lung cancer liquid biopsy exosomes CTC (circulating tumor cells) MiRNA ctDNA extracellular vesicles cellsearch NSCLC

1. Lung Cancer In Vein
Prof. Waheed Shouman
Chest Department
Zagazig University
Egypt

2. Rule Of Thumb:
Tissue
Is the Issue

4. A typical primary tumor contains billions of cells with
genetic mutations that drive them to grow, divide,
and invade the surrounding tissues.
Some of these cells slough off the edges of the tumor
mass and are swept away by the bloodstream or
lymphatic system

5. CTCs in blood was first reported by Thomas Ashworth
(1869), In a patient with metastatic tumors of
anterior wall of the chest and abdomen. He separated
circulating tumor cells (obtained from the saphenous
vein)

6. Circulating Tumor Cells (CTC):
Isolated single cancer cells in the blood or lymphatic
fluids
Disseminated Tumor Cells (DTC):
Isolated single or clusters of cancer cells in lymph
nodes or bone marrow
Circulating Tumor Microemboli (CTM):
Clusters or aggregates of CTCs

8. The term liquid biopsy was originally
introduced to define circulating tumor cells (CTCs).
Currently it is also used for circulating tumor DNA
(ctDNA) as well as for exosomes

11. The bloodstream is highly unfavorable to the survival
of tumor cells owing to physical shear forces, immune
surveillance due to the presence of immune cells,
apoptosis and anoikis
Half life of CTC is 1-2.4 hours

12. The size of CTCs can vary from 10 to more than 20 μm
in diameter. This is a large size compared to other
blood cells and can be used to separate them from
hematological cells
It has been demonstrated that among CTCs the size
can be variable from one cancer type to another

13. CTCs:
1. Role in screening and diagnosis of lung cancer
2. Prognostic in early cancer stages
3. Prognostic in advanced cancer stages
4. Prognostic in metastatic lung cancer
5. Predictor of drug resistance
6. Role in MRD

14. Minimal residual disease (MRD):
It is the presence of malignant cells in distant organs
that are undetectable by routine imaging and
laboratory tests used for tumor staging after curative
surgery of the primary tumor.
CTCs are considered surrogates of MRD and
potentially metastasis-initiating cells

16. Detection of CTC

17. A platform for capturing CTC should have the
following characteristics:
1. High sensitivity: detection of every single
circulating cancer cell
2. High specificity: no false positives in healthy
volunteers.
3. High purity: ability to isolate CTC with no
contaminating normal cells
4. Preservation of CTC viability, morphology, protein
and nucleic acids
5. Economical: low cost, high-throughput, minimal
operator time required.
6. Reproducible: no intra-operator or inter-operator
variability

23. FDA-approved method for detection of CTCs (2007):
CellSearch® System, separates the cells with magnetic
beads coated with anti-epithelial cell adhesion
molecule (EpCAM) antibody followed by flow
cytometry of cells captured with anti-cytokeratin
fluorescence
(In solid cancers, not Lung Cancer)
The only one approved

24. CellSearch captures CTCs by labeling them with
ferrofluid nanoparticles functionalized with anti-
EpCAM antibodies. These cells are then isolated via a
magnetic field
CellSearch ® system is considered the current “gold
standard” for clinical CTC enumeration, but its
sensitivity and capacity for downstream molecular
analysis is limited

25. Epithelial immunospot (EPISPOT)
It is an antibody-based method for quantification of live CTCs
by detection of CTCs which are capable of secreting proteins
such as cathepsin D, MUC1 and CK19 . Briefly, CTCs in blood
samples are enriched by negative selection using anti-CD45
immunomagnetic beads. The isolated CTCs are then cultured
in tissue culture plates pre-coated with antibodies which
capture the secreted protein of interest. The secreted
proteins are therefore immobilized around the CTC which
produce them and forms a ‘spot’ around it. After the
incubation period, the cells are removed and secreted protein
‘spots’ are detected by immunological techniques and
counted, where each spot corresponds to 1 live CTC

