Post on 17-Feb-2021
transcript
Liquid Biopsy: Current & Potential Uses
in Management of Lung Cancer
Dr. Anurag Mehta
RGCI & RC
Scour Blood or Body fluids
CTC
Enumerate
* Response to treatment
* Prognosis
Harvest
* Genetic alterations
Cf-NAs
* Druggable mutation
* Monitor response
* Progression & Secondary driver(s)
* Tumor burden
Exosomes TEP
What is “Liquid Biopsy”?
Liquid biopsies are thought to capture the entire tumor genome – powerful tool1. Lebofsky, R., et al., Circulating tumor DNA as a non-invasive substitute to metastasis biopsy for tumor genotyping and personalized
medicine in a prospective trial across all tumor types. Mol Oncol, 2015. 9(4): p. 783-90.2. Esposito, A., et al., Monitoring tumor-derived cell-free DNA in patients with solid tumors: clinical perspectives and research
opportunities. Cancer Treat Rev, 214. 40(5): p. 648-55.
Most crucial step• Cartridge based methods• Salt precipitation methods• QI Amp cf NA – superior results
If Qiamp kit from Qiagen is to be used for DNA
extraction, prefer PAXgene Blood DNA Tube for
collection of blood.
Quality of DNA Qubit
K3 EDTASTRECKPAXgene
Analysis Technique Description Advantages Disadvantages
Conventional Real-Time qPCR
DNA is amplified by repeated cycles of DNA,primer, and probe thermal denaturation,annealing, and DNA polymerization with aheat-tolerant DNA polymerase.
Low cost, relatively easily implemented.Low sensitivity for cfDNA, only interrogatesDNA for areas between the primersequences.
Scorpion ARMS PCR
A bi-functional PCR primer covalently linked toa probe with closely associated fluorophore anda quencher. Amplification will only happen ifthe 3’ primer nucleotides match the targetsequence. During the PCR reaction, thefluorophore and quencher become separate,giving a detectable fluorescence.
Relatively low cost and high sensitivity.Only identifies the specific sequences theprobes are designed to detect.
PNA-LNA PCR
Composed of an uncharged polyamidebackbone with attached bases that hybridizes tossDNA with high affinity allowing probesenhanced binding to dilute cfDNA sequencesthan standard PCR DNA probes.
High sensitivity, lower cost.Only identifies the specific sequences theprobes are designed to detect.
NGS
DNA is fragmented into millions of shortsegments, ligated to DNA adaptor molecules,segregated on a solid matrix, and sequenced inparallel by labeled nucleotides addition, andbioinformatically aligned into a genomicsequence.
Can target specific sequences, the exome,entire genome, detect all sequencevariations, rearrangements, copy numberchanges, and often gene fusions.
High cost, complex to implement, analysiscomplex and requires bioinformaticsanalysis.
ddPCRPartitions cfDNA into thousands of parallelindividual PCR reactions. Signal detectionmeans the target sequence is present.
High sensitivity, can analyze multipleanalytes simultaneously.
Only identifies the specific sequences theprobes are designed to detect.
A summary of the more commonly used techniques to analyze cfDNA. The different techniques are
described, and the specific advantages and disadvantages, and relative costs of each technique are briefly
summarized.
Junaid Ansari, Jungmi W. Yun, Anvesh R. Kompelli et. al. The liquid biopsy in lung cancer Genes & Cancer, Vol. 7 (11-12), November 2016
DiagnosisPredictive biomarker
MonitorPrognosticate
Resistance(T790M)
MonitorPrognosticate
Resistance(C797S)
Current treatment path: EGFR as a prototype
Can liquid biopsy answer these needs?
