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Multiplexed ICE COLD-PCR (MX-ICP): For Detection of Low-Level Mutations in Liquid Biopsies Katherine Richardson Vice President, Research & Development Transgenomic® Advancing Personalized Medicine 1 Transgenomic Background & Introduction Biopsy Learn more Ultra-sensitive mutation detection in circulating free DNA (cfDNA) Transgenomic® Advancing Personalized Medicine 2 Transgenomic Technologies that Enable Biomarker Discovery & Development of Molecular Diagnostics Transgenomic Technology Development Focus: Holds rights to IP on ICE COLD-PCR developed by Dr. Mike Makrigiorgos at the Dana Farber Cancer Institute Develops enrichment assays to enable sensitive mutation detection Supports oncology and immuno-oncology biomarker development Collaborates in biomarker discovery for colorectal, lung, melanoma and other cancers Holds additional IP for several targets including PIK3CA and FC gamma receptor 1997 WAVE® System WAVEMAKER™ Software – nucleic acid variation analysis 2002 2004 Navigator™ SURVEYOR® Software Nuclease Kits Automated Mutation Calling on the WAVE System 2010 REVEAL® ICE COLD-PCR Kits 2011 RAScan™ & RASseq™ CRC Somatic Mutation Detection Kits 2014 Multiplex ICE COLDPCR Coupled with NGS 2015 ICEme Mutation Enrichment Kit 3 Discovery Discovery (Novel Targets) • Identification of any and all mutations • Proof-of-concept • Assay Development Services Kits Services Kits (Off-the-shelf) • Targeted Sequencing • RNA Sequencing • microRNA Panels • Gene Expression • Methylation • SNP Genotyping • DNA Quantitation • Mutation Enrichment • Gene Panels • ICE COLD-PCR • Choice of exons/reactions • Technical Support hotline • RUO / 510K / CE-IVD • MX-PCR/ICP Multi-Disciplinary Team: Expert Knowledge and Expertise ICEme Kits Knowledgeable and Experienced Scientists. Discovery Services Expertise in ISO and QSR, CDx and CLIA/CAP. Medical device development expertise. Clinical, pharmaceutical and molecular genomics expertise. Kits Preclinical Studies, extensive expertise in AAALAC Regulatory Consultants with extensive expertise in CDx development. 4 Liquid Biopsies Traditional Tissue Biopsies are: Costly Invasive May Require lengthy workflows Tumor heterogeneity can be an issue Biopsy Ultra-sensitive mutation detection in circulating cfDNA ICE COLD-PCR is a technology exquisitely made for mutational analysis of Liquid Biopsy Circulating cell-free DNA (cfDNA) is a potential surrogate for the entire tumor genome Liquid Biopsy enables Precision Medicine (diagnosis, prognosis, and monitoring of cancer, or as predictive markers for the patient’s response to a targeted therapy) Transgenomic® Advancing Personalized Medicine 5 ICE COLD-PCR Overview Biopsy Learn more Ultra-sensitive mutation detection in circulating free DNA (cfDNA) Transgenomic® Advancing Personalized Medicine 6 ICE COLD-PCR Advantages Non-Allele Specific – Enriches ANY & ALL Variants Improves clinical outcome by use of non-invasive liquid biopsies for treatment & monitoring Works on any sample – tissue, plasma, cells, exosomes, FFPE, CTC’s Increased sensitivity of detection on any platform: Simple integration into existing workflow on NGS, ddPCR, Sanger Significantly improves sensitivity – 100 to 500 fold increase. Therefore enables Sanger confirmation of low level NGS mutations Exclusive license from Dana Farber