Title: Liquid Biopsies - The Future of Cancer

Early Detection

Presented By : Areeba Saeed

Roll # : MMMG-F23-01
Semester : 1st
Presented To : Dr Iqra Arooj
Course : Cancer Genetics
The Women University Multan
 Table of Contents

 Tissue Biopsy
 Liquid Biopsy: What is it?
 Liquid Biopsy vs Tissue Biopsy
 Analytes used in Liquid Biopsy
 FDA Approved liquid Biopsy Tests
 Conclusion
 BIOPSY

A biopsy is a sample of tissue taken from the body in order to examine it more
closely.
Biopsies are most often done to look for cancer.

Depending on the area, the procedure is usually done using a needle or surgical
incision.
 LIQUID BIOPSIES

Liquid Biopsies are minimally invasive tests that require a small amount of
liquid sample to detect intact cancer cells or materials that tumors or
metastatic lesions release into body fluids.

As it is a simple and non-invasive alternative to conventional surgical

biopsies, enabling doctors to discover a range of information about a tumor
through a simple blood sample.

Liquid biopsy has the potential to provide information about cancers without
invasive biopsy, using circulating biomarkers.
Liquid Biopsy Samples Include

 Blood(Plasma/Serum)

 Saliva

 Urine

 CSF
 COMPARISON

LIQUID Biopsy Vs TISSUE Biopsy

Minimally Invasive and less expensive Invasive and expensive

Less dependent on original tumor site since Specific to localized tumor site
tumors from both primary and metastatic
sites release DNA into bloodstream

Can capture tumor heterogeneity Assessment of tumor heterogeneity limited to

section of biopsy analyzed

Few copies of mutant ctDNA are sufficient for Limited amount of tissue may be obtained for
analysis immunohistochemical and genomic analysis
 COMPARISON

LIQUID Biopsy Vs TISSUE Biopsy

Easy to collect sample from blood Difficult to biopsy some organs

Allows for serial evaluation in absence of Not viable if primary tumor has been resected
primary tumor or metastases or if the tumor cannot be easily visualized via
imaging studies

Patients can tolerate serial blood draws for Serial biopsies are difficult to tolerate
evaluation; may led to greater compliance

May allow for evaluation of development of

resistance and May aid in early detection of cancer
Analytes used in Liquid Biopsies for detection
of Cancer

 Circulating Tumor Cells (CTCs)

 Circulating Tumor DNA
 Exosomes /Extracellular vesicles
 New addition: Tumor Educated Platelets
 1. CTCs

Serve as seeds for

CTCs are cancer cells subsequent growth of
that detach from a CTCs represent a rare additional tumors
(metastasis) at distant
primary tumor and cell population in the sites.
travel through the blood, typically less
bloodstream or than 10 cells/mL CTCs are primarily
detected in various
lymphatic system to compared with 1 metastatic carcinomas
other parts of the million WBCs/mL such as breast, prostate,
body. lung and colorectal
cancers
 SAMPLE COLLECTION

Cell saves preservative tube

 Made of negatively charged materials
 Contain stabilizing reagents
 Preventing blood cell breakdown for a period of about 5 days

Given the fact that most CTC isolation platforms necessitate that the blood
sample be processed within 96 hours after collection, one of the major
pitfalls of this approach is the inability to biobank the collected intact cells.
The initial applications of liquid biopsy in the cancer field were focused on CTCs.
CTCs have different molecular markers depending on the type of cancer. However,
since most cancers are of epithelial origin, there is a ‘universal’ epithelial molecular
marker, EpCAM, which can be used for CTC detection.
The expression of EpCAM varies with different cancer types and is mainly applied
to cancers such as breast and prostate which strongly express EpCAM. Since there
are currently only a limited number of molecular markers available, it is difficult to
define the entire CTC population.

The number of CTCs present in blood samples has been seen to correlate with clinical
staging, with the highest numbers generally found in patients with late-stage aggressive
cancers.
 2. Cell‑free DNA/Circulating Tumor DNA

cfDNA is the fragmented DNA found in cfDNA is released from cells mainly
biofluids released from both normal and through apoptosis (programmed cell
tumor cells into the circulatory system. death), necrosis (accidental cell death)
and active secretion from the tumor.