Diagnosis
Tissue biopsy - standard sample
1. Histological typing
2. Cannot presuppose the type of genetic alteration
3. Clinical trials
But, if the tissue is not available
1. Moribund patient, Serious comorbidities
2. Inaccessible site
3. Biopsy done but tissue is still inadequate
Stephen B. Solomon et. al. Core Needle Lung Biopsy Specimens: Adequacy for EGFR and KRAS Mutational Analysis. AJR Am J Roentgenol. 2010 January ; 194(1): 266–269
Can liquid biopsy surrogate for
tissue biopsy
1. EGFR mutations are fairly specific for
lung cancer & have not been identified
in any other cancer type
2. A positive biomarker can profoundly
change therapy and survival
3. FDA approved kit is available for
testing EGFR sensitizing mutations.
NGS on ct- DNA- all mutations
Correlation of EGFR mutation status between matched tissue and ctDNA
Study Method Matched Samples Results
Assess Study(Reck et al JTO 2016)
Variable 1162
Concordance = 89%Sensitivity = 46%Specificity = 97%PPV = 78%NPV = 90%
Meta-analysis ( 27 Studies)(Qiu M, Wang J et.al. Cancer Epidemiol Biomarker Prev2015; 24: 206-212
Variable 3110Sensitivity- 62%Specificity- 96%DOR- 38.7
IFUM(Douillard et al, JTO 2014)
Therascreen 652
Concordance = 94.3%Sensitivity = 65.7%Specificity = 99.8% PPV = 99%NPV = 94%
FASTACT-2(Mok et al Clin Cancer Res 2015)
Cobas 238
Concordance = 88% Sensitivity = 75%Specificity = 96%PPV = 94%NPV = 85%
What is the “Clinical utility” of plasma positivity for Primary EGFR mutations:
Study Method Matched Samples Results
IFUM(Douillard et al. JTO 2014)
Therascreen 652
Concordance = 94.3%Sensitivity = 65.7%Specificity = 99.8% PPV = 99%NPV = 94%
FASTACT-2(Mok et al. Clin Cancer Res 2015)
Cobas 238
Concordance = 88% Sensitivity = 75%Specificity = 96%PPV = 94%NPV = 85%
1. Tissue and plasma = similar response rates (76.9% and 70%).
2. cf NA is an appropriate sample when tumor tissue is unavailable
Tissue and plasma = similar clinical outcomes
Plasma false positive or tissue false negative?
Mok et. Al.
• ASSESS study➢ 25 patients = false +ve rate 22%: Likely to false tissue negative
➢ Where more sensitive and identical methods were used for both samples- results generally improved
➢ Real world data suggests that ctDNA is a feasible sample for EGFR mutation analysis.
Wan et al, Journal of Thoracic Oncology Vol. 12 No. 9: 2017
Of the 28 Tx Negative & ctDNA positive
10 + by ddPCR02 + in liver Mets15 + by NGS1 patient did not have adequate tissue for NGS
Actually these were tissue false negative
64/180 0f those who had ctDNA negative were found positive by ddPCR
Diagnosis
1. Possible in ~65- 70% cases
2 Additional positive cases can be detected ~ 2-3%
3. Treatment response to Tissue or plasma detected sensitizing
mutation is same
Tissue biopsy EGFR -ve
Perform liquid biopsy
Question to ponder: cases with tissue biopsy result negative but clinically &
demographically promising cases for EGFR mutation be further tested by plasma
testing ( heterogeneity/ technical)
2016 statement Explanation
Recommendation: In some clinical settings in which tissue is limited and/or insufficient for molecular testing, physicians may use a cell -free plasma DNA (cfDNA) assay for EGFR.
New Recommendation Statement(Liquid biopsy has been permitted as a valid diagnostic tool for Primary EGFR mutation testing)
Biomarker Monitoring
1. Numerically quantifiable
2. Runs parallel to tumor load
3. Assayed serially
4. Reflects response to therapy / development of resistance
5. Sample is easy to obtain - non invasively
6. Assay is reliable and accurate
Longitudinal Mutation tracking through Liquid Biopsy ?
