Recent diagnostics and pathophysiology of various cancers are seen from the perspective of molecular biology.

1. LIQUID BIOPSY
Dr Shashank Agrawal

2. Introduction
• Large amount of molecular data derived from Human
Genome Project and advances in Pharmacogenomics
has led to paradigm shift in oncology from “One size
fits all” to more precise medicine and has led to
development of “Personalised Medicine”.
• In view of limitations of traditional biopsies-
Invasiveness, complications and sometimes
impossible, also there cross-sectional behaviour (as
tumours and metastasis is dynamic a/c environment
and medications and there heterogenicity), a new field
analysing cancer derived components in circulation
and other body fluids attracts minds in oncology.

3. • Apoptotic or necrotic cancer cells release circulating cell-free DNA
fragments, designated as circulating tumor DNA (ctDNA), as well as
exosomes (EXOs), namely membrane-encapsulated subcellular
structures containing proteins and nucleic acids released by the
tumor cells.
• Primary tumor and metastatic sites are also able to exfoliate vital cells
that, once entered the bloodstream, are circulating tumor cells
(CTCs).
• Isolation of these tumor-derived components from peripheral blood
and other potential fluids and their genomic or proteomic assessment
represent a new diagnostic tool that has been called ‘liquid biopsy’.

4. Approach to liquid biopsy
• Cancer at any stage can shed tumor cells as well as fragments of
cancer-causing DNA into the blood system and may give good
indication of mutations in the tumor at the time of sampling.
• The more advanced the cancer, the more likely tumor cells and
cancer-causing DNA can be found in the blood.

6. Circulating Tumor Cells (CTCs)
• CTCs represent intact, viable non-hematological cells with malignant
features that can be isolated from blood.
• 1869, Ashworth described CTCs in cancer patients and its presence in
blood stream was demonstrated by Engell in 1955.
• CTCs are released into the bloodstream during metastatic spread of
the cancer through blood and are present as single cells or clusters.

7. CTC (cont.…)
• Even in patients with metastatic cancer, they occur on average at a
frequency of 1 in 100 million cells and are mixed with approximately
10 million leukocytes and 5 billion erythrocytes per 1 ml blood.
• And, due to apoptosis of CTCs, which begins soon after separation
from the tumor of origin, they are extremely fragile.
• Next generation sequencing (NGS) has revealed CTCs carry mutation
signatures that resemble the signatures of their primary tumors
including driver and druggable mutations like APC, KRAS, TP53,
ERBB3, FBXW7 and ERBB2.

8. Cell free DNA (cfDNA)
• Small DNA fragments found circulating in plasma or serum, as well as other
bodily fluids.
• The presence of cfDNA in blood plasma was discovered in 1948 by Mandel
and Metais.
• Plasma cfDNA usually are fragments of about 170– 500 bp, thought to
originate mostly from apoptotic cells.
• The total amount of ctDNA might be <0.01% of the total cfDNA
concentration.
• A major breakthrough came with the advancement of NGS technologies for
mutation detections which allowed for the sequencing of DNA and RNA
much more quickly and cheaply than the previously used Sanger
sequencing.

9. • Patients with distant metastases have a significantly higher level of
cfDNA compared to patients without metastases. Total mutation–
specific cfDNA decreases during treatment but the levels increase
later on in patients with recurrence.
• Initial studies, such as those performed by Diaz et al., suggest that
when both cfDNA and CTCs are present, cfDNA fragments
outnumbered CTCs by 50 to 1.
• In addition, a direct comparison of mutation detection on cfDNA vs
CTCs showed a higher abundance of the mutation on the cfDNA from
the same patient.

10. • An advantage of cfDNA is that they are relatively stable and can be
analyzed from bio-banked biofluids, such as frozen plasma.
• The most commonly used vacutainers for blood collection for
isolation of cfDNA are proprietary Streck Cell-Free DNA BCT and
CellSave tubes and standard K2EDTA tubes.
• Studies comparing the optimal conditions for blood collection and
storage temperature for cfDNA have found similar abundance and
stability for up to 6 h in all tube types with no effect on the yield.

