzing various
components in the blood, including Circulating Tumor
Cells (CTCs), cell-free nucleic acids (mainly ctDNA or
ctRNA), exosomes, micro-vesicles, and platelets, which
originate from primary tumours or metastatic sites.
Circulating Tumor Cells (Ctcs)
• CTCs, a key component of LB, are rare and heterogeneous
cancer cells that break away from the primary tumour,
adapting to different tissue environments.
• They are identifiable by their unique characteristics: round
or oval shape, visible nucleus within the cytoplasm, and
specific marker expressions (positive for epithelial markers
like EpCAM and cytokeratins, and negative for the white
blood cell marker CD45).
• CTCs vary in size, typically larger than other blood cells,
but can be similar to or smaller than white blood cells.
They exist in different forms, such as intact single CTCs,
apoptotic CTCs, and CTC clusters, adding to their
complexity in LB analysis.
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Application of Circulating Tumor Cells (CTCs) in Medical Oncology
• Circulating Tumor Cells (CTCs), a crucial element of Liquid Biopsy (LB), has significant potential in medical
oncology across all cancer stages, from diagnosis to treatment evaluation and understanding of drug resistance.
• The concentration of CTCs in the blood varies; while some patients may have a high count (up to 1000 in 1
mL), typically, a metastatic carcinoma patient has 5 to 50 CTCs per 7.5 mL of blood. The number of CTCs is
linked to cancer prognosis; a higher count often indicates a more aggressive disease, poor treatment response,
lower overall and disease-free survival rates, increased metastasis, and shorter relapse time
• CTCs offer insights into tumour characteristics, invasiveness, and responsiveness to therapy, even in cases
without detectable metastases.
• They are instrumental in guiding treatment decisions, selecting pharmacological targets, and understanding the
emergence of drug resistance. While CTCs are valuable biomarkers in LB, their detection, quantification, and
isolation require further refinement for clinical validation in various cancer types.
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Cell-Free Circulating DNA
 Cell-free circulating DNA (cfDNA) is a key component of liquid biopsy, released both passively by dying
cells and actively by living cells. It includes fragments like cfRNA and is notably found in higher levels in
cancer patients.
 The presence of cfDNA in the blood isn't exclusive to cancer, as it can also originate from normal cell
apoptosis and be elevated in various non-cancerous conditions.
 In cancer patients, cfDNA levels are typically higher, especially in advanced stages, due to reduced DNA
degradation activity. cfDNA offers valuable insights for medical applications, such as detecting fetal DNA and
monitoring cancer through the identification of genetic mutations and markers.
 Different methods have been developed for cfDNA extraction, including magnetic enrichment using charged
beads and silica column-based enrichment, which have varying efficiencies and fragment recovery rates.
 The analysis of cfDNA, particularly from tumor cells, is crucial in liquid biopsy for detecting specific genetic
alterations, which can guide targeted cancer therapy. This makes cfDNA a significant tool for understanding
and managing cancer at various stages.
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Circulating Tumor DNA (ctDNA)
 Circulating Tumor DNA (ctDNA) is a critical component in liquid
biopsy, existing as single- or double-stranded DNA in plasma or
serum. It is characterized by cancer-associated features like mutations
and methylation changes, indicating its tumour origin.
 The potential sources of ctDNA include apoptotic or necrotic tumor
cells, live tumor cells, and Circulating Tumor Cells (CTCs). The
release of ctDNA into the bloodstream occurs through two main
processes:
i. Passive Release: This involves the release of DNA fragments
during cell death, either through apoptosis (yielding fragments
around 166 bp) or necrosis (resulting in larger fragments from 320
bp to 1000 bp).
ii. Active Secretion: This process involves extracellular vesicles,
such as exosomes, releasing smaller DNA pieces (150–250 bp).
 The amount of ctDNA shed into circulation varies based on tumor
characteristics like location, size, and vascularity, leading to a wide
range in blood ctDNA levels (0.01%–90%). ctDNA has a short half-
life in the bloodstream, ranging from 16 minutes to 2.5 hours.
 While healthy individuals typically have a cfDNA concentration
around 30 ng/mL in plasma (ranging from 0 to 100 ng/mL), cancer
patients can have levels up to 1000 ng/mL, highlighting ctDNA's
potential as a biomarker for cancer detection and monitoring.