1. Early explorations have begun
2. Fastact 2 has shown value of Mutation tracking in prognostication.
Biomarker Monitoring
FASTACT 2: serial ctDNA at baseline, C3 and at PD
Clin Cancer Res; 21(14); 3196–203. 2015 AACR
Biomarker Monitoring
Positive versus negative plasma EGFR at C3
Clin Cancer Res; 21(14); 3196–203. 2015 AACR
pEGFR +ve at baseline (n=122)
pEGFR +ve at C3 (n=42)
pEGFR –ve at C3 (n=80)
ORR = 33%(14/42)
ORR = 66%(53/80)
OR=3.93 (95%CI 1.78 – 8.66)p=0.0007
Biomarker Monitoring
• Persistent plasma EGFR +ve is predictive of PFS and OS
Clin Cancer Res; 21(14); 3196–203. 2015 AACR
Biomarker Monitoring
Similar results from a retrospective Korean study
Onctarget.2016 Feb 9;7(6):6984-93.
JOINT IASLC - CHINESE SOCIETY FOR CLINICAL ONCOLOGY – CHINESE ALLIANCE AGAINST LUNG CANCER SESSION; JCES01.04Liquid Biopsy in Monitoring Dynamic Changes of Driver Genes in Advanced NSCLC
Qing Zhou
Phase III trial conducted from 2009 to 2014 comparing erlotinib with gefitinib in advanced NSCLC harbouring EGFR mutations in tumor (CTONG0901).
61 Cases MPFS: 11.1OS: 19.7
19 casesMPFS: 7.5OS: 16
Best response
Resistance
EGFR Acquired Resistance: Time of re-biopsy in acquired resistance in oncogene-driven NSCLC
Advanced NSCLC with
oncogene-driven cancer
EGFR mutation
RECIST response
Subsequent Disease
Progression
Clinical progression
1.Gandara DR et al. Clin Lung Cancer. 2014. 15:1; 1-6 2. Novello S et al. Annals of Oncology. 2016;27(suppl_5):v1-v27
RECIST progression Re-Biopsy
Mechanisms of Resistance post 1st line of EGFR TKIs
OsimertinibResistance
Various samples types may be used as a DNA source for mutation testing at disease progression
