Utility of liquid biopsies EQA Program Naples, IT Sidney A. Scudder, MD Director, Clinical Science 13 May, 2017 picture placeholder
Agenda cobas EGFR Mutation Test v2 Ring Trial SQI Semi Quantitative Index Inter-laboratory comparison LSR Avenio
cobas EGFR Mutation Test v2* FDA approved as a Companion Diagnostic in tissue and plasma for ex19del, L858R and T790M 42 MUTATIONS DETECTED AMPLIFICATION MMX TARGET MMX1 EX19Del; S768I; EX28/IC MMX2 MMX3 v.2 L858R; T790M; EX28/IC L861Q; G719A/C/S; EX20Ins; EX28/IC MN 7248563190 14 June 2017 page 3 CONFIDENTIAL, NOT FOR DISTRIBUTION Roche Molecular Diagnostics 2015
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
RING Trial cobas EGFR Test v2 Plasma Co-principal investigators Prof. Dr. E Dequeker Prof. Dr. N Normanno Prof. Dr. H van Krieken Study coordinator C. Keppens
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 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
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 * Only available ex-us 7 Confidential Not for Distribution - Roche 24Feb2016
Semi-Quantitative Index (SQI) Clinical studies NGs vs. SQI: Ex19Del L858R T790M Copies/mL vs. SQI: Marchetti - 2015 http://dx.doi.org/10.1097/jto.0000000000000643 R=0.99 R=0.97 R=0.97 COPIES / ml COPIES / ml COPIES / ml Keppens ESMO 2016
RING Trial Concordance of SQI values Intra-laboratory Repeatability Average CV over all 14 test sites Intra-laboratory Reproducibility 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 intralaboratory CV s
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) LOD LOD Roche Oncology LSRs not available in the U.S. 14 June 2017 page 10 Roche Molecular Diagnostics 2015
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
Agenda cobas EGFR Mutation Test v2 Ring Trial SQI Semi Quantitative Index Inter-laboratory comparison LSR Avenio
The BRAF/NRAS and KRASv2 LSRs Now for FFPET and Plasma samples MUTATIONS DETECTED 1,2 MUTATION COVERAGE 3 BRAF/NRAS Mutation Test (LSR) 36 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 BRAF: 96% melanoma, 98% CRC NRAS: 96% melanoma, 97% CRC KRAS Mutation Test v2 (LSR) 28 Mutations 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 99% in CRC, NSCLC, PDAC DNA INPUT 1,2 2mL Plasma 150ng DNA 150ng DNA SENSITIVITY 1,2 100 copies/ml for most common mutations 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 For Life Science Research Only (LSR). Not for use in diagnostic procedures. 2 KRAS Mutation Test v2 (LSR) Package Insert 3 COSMIC Database v80 Roche Oncology LSRs not available in the U.S. Roche Molecular Diagnostics 2016 14 June 2017 page 13
Same LSR workflow. Now for plasma samples. Expanding flexibility of the LSR Testing Workflow to allow cfdna testing Plasma Samples Added Growth curve visibility Ct values provided Web-based data analysis 8 hours Sample-to-result possible within a single laboratory shift Roche Oncology LSRs not available in the U.S. Roche Molecular Diagnostics 2016 14 June 2017 page 14
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 The SQI unit is only provided for Mutation Positive results A series of Unit values provides trend information for the amount of circulating tumor DNA in the blood Increasing Unit trend indicates increase in the amount of circulating tumor DNA in the blood CLINICAL SAMPLE 1 CLINICAL SAMPLE 2 r 2 = 0.99995 r 2 = 0.99983 SQI SQI Source: Internal study assessing concordance of SQI and % Mutation from in-house MiSeq method using cfdna isolated from plasma of melanoma patients Roche Oncology LSRs not available in the U.S. Roche Molecular Diagnostics 2016 14 June 2017 page 15
Sensitivity in Plasma Samples LOD 100-200 copies/ml WILD-TYPE COPY BACKGROUND OF 160,000 COPIES 0.1% mutation 100 MUTANT COPIES/mL BRAF V600E V600K V600R G466A G469A G469R G469V NRAS G12A G12C G12D G12R G13A Q61Ha Q61K Q61L Q61P 2 ml 0.2% mutation 200 MUTANT COPIES/mL BRAF V600E2 V600D K601E G466V NRAS G12S G12V G13C G13D G13R G13S G13V A18T Q61Hb Q61R K117Nc 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 wildtype background of 16,000 copies. NRAS A146T was detected at 500 mutant copies in a wild-type background of 16,000 copies. Roche Oncology LSRs not available in the U.S. Roche Molecular Diagnostics 2016 14 June 2017 page 16
