Advance lung liquid biopsy research with Next Generation ... · Advance lung liquid biopsy research...

Advance lung liquid biopsy research with Next Generation Sequencing

Nicola Normanno

ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUMORI

FONDAZIONE G. Pascale – NAPOLI SC Biologia Cellulare e Bioterapie

CENTRO RICERCHE ONCOLOGICHE MERCOGLIANO (AV)

Laboratorio di Farmacogenomica

ESMO 2017- THERMO FISHER SCIENTIFIC SYMPOSIUM

Blood-based biomarkers in cancer

Crowley Nat Rev Cancer 2013

With the term liquid biopsy we refer to the possibility to perform tumor molecular profiling by using tumor-derived biomarkers that can be isolated from the peripheral blood (or any other body fluid) of cancer patients

BIOMARKERS DNA RNA miRNA Proteins

SOURCES Cell-free CTC Exosomes Platelets

Challenges in cfDNA analysis

The absolute levels are low: few nanongrams per ml of plasma

The circulating cell-free DNA (cfDNA) contains both tumor-derived DNA (ctDNA) and normal DNA originating form dividing cells (blood cells, GI tract, skin)

The ctDNA is only a fraction (<0.1% to 50%) of the cfDNA

Levels are usually correlated with tumor burden and are higher in advanced cancer

Highly fragmented, typically 50-200bp range (165bp peak)

Short half-life

Highly sensitive techniques are required for cfDNA analysis

Luke JNCI 2014

Applications of cfDNA analysis Potential for screening or earlier diagnosis

Wan NRC 2014

EGFR mutations in NSCLC predict response to EGFR TKI

Sharma Nat Rev Cancer 2007 Mok NEJM 2009

Liquid biopsy for mutational profiling of NSCLC

Challenges with research sample availability • In Europe, a surgical specimen or a core-biopsy is available in

<50% of NSCLC cases • In approximately 25% of the cases there is no material

available for molecular profiling or the material is inadequate

• Almost 40% of the cases have available cytology specimens or small biopsies for molecular characterization

• In cases with cytology or small biopsies the samples might not be representative of the disease

Normanno WCLC 2015

The ASSESS trial: sensitivity of plasma-based assays for EGFR sensitizing mutations

Performance of four different plasma assays (38 plasma samples from the AURA trial)

Thress Lung Cancer 2015

Secondary Mutation in Gefitinib/Erlotinib/Afatinib-Resistant NSCLC

• A substitution of methionine for threonine at position 790 (T790M) in the kinase domain in exon 20 increases the ATP affinity of the EGFR

• ~50% of patients with acquired resistance to EGFR TKIs develop the T790M mutation

• Among 155 lung tumour specimens only 1 had pre-existing T790M without prior EGFR-TKI exposure

1. Pao PLos Med 2005; Kobayashi New Engl. J. Med. 2005; Yun PNAS 2008 2. Socinski MA, et al. The oncologist. 2016.

The efficacy of targeted therapy depends on

TUMOR HETEROGENEITY

Burrell Nature 2013

Genetic and phenotypic variation observed between tumors of different tissue and cell types, as well as between individuals with the same tumor type

Subclonal diversity observed within a tumor (tumors are formed of different clones with different genetic and molecular features)

Identification of novel candidate driver genes in lung adenocarcinoma

TCGA Nature 2014

Resistance to anti-EGFR agents

Fenizia Future Oncology 2015

cfDNA analysis recapitulate heterogeneity of disease

• Tumor biopsies obtained from 3 different regions of SCC and cfDNA isolated from plasma

• cfDNA profiling reveals heterogeneous mutations that could be missed by standard single-pass tissue biopsies

Jamal-Hanjani Ann Oncol 2016

T790M Mutation Heterogeneity

Suda Sci Rep 2015

Heterogeneity of resistance mechanisms to EGFR TKIs

Chabon Nat Comm 2016

BEAMing, Droplet digital PCR (ddPCR)

Assays – sensitivity vs. breadth

Courtesy of K. Thress BREADTH

(No. bases assayed per sample)

RELATIVE SENSITIVITY

(Ability to see increasingly rare mutant alleles)

10% (1/10)

1% (1/100)

0.1% (1/1000)

0.01% (1/10,000)

0.001% (1/100,000)

>109 10 1 102 103 104 105 106 107 108

QPCR

Kilobases Megabases Gigabases (genome)

100% NGS – Unselected whole genome

NGS – Exome

NGS – Targeted panels

Allele specific- and emulsion-PCR methods are highly sensitive but detect only point mutations and short indels on very few loci NGS allows query of many more loci (up to whole genome), detects point mutations, indels, CNV and fusions but at low sensitivity

?