11. Exosomes and circulating RNA
• Tumor cells actively release several species of cfRNAs, into the blood
including non-coding RNAs (e.g., microRNAs or miRNA, small nucleolar
RNAs, PIWI interacting RNAs, and long non-coding RNAs).
• Such species are enriched in exosomes and strongly resist RNases.
• High levels of exosomes are found in several bodily fluids from cancer
patients and because of their resistant nature, exosomes and miRNA can
be isolated from biofluid samples and stored for many years in the freezer.
• Their composition seems to reflect that of the parental cells and therefore
provide a novel type of biomarker for various patient scenarios.
• Like cfDNA, circulating miRNA seems to give a better picture of variations
present in the tumor than CTC.

12. • Recent studies have shown that plasma miRNA levels significantly
correlate with larger tumor size, chemoresistance, and recurrence of
tumors.
• A study of plasma miR-224 levels in patients with hepatocellular
carcinoma found that plasma levels could accurately predict presence
of small tumors which were less than 18 mm preoperatively.
• Similarly, the overexpression of miR-21 and its detection in plasma
contributed to chemo-resistance in esophageal squamous cell
carcinoma.
• Level trends of miRNA in plasma co-relates with disease activity as
well as treatment response.

13. Isolation and Analysis
• Initial limitations were difficulties in isolation of nucleic acid and
differentiating between normal and tumor derived material.
• Various new technologies and advances have been introduced for
proper assessment of tumor derived materials.

14. Current approaches for detection of liquid biopsy components from
blood can be divided into three different categories:
• Methods targeting specific druggable driver mutations or all possible
aberrations in cfDNA,
• Methods to isolate and identify CTCs, and
• Methods to isolate and identify exosomes,

15. Methods to isolate and identify CTC
CTCs in blood can be enriched using marker-dependent techniques:
CTCs can be
Positively selected in vitro or in vivo using antibodies to epithelial and/or
mesenchymal proteins (such as epithelial cell adhesion molecule (EPCAM)
and/or cytokeratins including CK8, CK18, CK19 and mesenchymal vimentin or
N-cadherin) or
Negatively selected for through depletion of leukocytes using anti-CD45
antibodies (endothelial cells, with markers such as CD146, and
hematopoietic stem cells, with markers such as CD34).
Positive enrichment of CTCs can also be performed in vitro using assays
based on CTC characteristics including size(6 μm to >20 μm), deformability ,
density and electrical charge.

16. • Following enrichment, the isolated CTCs can be identified using
immunocytological, molecular or functional assays.
• With immunocytological platforms, CTCs are identified by membrane
and/or intra-cytoplasmic staining with antibodies to epithelial,
mesenchymal, tissue-specific or tumor-associated markers. Most
common being CellSearch platform for Cytokeratin staining.
• AdnaTest EMT-2 (Qiagen) test is an immuno-magnetic bead
enrichment step targeting the surface proteins EpCAM, HER2 and
EGFR.
• Molecular and functional characterization need single CTC isolation-
Done using DEPArray platform.

17. • Molecular technologies enable the identification of CTCs using RNA-based
assays, such as quantitative reverse transcription PCR (qRT-PCR), RNA
sequencing or in situ RNA hybridization.
• Functional assays can be used to detect viable CTCs on the basis of their
biological activities, for example, the fluoro- Epithelial ImmunoSPOT
(EPISPOT) assay for certain proteins secreted or shed by CTCs and the
related EPISPOT in a drop (EPIDROP) technology that enables the detection
of single CTCs in microdroplets.
• The molecular characteristics of CTCs can be further explored at the DNA ,
RNA and protein level, and the functional properties of CTCs can be
investigated in vivo by injecting the cells into immunodeficient mice to
form patient-derived xenograft (PDX) models.

21. Potential Applications

22. Methods of cfDNA analysis

23. BEAMing, beads–emulsion–amplification–magnetics; biPAP,
bidirectional pyrophosphorolysis-activated polymerization; CAPP-Seq,
cancer personalized profiling by deep sequencing; ddPCR , droplet
digital PCR; NGS, next-generation sequencing.
Druggable,
Resistant,
New
mutations
etc

24. Commercial Kits
• “SiRe” panel to identify 568 clinically relevant mutations in six genes
(EGFR, KRAS, NRAS, BRAF, cKIT and PDGFRα) involved in NSCLC,
gastro-intestinal stromal tumor, metastatic colorectal carcinoma and
melanoma.
• Guardant360 platform has been designed to identify tumor-related
genomic alterations via complete exon sequencing in 73 different
genes in cfDNA from blood.
• FoundationACT Liquid Biopsy Assay sequences the exons of 315
cancer-associated genes and introns from 28 genes involved in
rearrangements.

