Pros and cons of

liquid biopsy:
Ready to replace
tissue?
2-Day Molecular Biologists
Symposium: Liquid biopsies

Federico Rojo
Enterprise | Interest
No disclosures.
Biological limitations for molecular testing:
Spatial and temporal (dynamic)
molecular heterogeneity in cancer
 Biological limitations for molecular testing:
Feasibility (and opportunity) for
re-biopsy in disseminated disease
• Waiting time
• Anatomical location is difficult for biopsy
• Sense of risk involved in re-biopsy
• Patient poor performance status
• Failure up to 20% of patients

Yoon, et al. Radiology 2012

Yu, et al. Clin Cancer Res 2013
Chouaid, et al. Lung Cancer 2014
Redig , et al. JAMA Oncol. 2016
Campo et al. J Clin Oncol 2016
 Circumvent biological limitations of molecular
testing by circulating biomarkers:
Liquid biopsy
Often tissue is not enough: Liquid biopsy
Main advantages: Tumor
Analysis of therapeutic targets and drug
• Rapid resistance–conferring gene mutations on
• Easy and accepted by patients peripheral blood samples:
• Understanding metastatic development
• Repeatable • Estimation of risk for metastatic relapse or
progression
• Avoid issues relater to tumor • Prediction of targeted therapy response
heterogeneity • Monitoring (minimal residual) disease
• Tracking secondary (‘acquired’) resistance
• Assessing intratumor heterogeneity
Main disadvantage:
• Sensitivity
Targets CTCs cfDNA cfRNA Platelets Exosomes
Selected viable tumor
Active secretion of
cells leaving actively Necrotic and apoptotic Necrotic and apoptotic Active incorporation of
Origins primary and/or tumor cells tumor cells exosomes
encapsulated particles
by tumor cells
metastasis
Fragmented genomes
Fragmented RNA
released from dying tumor Circulating
Definition Tumor cells
cells of primary and/or
released from dying Circulating platelets
encapsulated particles
tumor cells
metastasis
DNA, RNA (mRNA, RNA (mRNA, miRNA),
Analytes miRNA), protein
DNA RNA RNA (mRNA, miRNA)
protein
 Survey of ctDNA in human cancer
by tumor type and stage
Cases with detectable cDNA, Stage IV Cases with detectable cDNA,
localized vs metastatic

Bettegowda et al, Sci Tran Med Feb 2014

Liquid biopsy: Pros
Diagnostic applications of
blood-based molecular testing
Diagnostic applications of
blood-based molecular testing
Early Stage

Early Diagnosis
 Early stage: Early detection
Liquid biopsy direct detection of early
stage cancer
 CancerSEEK evaluates 8 proteins and mutations
in 1933 distinct genomic positions from 16 genes
 Sensitivity: 69-98% for the detection of five
cancer types (ovary, liver, stomach, pancreas, and
esophagus)
 Specificity: >99%

N=1005, non-metastatic ovary, liver,

stomach, pancreas, esophagus,
colorectum, lung and breast tumors
Diagnostic applications of
blood-based molecular testing
Early Stage

Prognosis
 Early stage: Prognosis
Prediction of relapse in early breast
cancer
ChemoNEAR study design

García-Murillas, I et al. Science Trans Med 2015

García-Murillas, I et al. SABCS 2016
Early stage: Prognosis
Prediction of relapse in early breast
cancer

García-Murillas, I et al. Science Trans Med 2015

García-Murillas, I et al. SABCS 2016
 Early stage: Prognosis
Role of liquid biopsy in predicting
post-operative recurrence in NSCLC
5 studies, 353 patients
DFS in surgical NSCLC

Ling, H et al. J Thorac Dis 2018

Diagnostic applications of
blood-based molecular testing
Early Stage

Residual
Disease
 Early stage: Minimal residual disease
ctDNA as prognostic marker for
colorectal cancer
RFS in stage II in early
colorectal cancer stage

N=250 stage II CRC

Post-surgery plasma Post-chemo plasma Clinical risk

Tie, J et al. Sci Trans Med 2016

Diagnostic applications of
blood-based molecular testing
Advanced Stage

Treatment selection
 Advanced stage: Treatment selection
ctDNA EGFR testing predicts TKI
benefit in NSCLC
Plasma EGFR mut+

