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