Utility of liquid biopsies

(1)

EQA Program Naples, IT

Sidney A. Scudder, MD Director, Clinical Science 13 May, 2017

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Utility of liquid biopsies

(2)

Agenda

cobas® EGFR Mutation Test v2 Ring Trial

SQI – Semi Quantitative Index Inter-laboratory comparison LSR

Avenio

(3)

cobas

^®

EGFR Mutation Test v2*

MUTATIONS

42

DETECTED

AMPLIFICATION

MMX TARGET

MMX1 EX19Del; S768I; EX28/IC MMX2 L858R; T790M; EX28/IC MMX3 v.2 L861Q; G719A/C/S; EX20Ins;

EX28/IC

FDA approved as a Companion Diagnostic in tissue and plasma for ex19del, L858R and

T790M

MN 7248563190

(4)

RING Trial

cobas® EGFR Test v2 Plasma

Evaluate the performance of the cobas^® EGFR Mutation Test v2

• contrived plasma samples

• cobas^® 4800 system

• EGFR exon 19 deletion, p.S7681, p.T790M, exon 20 insertion, p.L858R, and p.L861Q mutations.

• ^Objectives

^:

• Identification of the target mutation

• Demonstrate linearity of copy numbers(or percentage mutation) to the SQI (Semi-Quantitative Index) value

• Demonstrate the concordance of SQI values across multiple testing sites

(5)

• Co-principal investigators – Prof. Dr. E Dequeker – Prof. Dr. N Normanno – Prof. Dr. H van Krieken

• Study coordinator – C. Keppens

RING Trial

cobas® EGFR Test v2 Plasma

(6)

Cell-Line DNA

Combination 1 Cell-Line DNA

Combination 2 Cell-Line DNA

Combination 3 Cell-Line DNA Combination 4

Exon 19 Del L858R S768I L861Q

T790M T790M G719A Exon20 ins

RING Trial panel design

• Two 27 member panels

• 3 WT samples

• 24 samples with mutations

• HD plasma spiked with plasmid DNA

• Double EGFR mutations in 4 combinations

• Six target copies/mL

• Additional panel for ddPCR/NGS

(7)

Semi-Quantitative Index (SQI) Overview*

• Increase or decrease in the SQI value is reflective of a change in mutational load within a patient

• SQI value is only meaningful relative to a previous or future measurement in the same patient

• SQI value correlates to target mutation concentration in a fixed volume of plasma (2 mL)

• Cannot compare mutational load across patients

• Currently, no standard exists for quantifying EGFR-mutant ctDNA concentration in plasma

7 Confidential – Not for Distribution - © Roche 24Feb2016

* Only available ex-US

(8)

Semi-Quantitative Index (SQI) Clinical studies

http://dx.doi.org/10.1097/JTO.0000000000000643

Marchetti - 2015

Keppens – ESMO 2016

Ex19Del L858R T790M

COPIES / mL COPIES / mL COPIES / mL

Copies/mL vs. SQI:

NGs vs. SQI:

R=0.99 R=0.97 R=0.97

(9)

RING Trial

Concordance of SQI values

Intra-laboratory Repeatability

Intra-laboratory Reproducibility

• Average CV over all 14 test sites

• Average CV over 4 test repetitions is given

• CV’s for p.(G719A) are higher compared to the rest

• Lower cp/mL lead to higher CV.

• Inter-laboratory CV’s are higher compared to intra- laboratory CV’s

(10)

U.S.

LOD

RING Trial Results

Overall:

 98.0% correct EGFR status

 2.0% (52/2662) false-negatives

 0.06% (6/9314) false-positives

 0.8% of runs (12/1512) excluded due to protocol deviations, 0.2% (3/1512) technical failures (test level)

(11)

RING Trial

cobas® EGFR Test v2 Plasma Conclustions:

• Robust Performance of the cobas® EGFR Mutation Test v2 in plasma

• Within labs (repeatability)

• Between labs (reproducibility)

• No false positives for WT samples (specificity)

• Repeat testing for low SQI values may reduce the average variation.

• High correlation between Copies/mL and SQI allowing for

sequential estimates of mutational load in an individual patient

(12)

Agenda

Avenio

(13)

The BRAF/NRAS and KRASv2 LSRs

BRAF/NRAS

Mutation Test (LSR) 36 Mutations

KRAS

Mutation Test v2 (LSR) 28 Mutations

BRAF (11): V600E/E2/D/R/K, K601E, G466A/V, G469A/R/V

NRAS (25): G12A/C/D/R/S/V, G13A/C/D/R/S/V, A18T, A59D/T,

Q61Hc/Ht/K/L/P/R, K117Nc/Nt, A146T/V

KRAS (28): G12A/C/D/R/S/V, G13A/C/D/R/S/V, A59E/G/S/T, Q61E/Hc/Ht/K/L/P/R, K117Nc/Nt, A146P/T/V

MUTATIONS DETECTED^1,2

MUTATION COVERAGE³ ^BRAF:96% melanoma, 98% CRC

NRAS:96% melanoma, 97% CRC ≥99% in CRC, NSCLC, PDAC

DNA INPUT^1,2 _{150ng DNA} _{150ng DNA}

SENSITIVITY^1,2 ≥5% mutant FFPET DNA in a

background of wild-type DNA ≥1% mutant FFPET DNA in a background of wild-type DNA