25. Potential Applications

26. mRNA and Exosomes
• Isolation of Exosomes and mRNA based on specific centrifugation
techniques and electron microscope.
• miRNeasy mini and microKits (Qiagen) seems to the most popular kits
for isolation of exosome-incorporated miRNAs and cell-free miRNAs

27. Comparison

28. • To date, liquid biopsy is not a routine test in clinical practice, but its
potential applications are rapidly growing: from diagnostic genomic
profiling to the monitoring of radicality in surgical outcomes, from
evaluating either response or resistance to systemic treatments, to
quantifying minimal residual disease.

30. Esophageal Cancer (EC)
Screening
• Data suggests about 93% have no prior
screening and 40% presents with
advanced ds.
• CTC is a sensitive and minimally invasive
alternative tech to endoscopy.
• A small study of CTC-based liquid biopsy
differentiated squamous cell EC patients
from healthy controls with 86.3%
sensitivity and 90.3% specificity.
• ctDNA is less sensitive, as it is detectable
in only 58% of patients without
metastatic disease, however, sensitivity
increases with late disease stage.
Staging
• PET-CT and endoscopic ultrasound
• 50% individuals at curative intent
procedure present with hematogenous
spread at 5 years.
• CTC corresponds with higher stages but
also been detected in N0 ds.
• ctDNA helps in prognostication by
defining tumor biology. E.g. in SCC, MSH2
hypermethylation promoter was
associated with inferior DFS.
• Overall LB helps in stratifying high risk
individuals for refining need of
neoadjuent/adjuent therapies.

31. Response to Therapy/Surveillance
• Management is multimodal.
• Studies have shown post RT CTC positivity is associated with poor outcome.
• 13-39% patients have discordant mutation b/w primary and CTC affecting
personalised care.
• The Personalized Antibodies for GastroEsophageal Adenocarcinoma
(PANGEA) trial highlighted the significance of tumor heterogeneity and
the potential of ctDNA-based liquid biopsy to guide precision therapy.
• Another group demonstrated that acquired MET amplifications detected in
ctDNA can serve as markers of resistance to trastuzumab and novel HER-
kinase inhibitors in adenocarcinoma.

32. Gastric Cancer (GC)
• Both CTC (epithelial, intermediate and mesenchymal on EMT) and
ctDNA have been seen in GC.
• No screening protocols for average risk individuals at present.
• ctDNA and miRNA are promising and more sensitive then CTC.
• But due to overall low sensitivity can’t be considered replacement of
endoscopy.
• CancerSeek is the platform available for evaluating ctDNA.

33. Staging
• A study of 44 patients demonstrated that the CTC count at diagnosis
is predictive of advanced stage, peritoneal dissemination, presence of
metastases, and poor survival.
• Furthermore, a recent meta-analysis of 16 studies demonstrated that
quantitative ctDNA level correlated with the above outcomes .
• HER2 amplification or overexpression.
• A prospective study of 88 patients demonstrated discordance in HER2
amplification status between primary tumors and CTCs in patients
with metastatic GC.
• So LB can be used to tailor the treatment accordingly.

34. Response to Therapy/Surveillance
• Trastuzumab resistance in metastatic HER2-positive disease can be
monitored with serial liquid biopsies.
• A similar example of this is illustrated in a study that trialed a pan-
HER kinase inhibitor (afatinib) and found that resistance to this drug
was correlated with tumor cells that were lacking EGFR
amplification or with MET amplification.
• CTC levels in post-treatment patients can be a prognostic marker.

35. Pancreatic Adenocarcinoma (PDAC)
• In patients with PDAC, both CTCs and ctDNA have been identified in peripheral
blood.
• Enrichment is challenging in PDAC due to small numbers of CTC in peripheral
blood and pilot studies demonstrated that 7.5 mL whole blood is required for
adequate sensitivity; in addition, cell size-based isolation (ISET) was more
sensitive than the Cellsearch system.
• Following enrichment, CTCs are analyzed using immunocytochemistry, FISH, and
whole genome sequencing.
• A study of 34 patients demonstrated that PDAC patients released ctDNA into the
circulation at a rate that was orders of magnitude higher than healthy controls
and genomic and epigenetic mutations were detectable in 5 mL of whole blood.
• The optimal technique for enrichment and analysis of both CTCs and ctDNA in
PDAC is a topic of ongoing laboratory investigation.