1. Diaz LA, et al. J Clin Oncol. 2014;32(6):579-586.
2. Pirker R, et al. J Thorac Oncol. 2010;5(10):1706-1713.
3. Oshita F, et al. Br J Cancer. 2006;95(8):1070-1075.
4. Van Eijk R, et al. PLoS One. 2011;6(3):e17791.
5. Kimura H, et al. Br J Cancer. 2006;95(10):1390-1395.
6. Huang WL, et al. Biomed Res Int. 2015;2015:1-11.
7. Wakelee H, et al. J Clin Oncol. 2016;34(15_suppl):9001.
8. Yang J C-H, et al. J Clin Oncol. 2016;34(15 suppl):9002.
CT-DNA
Resistance
Plasma ctDNA for T790M from AURA Phase I study (Oxnard et al JCO 2016)
216 tumour and plasma matched samples
BEAMing
Sensitivity 70%
Specificity 69% = false positive rate 31% (18 patients)
Tumour +ve RR 62%Plasma +ve RR 63%
1. 5PR2. 8SD3. 5PD
Resistance
Plasma ctDNA for T790M from AURA Phase II study (Jenkins et al
JTO 2017)
416 tumour and plasma matched samples
Cobas ARMS
27/416 = plasma false positive(6%)Or
? Tissue false negative
Resistance
Jenkins et al JTO 2017
Plasma false positive = tissue false negative
Resistance
1. The data supports the new paradigm of using plasma
genotyping as a screening test for T790M
1. Tissue biopsy would only be required with no T790M
detected in plasma
?intervention
Treatment 1st line TKI
Molecular Progression T790M
Clinical / Radiological Progression
Time
ctD
NA
Response Monitoring Resistance
?Intervention
Start Osimertinib
Start Osimertinib
The APPLE Trial
Remon et al, Clin Lung Cancer 2017
Biomarker Monitoring T790M
Progression T790M commence Osimeritnib
6 weeks
Time
ctD
NA
Response Monitoring Resistance
Treatment 1st line TKI
Median PFS 10.9m vs 5.5m
ORR 70% vs 35%
Thress et al ASCO abs 2017
Further follow up till end point mutation -C797S
40 % develop C797S resistance mutation always with a detectable T790MThress et al, Nat Med 2015
1. Combination TKI potential therapeutic option if in ‘trans’ orientation
2. NGS
Arulananda et al, JTO 2017
Current Treatment Strategy for Advanced ALK- or ROS1-Positive NSCLC
1L
Crizotinib
2L
2nd gen ALK TKIPD PD 3rd gen ALK TKI
3L
ALK
ROS1
1L
Crizotinib
2L
Next gen ROS1 TKIPD PD
1L
CrizotinibTherapy
2L
2nd generation ALK TKIPD PD
Tailoring Treatment After a 2nd-Generation ALK TKI
REBIOPSY
1L
2nd generation ALK TKI PD
Lorlatinib
Ceritinib or Lorlatinib
Crizotinib
Alectinib or Lorlatinib
G1202R
I1171
F1174
L1198Fcompound
ALK-based combo-OR-
Chemo
WT
G2032R41%
D2033N6%
S1986F6%
WT47%
Crizotinib Resistance is Often Mediated by Secondary ROS1 Resistance Mutations
Gainor et al., JCO precision oncol, Aug 14 2017
Liquid biopsy has a role.
1. NGS based or ARMS assay are possible to use.
2. Guardant 360- ALK and ROS1 mutations
3. Resolution Bioscience Seattle. Good panel
4. Oncomine panel: Competent panel
5. Oncotype Seq- genomic health mutations
Guardant 360
SNV/Indel Fusions CNV Suppressors
AKT1
ALK
BRAF
EGFR
FGFR3
HER2 (ERBB2)
KRAS
NRAS
MAP2K1 (MEK1)
MET
MET exon 14 Skipping
PIK3CA
RET
ROS1
ALK
FGFR3
NTRK1
RET
ROS1
ALK
EGFR
FGFR1
HER2 (ERBB2)
JAK2
MET
MYC
PD-L1 (CD274)
RICTOR
TP53
Assay DNA/RNA Gene Selected SNV hotspots CNVs Fusions Extras
Oncomine
Lung cfTNA
Assay
DNA &
RNA
ALK
BRAF
EGFR
ERBB2
KRAS
MAP2K1
MET
NRAS
PIK3CA
RET
ROS1
TP53
>150 hotspots including:
EGFR: T790M, C797S,
L858R, Exon 19 del
KRAS: G12X, G13X, Q61X
BRAF: V600E
ALK: Exon 21-25
PIK3CA: E545K, H1047R,
E542K
METALK, RET,
ROS1
MET exon 14
skipping
Oncomine
Lung cfDNA
Assay
DNA
ALK
BRAF
EGFR
ERBB2
KRAS
MAP2K1
MET
NRAS
PIK3CA
ROS1
TP53
>150 hotspots including:
EGFR: T790M, C797S,
L858R, Exon 19 del
KRAS: G12X, G13X, Q61X
BRAF: V600E
ALK: Exon 21-25
PIK3CA: E545K, H1047R,
E542K
--- --- ---
Conclusions
• ctDNA allows rapid biomarker assessment for identification of primary driver
• ? Role as adjunct to tissue biopsy – point to think
• It is possible to monitor response to 1st line therapy. Requires further
affirmations. Domain of thinkable.
• Persistent elevation of plasma EGFR is prognostic for shorter PFS and OS
• Strong clinical trial data supports detection of T790M in plasma
• Role in monitoring T790M –prognosticate the response & PFS. Data has started
emerging
• Institutions are using NGS based approaches to identify kinase domain
mutations in ALK & ROS to define further treatment on development of
acquired resistance