1 BRAF/NRAS Mutation Test (LSR) Package Insert 2 KRAS Mutation Test v2 (LSR) Package Insert 3 COSMIC Database v80 For Life Science Research Only (LSR). Not for use in diagnostic procedures.

Now for FFPET and Plasma samples

2mL Plasma

≥100 copies/mL for most common mutations

(14)

Same LSR workflow. Now for plasma samples.

Expanding flexibility of the LSR Testing Workflow to allow cfDNA testing

≤8 hours

Sample-to-result possible within a single laboratory shift

Growth curve visibility

Ct values provided Plasma Samples Added

Web-based data analysis

(15)

SQI SQI

Concordance of the SQI to % Mutation

Strong correlation to percent mutation measured by NGS

The Semi-Quantitative Index(SQI) is listed as Unit on the report for each Mutation

A series of Unit values provides trend information for the amount of circulating tumor DNA in the blood The SQI unit is only

provided for Mutation Positive results

Increasing Unit trend indicates increasein the amount of circulating tumorDNA in the blood

r²= 0.99995 r²= 0.99983

CLINICAL SAMPLE 1 CLINICAL SAMPLE 2

Source: Internal study assessing concordance of SQI and % Mutation from in-house MiSeq method using cfDNA isolated from plasma of melanoma patients

(16)

Sensitivity in Plasma Samples

LOD 100-200 copies/mL

100 MUTANT COPIES/mL

200 MUTANT COPIES/mL

WILD-TYPE COPY BACKGROUND OF 160,000 COPIES

BRAFV600E V600K V600R G466A G469A G469R G469V

NRASG12A G12CG12D G12RG13A

Q61Ha Q61KQ61L Q61P

BRAFV600E2 V600D K601E G466V

NRASG12S G12VG13C G13DG13R G13S

G13VA18T Q61Hb Q61RK117Nc

Note: Select mutations were detected at higher levels of mutant copies. NRAS A59D, A59T, K117Nt, and A146V were detected at 200 mutant copies/mL in a wild- type background of 16,000 copies. NRAS A146T was detected at 500 mutant copies in a wild-type background of 16,000 copies.

≥0.1% mutation

≥0.2% mutation

2 mL

(17)

Results Comparison of LSR to NGS

Results show nearly 99% overall concordance to NGS

LSR RESULT N %

NMD 137 73.7%

MUTANT 49 26.3%

BRAF Mutant 39 21.0%

V600E/E2/D 34 18.3%

V600K 4 2.2%

V600R 1 0.5%

NRAS Mutant 8 4.3%

Q61X 4 2.2%

G12X 2 1.1%

G13X 2 1.1%

BRAF+NRAS Mutant 2 1.1%

V600E/E2/D + N Q61X 2 1.1%

TOTAL 186

MiSeq

+ -

LSR + 48 0

- 2 136

LSR and MiSeq Method Correlation Summary Summary of LSR Test Results

Positive Agreement: 48/50 (96.0%) Negative Agreement: 136/136 (100%) Overall Agreement: 184/186 (98.9%)

Method Comparison: Study Design

n = 128 n = 57

wt

n = 1

186 plasma samples collected

BRAF/NRAS Mutation

Test (LSR) In-House MiSeq Reference Method

cfDNA isolated

same eluate used for both

test methods

(18)

cobas® DNA SP Kit IsolationNA

FFPET PlasmaOr Sample Collection

BRAF/NRAS

FFPET KRAS v2

FFPET

EXISTING

BRAF/NRAS PLASMA NEW!