Cancer Institute 7 ICE COLD-PCR : A Unique Technology for Preferential Enrichment of Mutations Liquid Biopsy Pre-amplification Multiplex-PCR (Traditional PCR) Mutant 0 5 10 15 20 # Cycles 25 30 Mutant # PCR Products DNA Extraction & Amplification # PCR Products Wild-Type ICEme Kit 0 35 MX-ICP (Specialized Wild-Type Nested-PCR) 5 10 15 20 # Cycles 25 30 35 Analyze ICP Product on any Sequence Analysis Platform The Pre-amplification step increases the amount of starting material A small portion of the MX-PCR is used in the ICE COLD-PCR reaction to enrich for any and all mutations present After the ICE COLD PCR reaction, the samples can be analyzed on any downstream platform 8 ICE COLD-PCR Assay Input Sample from MX-PCR 5’ 5’ 5’ 5’ 5’ 5’ PO4 PO4 PO4 5’ Pol. Buffer, dNTP’s Step 1: Denature 5’ PO4 5’ 5’ PO4 5’ XXXXX 5’ 5’ XXXXX PO4 PO4 5’ 5’ 5’ 5’ PO4 5’ 5’ Step 5: Polymerase Extension 5’ 5’ 5’ 5’ Wild-Type Sequence Mutated Sequence PO4 Exp. Amp Analysis on platform of choice for mutation detection 5’ Linear Amp 5’ 5’ 5’ 5’ PO4 5’ Tc 5’ Step 4: PCR Primer Hybridize PCR Primer 1 5’ 5’ Step 3: Denature at Critical Temperature (Tc) Repeat 5’ 5’ 5’ Step 2: RS-oligo Hybridize PO4 PO4 5’ 5’ 5’ 5’ 5’ 5’ PO4 5’ PO4 PCR Primer 2 RS-oligo PCR Process that Enriches for ANY Mutation in the Region Interrogated by the RS-oligo. 9 MX-ICP Flexible Workflow Sanger Sequencing of Individual MX-ICP Assays Detects ANY and ALL of the possible mutations present Sample DNA MX-PCR to increase the amount of DNA present for multiple gene regions MX-ICP Enrichment of ANY and ALL mutations present in the gene regions amplified in the MX-PCR. NGS: Pool all MX-ICP Reactions/Sample then Analyze Detects ANY and ALL of the possible mutations present ddPCR Sufficient mutation-enriched DNA for detection of ANY and ALL possible mutations. The number of different mutations which can be detected is limited by the number of available ddPCR assays. 10 Detection of ALL sequence variations Selection of Exons Available # of COSMIC Alterations Detectable in MX-ICP Region Single Multiplex PCR amplification Detection of ALL sequence variations including point mutations and small insertions/deletions in: KRAS Exons 2, 3, and 4 Including codons 12, 13, 59, 61, 117 & 146 NRAS Exons 2, 3, and 4 Including codons 12, 13, 59, 61, 117 & 146 BRAF Exons 11 and 15 Including codons 469 & 600 PIK3CA Exons 9 and 20 Including codons 542, 545 & 1047 EGFR Exons 12, 18, 19, 20, and 21 Including codons 492, 719, All Exon 19 dels/complex mutations, 790, 797, 858 & 861 *No mutations listed in COSMIC but observed in Colorectal Cancer COSMIC Insertions/ Deletions COSMIC Mutations BRAF Exon 11 0 35 BRAF Exon15 20 53 EGFR Exon 12 0 3 EGFR Exon 18 5 44 EGFR Exon 19 >87 45 EGFR Exon 20 1 21 EGFR Exon 21 1 34 KRAS Exon 2 8 69 KRAS Exon 3 5 29 KRAS Exon 4A 0 4 KRAS Exon 4B 0 6 NRAS Exon 2 9 30 NRAS Exon 3 0 28 NRAS Exon 4A 0 0* NRAS Exon 4B 0 1 PIK3CA Exon 9 0 41 PIK3CA Exon 20 0 51 Genes/Exons Available 11 Genotyping Platforms ICP increases sensitivity on all platforms Platform Sensitivity Sanger ICP + Sanger Pyrosequencing ICP + Pyrosequencing NGS ICP + NGS SNaPshot BEAMing ddPCR 10 -20% ≥0.01% 5% Mutation Quantitative type ALL Yes Mut, Indel Yes Site Yes ≥0.05% Site Yes 3-5% ≥0.01% 5% ≥0.01% ≥0.01% ALL Mut, Indel Site Hotspot Hotspot Yes Yes Yes Yes Yes Fold Increase >1000 >100 >500 12 