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 Circulating Tumor DNA (ctDNA) serves as a vital biomarker in liquid biopsy (LB) for cancer diagnosis
and prognosis, offering advantages in sensitivity and clinical correlation. Key points for ctDNA
applications in medical oncology include:
 Enhanced Sensitivity and Real-Time Monitoring: ctDNA provides a more sensitive measure compared to
traditional plasma biomarkers. Its short half-life (less than 2 hours) allows for accurate, real-time
monitoring of tumor burden and therapy response, unlike protein markers with longer half-lives.
 Minimized Sampling Bias: ctDNA can be derived from all tumor lesions in the body, reducing sampling
bias associated with tissue biopsies which only represent a small tumor region.
 Genotyping and Personalized Therapy: ctDNA analysis facilitates genotyping, detecting genetic mutations
crucial for personalized cancer treatment. It reflects the genetic makeup of the entire tumor, enabling
dynamic monitoring of mutations throughout cancer progression.
 Heterogeneity of Tumor cfDNA: Tumor cfDNA, derived from apoptosis, necrosis, and active secretion of
proliferating cancer cells, is highly heterogeneous. This heterogeneity, combined with LB's ability to
capture the full spectrum of tumor DNA through repeated blood sampling, positions LB as a versatile tool
in all stages of cancer management, from diagnosis to tracking disease progression and treatment efficacy.
Application of ctDNA in Medical Oncology

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Cell-Free Circulating RNA
 Cell-free circulating RNA (cfRNA), though less abundant than cfDNA, is a significant component in liquid biopsy
(LB), encompassing ctRNA, microRNA (miRNA), messenger RNA (mRNA), and non-coding RNAs.
Circulating Tumor Messenger RNA
 Detection of Mutations and Gene Expression: ctRNA in blood aids in identifying gene mutations and provides
insights into the expression levels of key genes involved in cancer progression.
 PD-L1 Overexpression: ctRNA can be used to detect PD-L1 overexpression, a factor that accelerates tumor growth.
 PD-L1 suppresses tumor-specific T cells and enhances cancer cell survival. A high concordance between PD-L1
protein in tumor tissues and PD-L1 gene expression in plasma has been observed, making it a predictive marker for
anti-PD-L1 therapy.
 Telomerase Reverse Transcriptase mRNA (hTERT): Elevated levels of hTERT mRNA are common in various
tumors like breast and colon cancer, but absent in cancer-free individuals, indicating its potential as a tumor marker.
 Challenges in Using mRNA as a Biomarker: Despite its utility, using mRNA in LB faces challenges due to its
protection in extracellular vesicles, association with protein complexes, and its inherent instability, low abundance,
and susceptibility to contamination during processing.
 These factors limit the reproducibility and practicality of cell-free mRNA as a biomarker in LB.

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Circulating Tumor MicroRNAs
 Circulating Tumor MicroRNAs (miRNAs) are emerging as significant biomarkers in cancer detection and
management. Key points include:
 Oncogenic and Tumor Suppressor Roles: MicroRNAs can function as tumor suppressors or oncogenes,
providing cancer cells with a proliferative advantage and promoting metastasis.
 Release and Stability: Cells release miRNAs in association with RNA-binding proteins or within exosomes,
which protect them from RNase activity. In blood circulation and other biofluids, miRNAs are more stable and
abundant than mRNAs, especially in cancer patients.
 Diagnostic and Prognostic Value: miRNA expression levels are promising for early cancer diagnosis, prognosis,
and predicting therapy responses. Specific miRNAs like miR-145, miR-20a, miR-21, and miR-223 in non-small
cell lung cancer, and miR-10b and miR-373 in breast cancer, have been linked to cancer progression and
metastasis.
 Isolation and Analysis Challenges: Before analysis, miRNAs must be isolated from contaminants like platelets
and cell debris. This isolation is crucial due to the difficulties posed by cell-free miRNAs in cancer biomarker
applications.
 Detection Methods: miRNAs can be detected using quantitative RT–PCR (qRT–PCR), microarray, and deep
sequencing. qRT–PCR is sensitive and requires a low RNA concentration but relies on a standard curve and
primer design for specificity.
 These aspects highlight the potential and challenges of using circulating tumor miRNAs as biomarkers in cancer,
underlining their role in improving the understanding and treatment of the disease.