POS CTRL

sample 1 sample 2 NEG CTRL

sample 3 sample 4

sample 5

sample 6

Multiplex

Mix mutation and sample types

(19)

Agenda

Avenio

(20)

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AVENIO ctDNA Analysis Kits

Liquid biopsy somatic mutation tests

INTENDED USE For Research Use Only

SAMPLE TYPE ctDNA from 4mL plasma

MUTATION COVERAGE Includes multiple mutation types in one panel (SNV, CNV, fusions and indels) to allow for the detection of variants relevant in

guidelines, clinical research selection, and longitudinal monitoring of tumor burden

THROUGHPUT 16 samples (16 adapters) RESEARCH SUBJECTS

Subjects diagnosed with late stage solid tumors Focus on: Lung and Colorectal cancer; pan cancer

applications (e.g., breast cancer)

TECHNOLOGY/PLATFORM Illumina NextSeq* 500/550;

manual assay

SOFTWARE User interface and report

with secondary analysis (variant calls)

*NEXTSEQ is a trademark of Illumina. The NextSeq 500/550 instruments and associated sequencing reagents are manufactured and sold by Illumina and are not supplied by Roche

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

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Bringing together multiple technologies to simplify the workflow and improve turnaround time

Workflow Overview

The AVENIO ctDNA Analysis Kits provide labs with a comprehensive end-to-end solution

21

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Source: Data on file

Panel Overview

A portfolio of liquid biopsy options provide flexibility to match the right panel to the right research goal

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Analyze genes in NCCN Guidelines Analyze genes targeted in clinical trials Analyze genes in longitudinal

tracking Number of genes

Size 81kb 192kb 198kb

17 77 197 genes

Mutation Classes SNVs, CNVs, Indels, Fusions

Targeted Panel Expanded Panel Surveillance Panel

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23 14 June 2017

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U.S.

Roche Diagnostic Oncology Portfolio

3 3

6 1

4 2

5 5

1112

9 9 8

13

7

7) HEAD & NECK

• IHC: CINtec Histology, HPV (CE-IVD)

10) BLOOD³

• IHC: B-Cell/T-Cell Lymphoma Markers

• PCR: BCR-ABL

11) COLORECTAL

• IHC: CDX2, CK20, CK7, MMR, BRAF V600E

• IA: CEA, CA19-9

• PCR: KRAS

12) BLADDER

• IHC: SMA, CK 20, p53, CK 7, Uroplakin III

• IA: CYFRA 21-1

13) PROSTATE

• IHC: Basal Cell Cocktail, p63, ERG

• IA: PSA, fPSA

3) BREAST

• IHC/ISH: HER2, ER, PR, Ki-67

• IA: CA15-3

• PCR: PIK3CA (RUO)*

4) UPPER GI¹

• IHC/ISH: HER2 (CE-IVD)

• IA: CA19-9, CA72-4, AFP

1) BRAIN/CNS

• IHC: GFAP, ACTH, S100, Synaptophysin, LH, NSE, NF, NSE

5) KIDNEY

• IHC: EMA, PAX8, RCC

2) THYROID

• IHC: BRAF

• IA: Tg, Calcitonin

• PCR: BRAF

8) SKIN

• IHC: Triple Melanoma Cocktail, MITF, S-100

• IA: S100

• PCR: BRAF

6) GYNECOLOGICAL²

• IHC: CINtec PLUS & CINtec Histology

• IA: CA125, HE4, CA72-4, SCC

• PCR: HPV

9) LUNG

• IHC: ALK, TTF-1, Napsin A, CD 5/6, p63, etc.

• IA: proGRP, NSE, CEA, SCC, CYFRA 21-1

• PCR: EGFR, KRAS

1 Includes esophagus, liver, pancreas, stomach *RUO = research use only 2 Includes cervical, ovarian, uterine

3 Includes leukemia, lymphoma, myeloma

IHC = immunohistochemistry

ISH = in situ hybridization IA = immunoassay PCR = polymerase chain reaction

Utility of liquid biopsies

EQA Program Naples, IT

Sidney A. Scudder, MD Director, Clinical Science 13 May, 2017

Utility of liquid biopsies

Agenda

cobas

EGFR Mutation Test v2*

42

FDA approved as a Companion Diagnostic in tissue and plasma for ex19del, L858R and

T790M

RING Trial

cobas® EGFR Test v2 Plasma

• Objectives

RING Trial

cobas® EGFR Test v2 Plasma

RING Trial panel design

Semi-Quantitative Index (SQI) Overview*

Semi-Quantitative Index (SQI) Clinical studies

RING Trial

Concordance of SQI values

RING Trial

cobas® EGFR Test v2 Plasma Conclustions:

Agenda

The BRAF/NRAS and KRASv2 LSRs

Same LSR workflow. Now for plasma samples.

Expanding flexibility of the LSR Testing Workflow to allow cfDNA testing

Concordance of the SQI to % Mutation

Strong correlation to percent mutation measured by NGS

Sensitivity in Plasma Samples

LOD 100-200 copies/mL

Results Comparison of LSR to NGS

Results show nearly 99% overall concordance to NGS

Multiplex

Mix mutation and sample types

Agenda

AVENIO ctDNA Analysis Kits

Workflow Overview

The AVENIO ctDNA Analysis Kits provide labs with a comprehensive end-to-end solution

Panel Overview

A portfolio of liquid biopsy options provide flexibility to match the right panel to the right research goal

Roche Diagnostic Oncology Portfolio

• ^Objectives