36. Screening
• Unmet Need
• It takes 15 years from single mutation to metastatic PDAC.
• CTC is in all stage but low specificity.
• Four principal mutations have been identified in ctDNA as most
commonly found in PDAC: KRAS, CDKN2A, SMAD4, and TP53.
• A study combining the detection of ctDNA KRAS mutations with CA
19-9 levels differentiated PDAC (47 patients) from chronic pancreatitis
(31 patients) with a sensitivity of up to 98% and a specificity of 77%
• Need further validation.

37. Staging
• A meta-analysis of 16 studies regarding the use of CTCs and
disseminated tumor cells (CTCs in bone marrow) as prognostic
indicators demonstrated that detection of CTCs in peripheral blood
and CTCs in bone marrow is associated with both progression-free
and overall survival.
• As for the role of ctDNA, a meta-analysis of 18 studies including 1243
patients determined that the presence of ctDNA with KRAS or ERBB2
mutations in peripheral blood significantly impacted overall and
progression-free survival.
• Furthermore, specific mutations, hypermethylation, and a high
absolute quantity of ctDNA were associated with inferior survival.

38. Response to Therapy/Surveillance
• CTC positivity, which dropped from 80.5% of patients to 29.3% after a cycle
of fluorouracil-based chemotherapy in 41 patients with advanced PDAC.
• Berger et al. demonstrated that in 20 therapy naive patients with ctDNA-
positive (KRAS or TP53 mutations) metastatic PDAC who underwent
chemotherapy, detectable mutations significantly decreased during
treatment and the resulting levels correlated with progression-free
survival.
• One study demonstrated that specific CTC phenotypes (high expression of
CD44, CD133, and aldehyde dehydrogenase) are predictive of recurrence.
• Higher post-treatment levels of overall plasma ctDNA were found to
correlate with both recurrence and metastases.
• Furthermore, one study showed that ctDNA detected recurrences 6.5
months earlier than CT imaging among 77 patients.

39. Colorectal Cancer
• The role of liquid biopsy in colorectal cancer (CRC) is evolving rapidly.
• CTCs, ctDNA, and exosomal DNA have been isolated from the blood of patients with CRC.
• Location of the blood-draw impacts the sensitivity of liquid biopsy in CRC, as blood
from the portal system is enriched with tumor material compared to peripheral venous
blood.
• Detection limits reach as low as 3 CTCs per 2ml whole blood.
• CTCs in CRC patients frequently have a genotype distinct from tissue tumor cells, as
demonstrated by the up- or down-regulation of hundreds of genes involved in
metabolism, epithelial-to-mesenchymal transition, and cell survival.
• The half-life of ctDNA in CRC is roughly 2.5 h, allowing for a relatively rapid determination
of response to therapy.
• Both variant alleles and epigenetic markers (CpG island methylation) are relevant in the
detection and interpretation of ctDNA in CRC.

40. Screening
• CRC is unique among gastrointestinal malignancies in that a relatively
mature screening program exists for average-risk patients.
• Colonoscopic screening is effective yet invasive, associated with poor
compliance from patients, and is labor- and resource-intensive.
• Less invasive means of screening, such as fecal immunochemical tests and
DNA tests (e.g., Cologuard), have been developed and deployed clinically in
recent years.
• No liquid biopsy-based assays were recommended for CRC screening in
the 2018 Guideline Updates from the American Cancer Society, however,
as trial data are collected and as technology improves, such tests may
have a greater role in CRC screening in the future.
• Various platforms like CancerSEEK for ctDNA and FirstSight CTC based
assays, are under rapid evolution to increase sensitivity.