Tissue EGFR mut+

Plasma EGFR mut-

Mok, T et al. Clin Cancer Res 2015 Maemondo, M et al. NEJM 2010
 Advanced stage: Treatment selection
Statement paper from IASLC: role of
liquid biopsy in advanced NSCLC

Rolfo, C et al. J Thorac Oncol 2018

Biological limitations for molecular testing:
Spatial and temporal (dynamic)
molecular heterogeneity in cancer

Ichihara, E & Lovely, CM. Cancer Discov 2015

 Advanced stage: Treatment selection
T790M plasma detection and clinical
response to osimertinib
BEAMing dPCR plasma analysis (AURA trial, n=271 patients) for T790M, Del19, & L858R
Tumor T790M+ PlasmaT790M+
62% ORR 63% ORR
9.7m PFS 9.7m PFS

• Plasma T790M positive predicts for a high ORR and a prolonged PFS, identical to that
predicted by a tumor T790M positive result (Cobas)
Oxnard, GR et al. J Clin Oncol 2016
 Advanced stage: Treatment selection
Statement paper from IASLC: role of
liquid biopsy in NSCLC progression
during treatment with TKI

Rolfo, C et al. J Thorac Oncol 2018

 Advanced stage: Treatment selection
ESMO guidelines: role of liquid
biopsy in NSCLC progression during
treatment with TKI

Novello, S et al. Ann Oncol 2016

 Advanced stage: Treatment selection
Tumor Mutational Burden in Blood and
Atezolizumab efficacy in 2L+ NSCLC

Gandara, DR et al. Nat Med 2018

Diagnostic applications of
blood-based molecular testing
Advanced Stage

Monitoring Response
 Advanced stage: Prognosis
ctDNA as prognostic marker for CRC
in advanced stage
PLACOL study

ctDNA concentration at baseline ctDNA variation during follow-up

Garlan, F et al. Clin Cancer Res 2017

 Advanced stage: Prediction of benefit
Early ctDNA dynamics and clonal selection
with palbociclib for breast cancer
PALOMA-3 phase III trial in metastatic luminal breast
cancer with progression to AI

Relative change in PIK3CA and ESR1 after 15 days of

treatment predicts PFS on palbociclib+fulvestrant

O’Leary, B et al. Nat Comm 2018

Diagnostic applications of
blood-based molecular testing
Advanced Stage
Clonal Evolution:
Acquired Resistance
 Advanced stage: Secondary mutations
ESR1 mutations in metastatic luminal
breast cancer after AI

Tot, W et al. Nat Genetics 2013

Robinson, DR et al. Nat Genetics 2013 Schiavon, G et al. Science Trans Med 2015
 Advanced stage: Secondary mutations
ESR1 mutations in metastatic luminal
breast cancer after AI
SoFEA
Fulvestrant 250mg +
Luminal breast cancer Placebo
Prior sensitivity to non-
steroidal aromatase Fulvestrant 250mg +
inhibitor Anastrozole
N=723 Exemestane

Fribbens, C et al. J Clin Oncol 2016

 Advanced stage: Secondary mutations
Emergence of secondary mutations in
mCRC after anti-EGFR mAb therapy

Bettegowda et al, Sci Tran Med Feb 2014

 Advanced stage: Mutation tracking
Monitoring ctDNA levels in NSCLC treated
with anti-EGFR TKI therapy

Chabon, JJ et al. Nat Communications 2016 Thomson, JC et al. Clin Cancer Res 2016
Liquid biopsy: Cons
 Liquid biopsy: Limitations
Driver mutations in non-cancerous
persons
Low frequency TP53 mutations were also found Clonal hematopoiesis with somatic
in 100% of peripheral blood samples from 17 mutations in 10% of persons older than
women with and without ovarian cancer (none 65 years of age and in 1% younger than
with hematologic disorder) 50 years