While ctDNA is fragmented tumor DNA that

has been shed into the blood stream.

ctDNA released by solid tumor and % is very low

(0.01%-1%)
 cfDNA / ctDNA

Has a very short half life ranging from 15 minutes to several hours.

Stable in plasma at -80 C

Blood can be sampled in EDTA tubes but plasma has to be isolated and
stored at -80 C within 1 hour of collection.

Preservative tubes are used to stabilize cfDNA in blood up to 4 days at room temperature.

cfDNA are around 160 to 180 base pairs in length with a higher prevalence of mutations in the
smaller fragments. One of the main advantages of cfDNA is that it can be analyzed from bio
banked biological fluids (including frozen plasma or serum).
 ctDNA can be distinguished from normal cfDNA fragments through the
presence of epigenetic or genetic alterations including tumor-specific
methylation markers and somatic mutations.

DNA methylation is an epigenetic mechanism involving the enzymatic transfer

of a methyl group onto the carbon-5 position of cytosine to form 5-
methlycytosine.
DNA methylation occurs naturally in the body, however abnormal patterns of
DNA methylation have been identified as indicators of diseases such as cancer.
 Extra Cellular Vesicles

Small membranous particles, which can be found in the majority of bodily fluids—
especially blood.
There are three main categories of EVs; exosomes, micro vesicles (MVs) and apoptotic
bodies, which are differentiated on their size, content, function, release pathways and
biogenesis.
Isolated EVs from the biofluids of cancer patients have been reported to contain tumor
derived molecules.
EV’s have advantages over ctDNA and CTCs as a tool for liquid biopsy; they have a
double-layered membrane structure which makes them less easily degradable than nucleic
acids and they also maintain the original source of cellular biological information well.
 EV based blood biomarker classifiers based on
EV protein profiles have been used to detect
stage I and II pancreatic, ovarian and bladder
cancer

Limitation of EVs

Obtaining blood derived EVs with a high purity is difficult, as they

can be obscured by other components in blood such as cells,
lipoproteins and cfDNA
 Tumor Educated Platelets

Blood platelets can act as both local and systemic responders during cancer
metastasis and tumorigenesis.

TEPs are blood platelets which have been exposed to tumor induced platelet
“education”.

During this process, tumor cells can directly bind to the platelets “educating” them to
contribute in tumor progression and metastasis, resulting in altered platelet behavior. This
change can be utilized as a biomarker to differentiate pan-cancer and non-small cell lung
cancer (NSCLC) from healthy individuals.
TEPs can be used as a liquid biopsy for the detection of glioblastoma
(GBM) and sarcoma cancer.

Advantages of TEPs in comparison to other blood-based bio sources is related

to their abundance, easy isolation, and ability to process RNA in response to
external signals.
It has been shown that in most cancer patients the platelet RNA profiles are
affected, independent of the type of tumor.
 FDA-Approved Liquid Biopsy Tests

Cell Search® CTC

Blood test used for the Foundation One
identification, isolation,
Guardant360 Liquid CDx
and enumeration of
Roche Cobas CDx
EGFR Can identify
CTCs of epithelial can detect changes in alterations in >300
origin was approved by gene mutations to >60 different genes genes relevant in
the FDA for clinical use identify non-small (genomic alterations) lung, prostate,
to assess the prognosis cell lung cancer causing solid tumors; colorectal, ovarian &
of patients with (NSCLC) approved as breast cancers; all as
metastatic breast, companion diagnostic companion
colorectal and prostate diagnostic
cancer.
Conclusion: Is this the end of Tissue Biopsies?

“Tissue biopsy will remain At the moment, liquid

the gold standard for the
next couple of years. As
scientific knowledge
advances, researchers are
learning more about the
potential of liquid biopsies
to detect mutations.
biopsies are Liquid biopsies that
recommended when a can detect cancer
tissue biopsy is difficult, early will improve
such as in the case of patient prognosis and
lung cancer, or when the
original site of the
survival.
disease is unknown.

Liquid biopsy for early cancer detection is currently led by genetic testing of
tumor derived biomarkers with both high sensitivity and high specificity