Exosomes
• Exosomes are small extracellular vesicles (40-150 nm) originating from endosomes and secreted by all cell
types, playing a vital role in intercellular communication. Key points about exosomes include:
• Composition: Exosomes carry a diverse range of contents, including proteins (such as cytoskeletal,
transmembrane, and thermal shock proteins), lipids, enzymes (like GAPDH, ATPase, PGK1), and nucleic
acids (mainly mRNA, miRNA, and single- and double-stranded DNA).
• Reflection of Cell Origin: The content of exosomes varies based on their cell of origin. Tumor-derived
exosomes particularly reflect the characteristics and status of the original tumor cells, as they contain similar
cellular contents.
• Abundance in Body Fluids: Tumor cells release more exosomes compared to non-tumorigenic cells. These
exosomes are present in various body fluids, including blood, serum, urine, cerebrospinal fluid, and breast
milk.
• Isolation Methods: Exosomes are typically isolated through successive centrifugation and ultracentrifugation
steps, ensuring the removal of cells and contaminants. Factors influencing the purity of the isolated exosomes
include centrifugation speed, duration, temperature, and the rotor type. Additionally, magnetic beads coated
with antibodies against exosomal markers (e.g., CD9, CD63) are used for isolating specific exosome
populations, especially in small sample volumes.
• These aspects highlight the significance of exosomes in medical research and their potential applications in
understanding and treating diseases, particularly cancer.
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Application of Tumor-Derived Exosomes in Medical Oncology
• Liquid Biopsy (LB) using exosomal profiles offers promising potential for early
cancer diagnosis and monitoring the efficacy of anti-tumor therapies.
• Role of Exosomes in Cancer Progression: Exosomes significantly contribute to
various stages of breast cancer development by transferring genetic information
that stimulates tumor angiogenesis, reshapes the tumor microenvironment, and
promotes tumor growth and drug resistance.
• Exosomal DNA (exoDNA) and tumor-associated microparticles (taMPs)
provide stable and specific cancer markers.
• Detection of Cancer-Specific Mutations: In pancreatic ductal adenocarcinoma
(PDAC), specific mutations like KRASG12D and TP53R273H are detectable in
circulating exosomal DNA. Glypican 1 (GPC1)-positive serum exosomes are
indicative of these mutations.
• Exosomal Markers in Different Cancers: High levels of GPC1 are observed in
breast cancer. In glioblastoma, serum exosomes show increased levels of
Epidermal Growth Factor Variant III (EGFRvIII) and miRNA-21. Additionally,
in advanced melanoma, proteins such as tyrosinase-related protein-2 (TYRP2),
very late antigen 4 (VLA-4), heat-shock protein 70 (HSP70), and HSP90 are
found elevated in circulating tumor exosomes (TEXs).
• These insights underscore the importance of exosomes in liquid biopsy for
providing crucial information about cancer status and progression, aiding in the
development of targeted therapies.
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Microvesicles
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• Microvesicles, larger than exosomes (100-1000 nm), mirror the proteomic profile of their originating cells,
making them valuable in cancer research. Key points include:
• Origin and Composition: Microvesicles originate from various cell types, including hematopoietic, endothelial,
mesenchymal stem cells, and cancer cells. Their membrane composition closely resembles that of the parent
cell.
• Role in Tumor Progression: Through the transfer of molecular contents, microvesicles can significantly alter
the functions of recipient cells. They play a crucial role in tumor development and progression by facilitating
intercellular communication that contributes to immune suppression, angiogenesis, and metastasis.
• Potential Therapeutic Targets: Due to their involvement in cancer progression, aspects of microvesicles'
biogenesis and function are being explored as potential targets for anti-cancer therapies.
• Impact in Breast Cancer: In breast cancer, tumor-associated macrophages release microvesicles containing
miRNAs that enhance the aggressiveness of cancer cells.
• These aspects highlight microvesicles' importance in understanding and potentially treating cancer, offering
insights into the complex mechanisms of tumor development and progression.

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 The use of tumor-altered blood platelets as biomarkers in Liquid Biopsy (LB) is a recent development,
providing valuable insights for clinical cancer diagnosis. Key points include:
 Platelet Tumor Education: Blood platelets interact with cancer cells, resulting in their "education" by absorbing
tumor-associated biomolecules. This process not only influences gene expression in tumor cells but also alters
the RNA profile of the platelets.
 Uptake of Tumor Materials: Platelets can incorporate circulating mRNAs and proteins from the tumor
microenvironment. They also sequester extracellular vesicles from cancer cells containing tumor-specific
RNA.
 Detection of Cancer-specific Changes: In metastatic lung cancer patients, platelets exhibit differential splice
isoforms of the NAD-dependent deacetylase sirtuin-2 (SIRT2), distinct from those in healthy individuals.
 High Diagnostic Accuracy: mRNA sequencing of platelets "educated" by various cancer types (including
glioblastoma, non-small cell lung cancer, colorectal, pancreatic, breast, and hepatobiliary cancers)
differentiates cancer patients from healthy individuals with 96% accuracy.
 Tumor-educated platelets (TEPs) can predict and screen for cancers like NSCLC, offering potential for early
detection and treatment outcome prediction.
Platelet