Krimmel, JD et al. Proc Natl Acad Sci 2016

Fernandez-Cuesta, L et al. EBioMedicine 2016 Genovese, G et al. N Engl J Med 2014
n future include sampling of ctDNA originating from stage IV lung cancers were diagnosed in
multiple micrometastatic tumours each too the LDCT group, suggesting that LDCT
Liquid biopsy: Limitations
e burden
ell-free
small to be apparent on CT imaging but
with a combined burden sufficient to result
generally enabled the identification of
lung cancers at an earlier stage than was
ology in detectable levels of ctDNA and/or a possible with chest radiography (resulting
ning Correlation between abundance of
z et al.41
shift in tumour cell death dynamics towards
a biology that is more conducive to ctDNA
in stage shifts), increasing the feasibility of
cure44. This finding suggests that, in order
py-
ctDNA and tumor stage
cer and
uivalent
release in the metastatic setting. to demonstrate superior sensitivity to chest

utant
um.
of ctDNA
also
early
42,43
.
ogical
through
with the
ons
PCR
he MAFs
umour
ur
ship
stimation
mean
0.008% Fig. 3 | The correlation between the abundance of ctDNA, tumour volume, tumour diameter, and
T stage. This figure demonstrates the correlation between estimated mean clonal mutant allele fre-
useAbbosh, C et al. Nat Rev 2018
quency (MAF) in circulating tumour DNA (ctDNA) isolated from plasma, tumour volume, and predicted
 Liquid biopsy: Limitations
Proportion of advanced cancer patients
with less than 2% of VAF in cfDNA

Pecuchet, N et al. Clin Chem 2016

 Liquid biopsy: Limitations
Correlation between abundance of
ctDNA and sensivity of the test
Liquid biopsy has lower sensitivity than tumor biopsy for rare variants:
- detection of alterations in peripheral fluids rather than the tumor itself (volume dilution)
- background of non-altered cfDNA from cellular sources other than tumor (other co-
morbidities such as sepsis, abundant cfDNA may be circulating that is derived from non-
cancerous cell compartments)

Abbosh, C et al. Nat Rev 2018

 Liquid biopsy: Limitations
Low abundance of ctDNA in certain
clinical situations
Very low release of tumor DNA into plasma in some patients with cancer:
- some lesions, e.g., brain metastases, may shed little to no ctDNA into the circulation
- particularly frequent in cancers of the central nervous system, potentially because the
blood-brain barrier blocks release of tumor DNA into the systemic circulation

García-Murillas, I et al. Science Trans Med 2015

García-Murillas, I et al. SABCS 2016 Chetan Bettegowda, C et al. Sci Transl Med 2014
 Advanced stage: Limitations
Clinical impact of ‘false positive’
T790M plasma
Plasma detection
EGFR T790M
Plasma from AURA trial sent for BEAMing
Paired tumor and plasma available for 216 patients

47 T790M+ 18 T790M+ in
16.5mos in tumor, plasma, not 4.3mos
mPFS not plasma 111 tumor mPFS
T790M+ in
tumor and
plasma
T790M+ in tumor: 9.2mos T790M+ in plasma:
62% RR, 10m PFS mPFS 63% RR, 10m PFS

40 patients T790M- tumor and plasma

2.8mos mPFS

Oxnard, J et al. J Clin Oncol 2016

 Liquid biopsy: Limitations
Standardization and clinical utility
 Efficiency and reproducibility of pre-analytical and analytical steps are critical to
allow reliable quantitation of variant ctDNA
 Lack of standardization: The best unit for quantifying DNA burden is not
established
 Definition of clinically-relevant threshold in each tumor type and clinical setting
 ctDNA responses do not always parallel imaging-based responses
 Clinical utility has not been established: no evidence that treatment on the basis
of the detection of ctDNA improves outcome. Prospective randomized trials have
failed to demonstrate survival benefits from screening for occult recurrences in
breast and ovary cancers, although there are data to suggest they are helpful in
colorectal and prostate cancers
Reinert, T et al. Gut 2016
Diehl, F et al. Nat Med 2008
Garcia-Saenz, JA et al. BMC Cancer 2017
Bardelli, A & Pantel, K. Cancer Cell 2017