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EQA ProgramNaples, ITSidney A. Scudder, MDDirector, Clinical Science13 May, 2017

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Utility of liquid biopsies

Agenda cobas® EGFR Mutation Test v2

Ring Trial

SQI – Semi Quantitative Index

Inter-laboratory comparison

LSR

Avenio

14 June 2017 page 3 CONFIDENTIAL, NOT FOR DISTRIBUTION

Roche Molecular Diagnostics © 2015

cobas® EGFR Mutation Test v2*

42 MUTATIONS

DETECTED

AMPLIFICATIONMMX 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

RING Trialcobas® 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

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

• Study coordinator– C. Keppens

RING Trialcobas® EGFR Test v2 Plasma

Cell-Line DNACombination 1




Exon 19 Del L858R S768I L861Q

T790M T790M G719A Exon20 ins

RING Trialpanel 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

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

Semi-Quantitative Index (SQI)Clinical studies

http://dx.doi.org/10.1097/JTO.0000000000000643Marchetti - 2015

Keppens – ESMO 2016

Ex19Del L858R T790M

CO

PIES

/ m

L

CO

PIES

/ m

L

CO

PIES

/ m

L

Copies/mL vs. SQI:

NGs vs. SQI:

R=0.99 R=0.97 R=0.97

RING TrialConcordance of SQI values

Intra-laboratory Repeatability

Intra-laboratory Reproducibility

• Average CV over all 14 test sites

• Average CV over 4 testrepetitions 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

14 June 2017 page 10

Roche Molecular Diagnostics © 2015Roche Oncology LSRs not available in the U.S.

LOD

LOD

RING TrialResults

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)

RING Trialcobas® EGFR Test v2 PlasmaConclustions:

• 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


Ring Trial



LSR

Avenio

Roche Oncology LSRs not available in the U.S. | Roche Molecular Diagnostics © 2016 | 14 June 2017 page 13

The BRAF/NRAS and KRASv2 LSRs

BRAF/NRASMutation Test (LSR)

36 Mutations

KRASMutation 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 DETECTED1,2

MUTATION COVERAGE3 BRAF: 96% melanoma, 98% CRC

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

DNA INPUT1,2 150ng DNA 150ng DNA

SENSITIVITY1,2 ≥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 Insert2 KRAS Mutation Test v2 (LSR) Package Insert

3 COSMIC Database v80For Life Science Research Only (LSR). Not for use in diagnostic procedures.

Now for FFPET andPlasma samples

2mL Plasma

≥100 copies/mL for most common mutations


Same LSR workflow. Now for plasma samples.Expanding flexibility of the LSR Testing Workflow to allow cfDNA testing

≤8 hoursSample-to-result possible within a single laboratory shift

Growth curve visibility

Ct values provided

Plasma Samples Added

Web-based data analysis


SQI SQI

Concordance of the SQI to % MutationStrong 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 increase in

the amount of circulating tumor DNA

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


Sensitivity in Plasma SamplesLOD 100-200 copies/mL

100 MUTANT COPIES/mL

200 MUTANT COPIES/mL

WILD-TYPE COPY BACKGROUND OF 160,000 COPIES

BRAFV600EV600KV600RG466AG469AG469RG469V

NRASG12AG12CG12DG12RG13A

Q61HaQ61KQ61LQ61P

BRAFV600E2V600DK601EG466V

NRASG12SG12VG13CG13DG13RG13S

G13VA18TQ61HbQ61RK117Nc

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% mutation2 mL


Results Comparison of LSR to NGSResults 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 MiSeqReference Method

cfDNA isolated

same eluateused for both test methods


cobas® DNA SP Kit

NAIsolation

FFPETOr

Plasma

Sample Collection

BRAF/NRASFFPET

KRAS v2FFPET

EXISTING

BRAF/NRASPLASMA

NEW!

POS CTRL

sample 1

sample 2

NEG CTRL

sample 3

sample 4

sample 5

sample 6

MultiplexMix mutation and sample types


Ring Trial



LSR

Avenio

Do Not Distribute | 25 April 2017 | page 20 | © Roche

20

AVENIO ctDNA Analysis KitsLiquid biopsy somatic mutation tests

INTENDED USEFor Research Use Only

SAMPLE TYPEctDNA from 4mL plasma

MUTATION COVERAGEIncludes 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

THROUGHPUT16 samples (16 adapters)

RESEARCH SUBJECTSSubjects diagnosed with late stage solid tumors

Focus on: Lung and Colorectal cancer; pan cancer applications (e.g., breast cancer)

TECHNOLOGY/PLATFORMIllumina NextSeq* 500/550;

manual assay

SOFTWAREUser 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.

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

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


21For Research Use Only. Not for use in diagnostic procedures.

Source: Data on file

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

22


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

Analyze genes inNCCN Guidelines

Analyze genes targetedin 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

2314 June 2017

14 June 2017 page 24

Roche Molecular Diagnostics © 2015Roche Oncology LSRs not available in the 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 only2 Includes cervical, ovarian, uterine3 Includes leukemia, lymphoma, myeloma

IHC = immunohistochemistryISH = in situ hybridization

IA = immunoassayPCR = polymerase chain reaction

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