26. The HD-CTC fluid biopsy assay:
Identifies CTCs without using surface protein-based
enrichment, instead using sophisticated imaging and
software algorithms to identify and present CTCs as high
definition (HD) diagnostic pathology quality images.
CTC Chip (the iChip):
Also does not rely on the presence of EpCAM or other
known tumor antigens on the cell surface of CTCs, taking
the approach of combining microfluidics with sequential
negative and positive enrichment methods on a
herringbone microchip.
Both assays (HD-CTC and iChip) have improved sensitivity
over the CellSearch ® system and can provide CTCs in an
ideal format for downstream

27. Microfluidic :
“the science and technology of systems that process
or manipulate small (10−9 to 10−18 L) amounts of fluids
using channels with dimensions of tens to hundreds
of micrometers”

29. Culture of CTCs:
Culturing CTCs in vitro/in vivo could facilitate drug
development.
Owing to the rarity of CTCs, it is necessary to culture
CTCs and establish cell lines. Most isolated CTCs are
not capable of dividing in situ.
However, viable CTCs have been shown to proliferate
in cell culture in response to stem cell growth factors
such as EGF and FGF2

31. Challenges for CTCs analysis:
Technical
standard technique, rarity and capacity for single cell study
Statistical
specificity,
sensitivity,
reproducibility
Biological
(immune surveillance, EMT, molecular and cellular
heterogeneity, and relation to cancer stem cells, CTCs
clustering, rarity and short half life )

32. CTCs Rarity of Occurrence
CTCs are estimated to be present as few as 1 in every
109 blood cells

33. CTC Heterogeneity
The rarity of CTCs hinders the discovery of unique CTC
biomarkers for diagnosis and therapy. A limited
number of markers are used for the detection of CTCs,
and a combination of unique antigens must be
identified for the detection CTCs.
Most CTC identification uses epithelial markers
(EpCAM and cytokeratins) or organ-specific markers

35. Limitations of CTC in biomarker studies
As some patients do not develop hematogenous
metastases, it is possible that a subgroup of patients
will never have CTC despite suffering rapidly fatal
disease
Not all patients with no detectable CTC or a low CTC
count have a good prognosis
Heterogeneity is well described in epithelial
malignancies. It is possible that as tumors progress,
they retain initiating or early events that are
uniformly present in all metastases while later
changes are more likely to be heterogeneous

36. Epithelial mesenchymal transformation (EMT/MET)
EMT has a major role in the initial step of the
metastatic cascade, where, through EMT, some tumor
cells acquire the ability to invade the basement
membrane and surrounding stroma and then
intravasate

37. Epithelial mesenchymal transformation (EMT/MET)
Inducers: (of many and more waiting)
HGF, FGF-1, EGF, TGF-α, plus other inflammatory
cytokines and hypoxia
CTCs with low or absent expression of EpCAM are
easily missed. Thus, tumor cells that have undergone
EMT, and which are probably the most aggressive, are
not detectable with CellSearch® technology

40. Extracellular Vesicles (EVs)
They are known to transport messages from donor
cells to recipient cells
EVs types are based primarily on size (and secondarily
on protein content):
1. Apoptotic bodies, range from 1 to 5 μm
2. Microvesicles (MVs) have a size range from 1 μm to
100 nm
3. Exosomes, have a size range of 30–100 nm

42. Roles of EVs in cancer progression
Carriers of macromolecules for intercellular
communication between cancer cells
EVs facilitate intercellular communication between
cancer cells and microenvironmental cells to promote
tumor progression

45. EVs in cancer management:
1. Biomarker for cancer diagnosis (contents and
amount)
2. Target EVs in cancer
3. EV-based cancer vaccine
4. EV-based drug delivery system

47. Isolation of EVs:
Differential centrifugation (the most common)
Density gradient centrifugation
Ultrafiltration
Polymer-based precipitation
Size-exclusion chromatography (SEC)

48. Exosomes
Exosomes are vesicles actively released by all cells,
which carry RNA, DNA, and proteins and function as
intercellular messengers
Exosomes are extremely abundant in all biological
fluids and can be isolated from serum, plasma, saliva,
urine, and cerebrospinal fluid
They are stable carriers of genetic material and
proteins from their cell of origin