Philip C. Mack, PhD, UC Davis Comprehensive Cancer Center and California Northstate University

ctDNA utility in under-genotyped non-squam NSCLC

Tissue Genotyping Status

383 of 1288 (30%)

Biomarker Positive

879 (68%) Quantity Insufficient (QNS) or

Undergenotyped (UG)

ctDNA NGS Increased Biomarker Yield by 65%

ctDNA analysis

identified 252 additional actionable biomarkers

(19% of 1288) (29% of 879)

not previously detected in tissue QNS/UG cases

The cost of the Guardant Health panel is 5600$

Philip C. Mack, PhD, UC Davis Comprehensive Cancer Center and California Northstate University

5

Updated from Lanman et al. 2015 PLoS One

Mutant Allele Fraction (MAF) is typically very low P

erce

ntag

e of

var

iant

s

MAF (cell-free DNA %)

All variants detected

heterozygous SNPs

homozygous SNPs

somatic SNVs

MAF (cell-free DNA %)

Per

cent

age

of v

aria

nts

Reported somatic variants

Min: 0.03% 25th: 0.19% Median: 0.44% 75th: 2.02% Max: 97.62% Mean: 3.27%

Differentiation of Somatic vs. Germline Variants

Half of Variants reported occur below 0.44% MAF

NGS for Liquid Biopsies: Sensitivity vs Specificity

Newman Nat Biotechnol 2016

Duplex NGS (complementary strand

variant matching)

Molecular barcoding

“Data polishing”

For research use only. Not for use in diagnostic procedures

Safe-Sequencing System “Safe-SeqS”

Kinde PNAS 2011 Bettegowda Sci Transl Med 2014

• Assignment of a unique identifier (UID) to each DNA template molecule

• Amplification of each uniquely tagged template molecule to create UID families

• Redundant sequencing of the amplification products

• PCR fragments with the same UID are considered mutant (“supermutants”) only if ≥95% of them contain the identical mutation

Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq)

• Panel targeting recurrently mutated regions in NSCLC

• Diverse mutation types: point mutations (SNVs), indels, copy number variants (CNVs), and rearrangements

• Optimized library preparation for cfDNA and low inputs (ng)

• Custom bioinformatics for variant detection <0.1% (≈0.02%)

• Ultra-deep sequencing: >10,000X

Newman Nat Med 2014

Barcoding and Polishing Eliminate Distinct Populations of Background Errors of CAPP-Seq

Newman Nat Biotechnol 2016

Integrated digital error suppression (iDES)-enhanced CAPP-Seq detected tumor-derived DNA down to 0.0025% (2.5 in 105 molecules)

24 For Research Use Only. Not for use in diagnostic procedures.

Oncomine cfDNA assays

Thermo Fisher All Rights Reserved .


Oncomine Lung cfDNA Assay

Content developed in collaboration with the OncoNetwork consortia Assays designed to detect

• Primary tumor drivers • Resistance mutations

169 hotspot mutations like EGFR: T790M, L858R, Exon19 del, C797S KRAS: G12X, G13X, Q61X ALK: 1151Tins, L1152R, C1156Y BRAF: V600E Detect rare variants present down to 0.1% with

Mean Sensitivity – 90% Mean Specificity – 98%

ALK BRAF EGFR ERBB2 KRAS MAP2K1

MET NRAS PIK3CA ROS1 TP53

Gene List

Thermo Fisher All Rights Reserved .


Core Technology and Variant Analysis Method

cfDNA molecule with tumor variant Wild type cfDNA molecule

Variant

Gene Specific Primer Molecular tags

Assay

Analysis

Amplification, tagging and sequencing

Variant calling


Oncomine Lung cfDNA Assay - R&D reproducibility results

Test site Users Sample Sensitivity Specificity

sensitivity each usr average ( n=4)

average ( n=16) specificity usr average

( n=4) all average

( n=16)

Site A usr1

0.5%MM

100

100

100

98

98

98

100 99 100 97 100 100

Site B

usr2

100

100

98

98 100 97 100 98 100 97

usr3

100

100

99

99 100 98 100 99 100 98

usr4

100

100

99

99 100 100 100 99 100 97

Site A usr1

0.1% MM

95

90

90

99

99

98

85 98 88 99 93 99

Site B

usr2

90

92

99

98 88 99 95 99 95 98

usr3

93

93

99

98 90 97 95 99 95 99

usr4

85

85

99

98 98 98 88 99 70 97


Sample Repeat

Sensitivity Specificity

single point in run average ( n=4)

all runs average ( n=12) single point in run average

( n=4) all runs average

( n=12)