Results Comparison of LSR to NGS Results show nearly 99% overall concordance to NGS Method Comparison: Study Design Summary of LSR Test Results 186 plasma samples collected cfdna isolated n = 128 n = 57 n = 1 wt 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 LSR and MiSeq Method Correlation Summary same eluate used for both test methods BRAF/NRAS Mutation Test (LSR) In-House MiSeq Reference Method LSR MiSeq + - + 48 0-2 136 Positive Agreement: 48/50 (96.0%) Negative Agreement: 136/136 (100%) Overall Agreement: 184/186 (98.9%) Roche Oncology LSRs not available in the U.S. Roche Molecular Diagnostics 2016 14 June 2017 page 17
Multiplex Mix mutation and sample types EXISTING NEW! Sample Collection NA Isolation BRAF/NRAS FFPET KRAS v2 FFPET BRAF/NRAS PLASMA POS CTRL NEG CTRL sample 1 FFPET Or Plasma cobas DNA SP Kit sample 2 sample 3 sample 4 sample 5 sample 6 Roche Oncology LSRs not available in the U.S. Roche Molecular Diagnostics 2016 14 June 2017 page 18
Agenda cobas EGFR Mutation Test v2 Ring Trial SQI Semi Quantitative Index Inter-laboratory comparison LSR Avenio
AVENIO ctdna Analysis Kits Liquid biopsy somatic mutation tests INTENDED USE For Research Use Only RESEARCH SUBJECTS Subjects diagnosed with late stage solid tumors Focus on: Lung and Colorectal cancer; pan cancer applications (e.g., breast cancer) THROUGHPUT 16 samples (16 adapters) SOFTWARE User interface and report with secondary analysis (variant calls) 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 TECHNOLOGY/PLATFORM Illumina NextSeq* 500/550; manual assay *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. 20 Do Not Distribute 25 April 2017 page 20 Roche
Workflow Overview The AVENIO ctdna Analysis Kits provide labs with a comprehensive end-to-end solution Bringing together multiple technologies to simplify the workflow and improve turnaround time For Research Use Only. Not for use in diagnostic procedures. 21 Do Not Distribute 25 April 2017 page 21 Roche
Panel Overview A portfolio of liquid biopsy options provide flexibility to match the right panel to the right research goal Targeted Panel Expanded Panel Surveillance Panel Analyze genes in NCCN Guidelines Analyze genes targeted in clinical trials Analyze genes in longitudinal tracking Number of genes 17 77 197 genes Size 81kb 192kb 198kb Mutation Classes SNVs, CNVs, Indels, Fusions Source: Data on file For Research Use Only. Not for use in diagnostic procedures. 22 Do Not Distribute 25 April 2017 page 22 Roche
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Roche Diagnostic Oncology Portfolio 1) BRAIN/CNS IHC: GFAP, ACTH, S100, Synaptophysin, LH, NSE, NF, NSE 2) THYROID IHC: BRAF IA: Tg, Calcitonin PCR: BRAF 3) BREAST IHC/ISH: HER2, ER, PR, Ki-67 IA: CA15-3 PCR: PIK3CA (RUO)* 4) UPPER GI 1 IHC/ISH: HER2 (CE-IVD) IA: CA19-9, CA72-4, AFP 1 2 3 3 4 5 5 6 7 9 9 11 12 13 8 7) HEAD & NECK IHC: CINtec Histology, HPV (CE-IVD) 8) SKIN IHC: Triple Melanoma Cocktail, MITF, S-100 IA: S100 PCR: BRAF 9) LUNG IHC: ALK, TTF-1, Napsin A, CD 5/6, p63, etc. IA: progrp, NSE, CEA, SCC, CYFRA 21-1 PCR: EGFR, KRAS 10) BLOOD 3 IHC: B-Cell/T-Cell Lymphoma Markers PCR: BCR-ABL 5) KIDNEY IHC: EMA, PAX8, RCC 11) COLORECTAL IHC: CDX2, CK20, CK7, MMR, BRAF V600E IA: CEA, CA19-9 PCR: KRAS 6) GYNECOLOGICAL 2 IHC: CINtec PLUS & CINtec Histology IA: CA125, HE4, CA72-4, SCC PCR: HPV 12) BLADDER IHC: SMA, CK 20, p53, CK 7, Uroplakin III IA: CYFRA 21-1 1 Includes esophagus, liver, pancreas, stomach *RUO = research use only 2 Includes cervical, ovarian, uterine 3 Includes leukemia, lymphoma, myeloma 14 June 2017 page 24 Roche Oncology LSRs not available in the U.S. Roche Molecular Diagnostics 2015 13) PROSTATE IHC: Basal Cell Cocktail, p63, ERG IA: PSA, fpsa IHC = immunohistochemistry ISH = in situ hybridization IA = immunoassay PCR = polymerase chain reaction