50. Contents of Exosomes:
DNA (including mcDNA) (inconsistent data)
mRNA
MiRNA
Proteins
Lipids

54. Circulating Tumor DNA (ctDNA)
The presence of circulating cell-free DNA (cfDNA) in the blood
of healthy individuals was firstly reported by Mandel and
Metais in 1948
The high proliferation of tumors leads to a higher degree of
necrosis, with increased release of tumor-related cfDNA,
named circulating tumor DNA (ctDNA)
Discriminating ctDNA from normal cfDNA is possible since
tumor DNA is characterized by the presence of mutations
These somatic mutations are present only in the genomes of
cancer cells or precancerous cells

55. Despite its short half-life, the absolute abundance of
cfDNA is significantly higher than CTCs, corresponding
to several hundreds to thousands of genome
equivalents per ml of blood (compared to <10 CTCs
per ml of blood).

56. ctDNA application in lung cancer
1. Early diagnosis
2. Detection of tumor heterogeneity
3. Detection of emerging molecular and drug
resistance
4. Monitoring tumor burden
5. Detection of MRD

57. The ability to measure ctDNA holds great promise
for diagnosing and monitoring cancer, and in
particular the ability to perform serial blood sampling
would afford an easy and rapid method of tracking
disease
Real-time PCR has been used to measure total levels
of cfDNA, and can provide relative quantification of
the amount of ctDNA using probes specific for
mutations or other alterations

58. There is limited sensitivity of real-time PCR for
quantification of ctDNA, in cancer patients, as
mutations are present at low allelic frequencies
Among newer technologies, digital PCR and next
generation sequencing (NGS) are among the most
common techniques employed

59. FDA-Approved Tests
Circulating tumor DNA (ctDNA) test
FDA approved the cobas® EGFR Mutation Test v2 in
2016 for ctDNA. It is currently the only one approved for
ctDNA testing
It is used in NSCLC to look for mutations in one specific gene –
EGFR –and indicates whether drugs that target these
mutations, such as erlotinib, could work against the cancer
It has limitations, as it may produce a false negative result.
The FDA recommends a tissue biopsy if the liquid biopsy is
negative (i.e., if it does not detect an EGFR mutation).

61. MicroRNAs:
MicroRNAs (miRNAs) are small noncoding, naturally
occurring RNA molecules that posttranscriptionally
modulate gene expression and determine cell fate by
regulating multiple gene products and cellular
pathways

64. Tumor educated platelets (TEPs)
Platelets interact with tumor cells and affect tumor
growth, invasion and establishment of distant
metastasis
The interaction with tumor cells relies on the
sequestration of tumor-associated biomolecules by
platelets. Indeed, platelets can directly ingest
circulating mRNA
TEP mRNA profiles also distinguished healthy donors
and patients with specific types of cancer

65. Highlights of liquid
biopsy in lung cancer

66. Liquid biopsy in NSCLC

69. In lung cancer, patients with more than 5 CTCs/7.5 ml
blood has lower progression free survival (PFS) and
overall survival (OS) compared to those with less than
5 CTCs (2.7 vs. 7 months)

70. A powerful example of the application of CTC
genotyping to targeted cancer therapy is provided by
the subset of NSCLC that is responsive to selective
kinase inhibitors of the EGFR.
Only 10% of NSCLC have somatic activating mutations
in EGFR

71. In NSCLC patients the total count of CTCs before
chemotherapy initiation is associated with staging
(higher detectable number of cells in stage IV
patients)
The progression-free survival (PFS) and overall
survival (OS) seem to be correlated to CTC count
The reduction of CTC count higher than 89% after one
chemotherapy cycle improves prognostic accuracy
and is associated with lower risk of death.
Furthermore CTC count is strongly associated with the
number of metastatic sites

72. CTCs were detected by ISET (Isolation by SizE of
Tumor cells) in 3% of patients with COPD (5 of 168
patients). The annual surveillance of the CTC-positive
COPD patients by CT-scan screening detected lung
nodules 1 to 4 years after CTC detection, leading to
prompt surgical resection and histopathologic
diagnosis of early-stage lung cancer, whereas, no CTCs
were detected in the control smoking and
nonsmoking healthy individuals

78. Rule Of Thumb:
Tissue
Is the Issue.

79. Is Tissue the Issue?
There May be No Rules
Of Thumb

80. Thank You