0.5%MM

run1

100.0%

100.0%

100.0%

99.2%

99.6%

99.6%

100.0% 100.0% 100.0% 99.2% 100.0% 100.0%

run2

100.0%

100.0%

100.0%

99.4% 100.0% 99.2% 100.0% 99.2% 100.0% 99.2%

run3

100.0%

100.0%

100.0%

99.6% 100.0% 100.0% 100.0% 99.2% 100.0% 99.2%

0.1%MM

run1

97.5%

97.5%

98.3%

100.0%

99.8%

99.8%

97.5% 100.0% 97.5% 99.2% 97.5% 100.0%

run2

100.0%

99.4%

99.2%

99.8% 100.0% 100.0% 97.5% 100.0%

100.0% 100.0%

run3

100.0%

98.1%

100.0%

99.8% 95.0% 99.2% 97.5% 100.0%

100.0% 100.0%

Oncomine Lung cfDNA Assay – R&D repeatability results


OncoNetwork Consortia Prof. Harriet Feilotter

Dr Paul Park Queen's University, Ontario

Canada

Dr. Jose Costa IPATIMUP, Medical Faculty of

Porto. Portugal

Marjolijn J.L. Ligtenberg Radboud University Nijmegen Medical Centre, Netherlands

Dr. Nicola Normanno Centro Ricerche Oncologiche

Mercogliano, Italy

Prof. Orla Sheils St James's Hospital Dublin,

Ireland

Prof. Ian Cree UHCW

United Kingdom

Prof. Pierre Laurent Puig Université Paris Descartes,

HEGP Paris, France Prof. Aldo Scarpa ARC-NET University of

Verona Italy

Dr. Henriette Kurth VIOLLIER AG Basel ,

Switzerland

Prof. Kazuto Nishio Faculty of Medicine, Kinki University Osaka, Japan

Cecily P. Vaughn ARUP- Institute for Clinical and

Experimental Pathology Utah, USA

Dr. Michael Hummel Institute of Pathology Charité

Berlin, Germany


• Each lab performed two runs using Oncomine Lung cfDNA Assay on Horizon Multiplex cfDNA Reference Standard Set.

• Each run included 5%, 1% and 0.1% and WT samples.

Multicenter study- repeatability and reproducibility

• Horizon controls at different percentages and WT in a duplicate run Repeatability

• Same experiment in 11 laboratories Reproducibility


Mutations Screened In the Multicenter Study

Additional WT level variants in cell line detected by the cfDNA assays include:

EGFR p.G719S; PIK3CA p.H1047R; MAP2K1 p.Q56P; BRAF p.V600E These variants should be expected at approximately 20-40% AF

Multiplex I cfDNA Reference Standard Set (5%, 1%, 0,1%, 0%)


Lab Sensitivity Specificity NPV PPV

ARCNET 89,58% 99,53% 99,61% 87,76%

CROM 95,83% 100,00% 99,84% 100,00%

IPATIMUP 95,83% 100,00% 99,84% 100,00%

Radboud 89,58% 99,84% 99,61% 95,56%

Queens 97,92% 99,92% 99,92% 97,92%

St James 95,83% 100,00% 99,84% 100,00%

UHCW 95,83% 99,76% 99,84% 93,88%

Charite 95,83% 99,76% 99,84% 93,88%

Viollier* 97,50% 99,82% 99,91% 95,12%

HEGP 93,75% 99,69% 99,76% 91,84%

Kindai* 95,83% 99,69% 99,84% 92,00%

Total 94,81% 99,82% 99,80% 95,93%

Sensitivity and Specificity at overall results


Lab Sensitivity Specificity NPV PPV

ARCNET 68,75% 100,00% 98,43% 100,00%

CROM 87,50% 100,00% 99,37% 100,00%

IPATIMUP 87,50% 100,00% 99,37% 100,00%

Radboud 68,75% 99,68% 98,43% 91,67%

Queens 93,75% 100,00% 99,68% 100,00%

St James 87,50% 100,00% 99,37% 100,00%

UHCW 87,50% 100,00% 99,37% 100,00%

Charité 87,50% 100,00% 99,37% 100,00%

Viollier 87,50% 100,00% 99,37% 100,00%

HEGP 81,25% 99,36% 99,05% 86,67%

Kindai 87,50% 99,68% 99,37% 93,33%

Total 83,93% 99,88% 99,19% 99,13%

Sensitivity and Specificity at 0.1% detection limit


Sensitivity and specificity – OncoNetwork

Allele Frequency Sensitivity Specificity

0.1%-5% 94.81% 99.82%

0.1% 83.93% 99.88%

Data collected from 11 laboratories: UHCW, CROM, ARCNET, IPATIMUP, Radboud University, Queen’s University,