41. Staging
• Doubtful role of adjuent CT in stage 2.
• A meta-analysis of 11 studies demonstrated that ctDNA positivity at the
time of diagnosis is associated with inferior overall survival in colon
cancer, however, the strength of this association varies with timing and
technique.
• Exosomal mircoRNA-21 positivity was correlated with poor disease-free
and overall survival in stage II and III colon cancer.
• Post-resection positivity of ctDNA have proven role in deciding CT in stage
2.
• Ill defined role of CRS/HIPEC unless patient is symptomatic.
• A pilot study of 14 patients suggested that postoperative CTC-positive
patients with suspicion of peritoneal disease would benefit from
CRS/HIPEC in terms of overall survival.

42. Response to Therapy/Surveillance
• Liquid biopsy has demonstrated promise in detecting minimal residual
disease or recurrence during the surveillance of colon cancer.
• Standard post-treatment surveillance requires the use of non-specific
serum markers (CEA), endoscopy, and an abdominopelvic CT scan.
• A well-designed study of 230 patients determined that ctDNA with
either a KRAS, TP53, or APC mutation was sufficient evidence of
residual disease after curative-intent therapy.
• Supporting this, a recent prospective study of 125 stage I–III colon
cancer patients reported that, following definitive treatment, patients
with persistent ctDNA during surveillance had a 40 times higher risk
of recurrence that those with negative ctDNA .

43. • Most patients with CRC are treated with 5-fluorouracil-based
chemotherapy. Targeted agents such as panitumumab and cetuximab
are routinely added to these regimens if the tumor is RAS wild-type.
• However, most patients develop resistance to these agents.
• One well-designed study demonstrated that ctDNA-based liquid
biopsy was extremely sensitive (>85%) for primary resistance to HER2
inhibitors .
• A randomized controlled trial of 254 patients used ctDNA to identify
a subgroup of patients with anti-EGFR resistance that benefited
from alternative targeted therapy.

44. • Response to systemic therapy could also be monitored with CTC-based
liquid biopsy and persistent positivity following adjuvant mFOLFOX in stage
III colon cancer patients was a poor prognostic factor.
• Advances in regional therapy for rectal cancer in particular have made
distant recurrences the primary cause of mortality.
• A well-designed cohort study with 159 patients demonstrated that the
presence of ctDNA after resection is indicative of residual disease and a
high probability of recurrence. In these patients, treatment with adjuvant
systemic chemotherapy significantly improved OS .
• Another study of 130 patients with stage I–III CRC suggested that serial
ctDNA analysis with ultradeep sequencing could predict recurrence as early
as 30 days after resection, which is much sooner than the capabilities of
current radiographic, endoscopic, and CEA-based surveillance.

45. LIMITATIONS
• Technically demanding.
• Rapid EMT
• Benign and premalignant conditions of various cancers express
alterations in BRAF, RAS, EGFR, HER2, FGFR3, PIK3CA, TP53,
CDKN2A,and NF1/2 genes as compared to malignant tissues.
• Blood processing delays, storage, temperature, agitation of the
sample and shipment are also a major source of variability between
samples.

46. Summary
• As a screening tool, liquid biopsy has the potential to save the cost and morbidity
of invasive endoscopic screening procedures in diseases such as CRC and improve
early detection of diseases such as pancreatic adenocarcinoma.
• It appears ctDNA-based liquid biopsy is generally more sensitive for the early
detection of gastrointestinal cancers than CTC-based assays.
• While surgical techniques and outcomes continue to improve, procedures such as
esophagectomy, gastrectomy, and pancreaticoduodenectomy still come with a
high risk of morbidity and mortality.
• The ability to accurately stage patients with micrometastatic disease could save
patients, who would not benefit from upfront resection, the morbidity inherent
to such operations.
• Presence of CTCs may be an indicator of unresectability in certain
gastrointestinal tumors, whereas the presence of ctDNA in determining
resectability is less clear.

47. • Regarding the selection of systemic therapy regimens, ctDNA is generally
more predictive of response to therapy and more useful in identifying
actionable mutations for targeted therapy.
• Improvements in surveillance would benefit thousands of patients each
year with locally advanced disease who may be candidates for systemic or
extensive regional therapy, including cytoreductive surgery and
hyperthermic intraperitoneal chemotherapy (HIPEC).
• LB is a example of benefit derived from academic and industrial sector.
• One promising example is the Circulating Cell-free Genome Atlas study—a
collaboration between GRAIL, Inc. and major academic institutions.

49. Thank You