Applications of Liquid Biopsy’s Biomarkers
 Liquid Biopsy (LB) biomarkers have significant applications in clinical oncology, with promising results for
various cancer types. Key applications include:
 Lung Cancer: LB can detect PD-L1 expression in Circulating Tumor Cells (CTCs) or white blood cells, aiding
in the application of targeted therapies like anti-PD-L1 immunotherapy and EGFR tyrosine kinase inhibitors.
 Colorectal Cancer: Genotyping studies show over 90% concordance between KRAS mutations in tumor tissue
and ctDNA, supporting the use of ctDNA LB as a viable alternative for tissue tests.
 Prostate Cancer: The AR variant 7 (AR-V7) biomarker, linked to more aggressive, castration-resistant tumors
and treatment resistance, can be detected in plasma proteins using an automated capillary nano-immunoassay,
though not yet routinely used in clinical practice.
 Melanoma: ctDNA is a promising biomarker for monitoring treatment with BRAF/MEK inhibitors in
metastatic melanoma, useful in predicting response and resistance.
 Breast Cancer: Digital PCR (ddPCR) assays based on ctDNA detection have demonstrated clinical utility in
identifying HER2 mutations.
 Pancreatic Cancer: Analysis of KRAS mutations via ctDNA shows greater diagnostic power than traditional
markers like CEA, CA19-9, and CA125, though not yet standard in clinical practice.
 These applications highlight LB's growing role in cancer diagnosis, prognosis, and treatment selection,
offering less invasive and more precise alternatives to traditional methods.
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Advantages
 profile, which is often missed in a single tissue biopsy. Liquid biopsies
provide a less invasive method compared to tissue biopsies, reducing
patient discomfort and risk.
 Useful for real-time tracking of tumor dynamics and treatment response,
particularly during follow-up.
 Liquid biopsies can be applied in various cancer types and stages.
 Tumor Heterogeneity Representation: They can capture the diverse
tumor
Disadvantages
 Sensitivity and Early Detection: Detecting analytes like CTCs and
ctDNA is challenging, especially in early-stage cancers where their levels
are often low.
 Low Specificity and False Positives: There's a risk of high false positive
rates in cancer screening, leading to unnecessary procedures and distress.
 Clonal Hematopoiesis: cfDNA may include alterations from clonal
hematopoiesis, potentially leading to incorrect diagnoses.
 Quantity and Quality of Samples: Ensuring sufficient quantity and
quality of cfDNA for analysis is crucial, requiring careful extraction and
processing methods.
 Cost and Accessibility: The high cost and technical demands of liquid
biopsy procedures limit their widespread use in clinical practice.
Advantages and Disadvantages of Liquid Biopsies

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Future Perspectives in Liquid Biopsy
 Growing Clinical Relevance: Liquid biopsy is increasingly recognized for its therapeutic importance in cancer
care, with FDA approvals for ctDNA-based NGS diagnostic assays in various cancers.
 Quality and Quantity of ctDNA: Ensuring appropriate ctDNA sample quality and quantity is crucial, requiring
careful handling to avoid contamination and optimized extraction methods for high yields.
 Advanced Detection Methods: Highly sensitive techniques like ddPCR, NGS, and BEAMing are essential due
to the low concentration and fraction of ctDNA in circulation.
 CTC Analysis Potential: CTCs, though rare and costly to isolate, offer additional insights into intra-patient
heterogeneity and treatment resistance, and could predict future metastatic sites.
 Exosomal Research: Exosomal nucleic acids and proteins are key in cancer diagnosis and progression, yet
research on exosomal lipids and metabolites remains limited.
 Improvement in Exosome Isolation: Enhancing exosome isolation and characterization techniques is a priority,
alongside establishing standardized methods for exosome-based diagnostics.
 Proteomics Advancements: Advances in proteomics, including improved detection resolution and high-quality
antibodies, are crucial for identifying low-abundance, organ-specific biomarkers.
 Single-Cell Proteomics: Emerging single-cell proteomics, especially in MS and surface protein phenotyping,
opens new avenues for cancer biomarker discovery, particularly in immunotherapy.

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 LB analyzes tumour-derived material like Circulating Tumor Cells (CTCs), cell-free circulating nucleic acids,
exosomes, microvesicles, and platelets from various body fluids (blood, urine, saliva, cerebrospinal fluid, CSF,
and pleural effusion).
 These components provide a comprehensive molecular analysis, reflecting the genetic profile of primary and
metastatic tumours, enabling real-time monitoring of tumour dynamics.
 LB addresses challenges posed by tissue biopsies, such as limited accessibility and clonal heterogeneity of
tumours.
 LB offers a less invasive method for identifying molecular biomarkers, and capturing real-time tumor
heterogeneity, and can be repeated as necessary.
 LB has the potential to establish molecular profiles for a range of clinical applications, aligning with the goals of
precision medicine.
 Despite its potential, LB faces challenges in technical complexity and the need for validation and standardization.
Its clinical utility and comparability with tissue biopsy results are areas of ongoing research.
 liquid biopsies offer significant advantages in terms of patient care and monitoring, they face challenges related
to sensitivity, specificity, and technical and financial feasibility, particularly in early cancer detection and routine
clinical use
Conclusion

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Innovations in Liquid Biopsies for Precision Cancer Diagnosis

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