St James Hospital, Violler, HEGP, Kinday University, Charite’ Hospital

Horizon - Multiplex I cfDNA Reference Standard Set


Reproducibility at 5% limit detection

EGFR E746_A750del ELREA

PIK3CA E545K

KRAS G12D

n=22 n=22

n=21


Reproducibility at 1% allele frequenci


PIK3CA E545K

KRAS G12D

n=22

n=22

n=21



PIK3CA E545K

KRAS G12D

n=20

n=20

n=15

Reproducibility at 0.1% limit detection


0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

Alle

le F

requ

ency

5% ARCNET

CROM

IPATIMUP

St Radboud

Queens

St James

UHCW

Charite

Viollier

HEGP

Kindai

EGFR – T790M at 5%, 1% and 0,1% allele frequency

0.00

0.20

0.40

0.60

0.80

1.00

1.20

1.40

1.60

Alle

le fr

eque

ncy

1% ARCNET

CROM

IPATIMUP

St Radboud

Queens

St James

UHCW

Charite

Viollier

HEGP

Kindai

0.00

0.05

0.10

0.15

0.20

0.25

0.30

Alle

le F

requ

ency

0,1% ARCNET

CROM

IPATIMUP

St Radboud

Queens

St James

UHCW

Charite

Viollier

HEGP

Kindai


Oncomine™ cfDNA Assays – Content

35 amplicon panel for Lung

Covering key hotspot mutations in 11 genes

169 Hotspot SNVs & indels

Oncomine™ Lung cfDNA Assay



48 amplicon panel for colon (gastrointestinal) cancer type(s)



Oncomine™ Colon cfDNA Assay

AKT1 BRAF CTNNB1 EGFR ERBB2 FBXW7 GNAS

KRAS MAP2K1 NRAS PIK3CA SMAD4 TP53 APC

26 amplicon panel for Breast



Oncomine™ Breast cfDNA Assay

AKT1 EGFR ERBB2 ERBB3 ESR1

FBXW7 KRAS PIK3CA SF3B1 TP53

Thermo Fisher All Rights Reserved

40

Coming Soon – Enhanced Content to Lung & Breast cfDNA Assays

58 amplicon + 49 Fusion Assays + 3 MET Exon Skipping Assays for new Lung Assay

Covering: • Key hotspot mutations in 11 genes • Fusions – ALK, RET, ROS1 • CNV – MET • MET exon 14 skipping

SNV LOD down to 0.1% with 20 ng input Same Sensitivity & Specificity for SNVs Single library from both DNA & RNA

76 amplicon panel for new Breast Assay

Covering: • Key hotspot mutations in 10 genes • CNVs – CCND1, ERBB2, FGFR1 • More complete coverage of TP53 • de novo variant calling across TP53

SNV LOD down to 0.1% with 20 ng input Same Sensitivity & Specificity for SNVs Single library to detect SNVs and CNVs

• Designed to detect primary tumor drivers and resistance mutations • From a single tube of blood

New Oncomine™ Lung cfTNA Assay



New Oncomine™ Breast cfDNA Assay

AKT1 EGFR ERBB2 ERBB3 ESR1

FBXW7 KRAS PIK3CA SF3B1 TP53

Thermo Fisher All Rights Reserved For Research Use Only. Not for use in diagnostic procedures.

41

Lung enhanced panel – Fusion and MET skipping (+) controls

Assay ID Type 1% rep 1 1% rep 2 0.5% rep 1 0.5% rep 2 MET-MET.M13M15 RNAExonVariant 740 787 382 382

MET.E11E12.WT RNAExonVariant 628 640 297 323 MET.E6E7.WT RNAExonVariant 893 986 375 477

TBP.ENCTRL.E3E4 ProcControl 286 334 208 272 HMBS.ENCTRL.E8E9 ProcControl 275 383 161 183

Assay ID Type 1% rep 2 1% rep 1 0.5% rep 2 0.5% rep 1 0.25% rep 2 0.25% rep 1 CCDC6-RET.C1R12.COSF1271 Fusion 52 40 37 16 16 20

EML4-ALK.E6A20 Fusion 9 19 5 0 7 3 MET-MET.M13M15 RNAExonVariant 0 1 0 0 0 0

MET.E11E12.WT RNAExonVariant 704 669 370 368 147 212 MET.E6E7.WT RNAExonVariant 1110 1113 633 567 226 306

SLC34A2-ROS1.S4R32.COSF1196 Fusion 209 194 83 80 31 51

TBP.ENCTRL.E3E4 ProcControl 273 208 197 198 112 133 HMBS.ENCTRL.E8E9 ProcControl 231 284 161 191 133 157

Tri-Fusion positive samples

MET exon skipping positive samples

*Tri-Fusion or MET exon skipping positive RNA was spiked into cell free total nucleic acid at the indicated frequencies. Values in table represent molecular

coverage for each amplicon indicated

42

Expanded Variant Type Detection with cfDNA Lung Panel

MET CNV + Fusion/MET Exon 14 Skipping at 0.5% titration

CNV Analysis

Fusion/MET exon Skipping Analysis

Note: Expected MET CNV ratios (w/significant p-values) confirmed. Expected fusion/exon skipping/control targets confirmed

For Research Use Only. Not for use in diagnostic procedures.

43

Results from 4 Retrospective Samples

Sample Tissue at diagnosis Plasma at progression after TKI Detection Method

9308 Not available p.Glu746_Ala750del Therascreen/ ddPCR

7947b p.Glu746_Ala750del p.Glu746_Ala750del Therascreen/ ddPCR

7679b p.L858R L858R and T790M Therascreen/ ddPCR

8151 p.L858R L858R and T790M Therascreen/ ddPCR

Oncomine Lung Cell-Free Total Nucleic Acid Assay results*

9308

7679b

7947b

8151

For Research Use Only. Not for use in diagnostic procedures.

Liquid biopsy can represent temporal and spatial heterogeneity in cancer progression

Burrell & Swanton Mol Oncol 2014

Normanno J Cell Biochem 2013

The future of biomarker testing in cancer

Laboratory of Pharmacogenomics Anna Maria Rachiglio Matilde Lambiase Francesca Fenizia Raffaella Pasquale Claudia Esposito Cristin Roma Laura Forgione Rino E. Abate Alessandra Sacco Alessia Iannacone Francesca Bergantino

Cell Biology and Biotherapy Unit Antonella De Luca Amelia D’Alessio Monica R. Maiello Marianna Gallo Daniela Frezzetti Nicoletta Chicchinelli Michele Grassi

CENTRO RICERCHE ONCOLOGICHE MERCOGLIANO (AV)

Laboratorio di Farmacogenomica

ISTITUTO NAZIONALE PER LO STUDIO E LA CURA DEI TUMORI

FONDAZIONE G. Pascale – NAPOLI

Surgical Pathology Unit Gerardo Botti Fabiana Tatangelo

External Collaborators Carmine Pinto, IRCCS Reggio Emilia Domenico Galetta, IRCCS, Istituto Tumori "Giovanni Paolo II ", Bari Bruno Daniele, Ospedale G. Rummo, Benevento Francesco Ferraù, Ospedale San Vincenzo, Taormina Vienna Ludovini e Lucio Crinò, Ospedale S. Maria della Misericordia, Perugia

Dept. Thoracic Oncology Gaetano Rocco Alessandro Morabito

Clinical Trial Unit Francesco Perrone Maria C. Piccirillo

Acknowledgments

OncoNetwork Consortium

Jose Costa - IPATIMUP, Medical Faculty of Porto

Orla Sheils - St James's Hospital, Dublin

Pierre Laurent Puig - Université Paris Descartes, Paris

Michael Hummel - Institute of Pathology, Charité, Berlin

Aldo Scarpa - ARC-NET University of Verona

Kazuto Nishio - Faculty of Medicine, Kinday University, Osaka

Henriette Kurth - VIOLLIER AG, Basel

Marjolijn Ligtenberg - Radboud University, Njimegen

Ian Cree - UHCW, Warwick

Harriet Feilotter - Queen's University, Ontario

Chris Allen

Kelli Bramlett

Dima Brinza

Tom Bittick

Andrea Luchetti

Rosella Petraroli

Thermo Fisher Scientific and its affiliates are not endorsing, recommending, or promoting any use or application of Thermo Fisher Scientific products presented by third parties during this seminar. Information and materials presented or provided by third parties are provided as-is and without warranty of any kind, including regarding intellectual property rights and reported results. Parties presenting images, text and material represent they have the rights to do so.

Advance lung liquid biopsy research with Next Generation ... · Advance lung liquid biopsy research...

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