Validation Data Generated using ICE COLD-PCR Biopsy Learn more Ultra-sensitive mutation detection in circulating free DNA (cfDNA) Transgenomic® Advancing Personalized Medicine 13 Initial Mutation (%) 1% 0.10% 0.01% 0.001% 100 10 1 0 45 5 0 200 100 10 1 400 152 600 303 455 800 152 1,000 30 3 0 1,200 500 330 150 100 50 33 10 15 2 0 1,000 1,400 15 2 Mutations in Starting Material 1,600 DNA (ng) # Genomic Equivalents 151,515 100,000 45,455 30,303 15,152 10,000 3,030 30 3 0 0 1,515 Number of Genomic Copies and Mutations Present in Starting Material 0 500 ng 330 ng 150 ng 100 ng 50 ng 33 ng 10 ng Amount of Starting DNA (ng) 33 ng: Theoretical Limit of Detection for 0.01% 150 ng: Robust detection of 0.01% mutation using MX-ICP 330 ng: Robust detection of 0.01% mutation using ddPCR 14 MX-ICP plus Sanger Enhances Mutation Detection of EGFR Exon 19 Deletions Input Mut Output Input Mut WT 1% del 100% del 0.1% del WT 0.1% del 95% del 0.01% del WT 0.01% del 60% del WT WT WT 1% del Standard PCR, M13-Based Sequencing ICE COLD-PCR w/Sanger Output WT 15 MX ICP Enhances Mutation Detection by NGS: Resulting in Increased Sensitivity of NGS EGFR is one of the most common mutations present in up to 15-20% of all lung cancer patients* % MT (sensitivity) refers to the concentration of mutant DNA in the total DNA sample Next Gen Without MX-ICP % MT % MT 0% 0% 20% 5% 1% 0.5% 0.1% 0.05% 0.01% 0.005% Next Gen With MX-ICP ✔ ✔ No del No del No del No del No del No del 20% ✔ ✔ ✔ ✔ ✔ ✔ ✔ 5% 1% 0.5% 0.1% 0.05% 0.01% 0.005% No del EGFR * Present is 14-20% of lung cancer in Caucasians & 40-50% in Asian: Journal of Clinical Oncology David a Eberhard et al Vol 23. Number 25 Sept 2015 16 Droplet Digital PCR and MX-ICP of EGFR T790M: Bio-Rad QX200 150 ng of starting DNA containing 0.1 or 0.01% EGFR Exon 20 T790M Mutation Enriched using MX-ICP prior to ddPCR Amount of Mutation in Starting Sample Dilution of Starting MX-ICP product Necessary 1 / 100,000 1 / 1 Million 1/ 10 Million AS = Droplets containing Mutations 0.1% T790M 22% Mutation 25% Mutation 22% Mutation WT = Wild-Type Droplets WT + AS = Dual Occupancy Droplets 0.01% T790M 3% Mutation 5% Mutation 7% Mutation Less DNA Needed = Less Plasma = Less Blood 17 Monitoring Mutations in cfDNA from a Patient: Example of Effective and Improved Treatment Detects actionable Mutations in DNA from plasma of patients prior to tumor growth Supports determination of when to start treatment and monitoring of response BRAF-Targeted Therapy 1 0.8 0.6 V600E decreasing in cfDNA 0.4 0.2 1.4 1.2 1 0.8 0.6 0.4 0.2 0 9/5/13 8/5/13 7/5/13 6/5/13 5/5/13 4/5/13 3/5/13 2/5/13 1/5/13 12/5/12 11/5/12 9/5/12 10/5/12 0 9/1/13 8/1/13 7/1/13 6/1/13 5/1/13 4/1/13 3/1/13 2/1/13 1/1/13 12/1/12 11/1/12 10/1/12 Tumor burden decreasing 9/1/12 Tumor Burden % cfDNA BRAF V600E % No Therapy 18 Improvement of Mutation Detection using MX-ICP: One Patient; Multiple Mutations, Pre- and Post- Treatment Monitoring 100 90 90 Amount of Mutation Detected (%) 90 80 70 SANGER without ICP (No Treatment) SANGER with ICP (No Treatment) SANGER with ICP (BRAF-Inhibitor) 60 50 50 40 40 35 30 25 23.7 20 10 0 10.8 10 00 0 Wild-Type Sequence V600 Q61 G12 E542 19 Patient Monitoring using ICP – Plasma from Stage IV CRC (Tumor Analysis not Performed at Transgenomic) Tumor Status/FFPE G12C G12D G12D NVD Without ICE COLD-PCR With ICE COLD-PCR G12C G12C NVD G12D NVD NVD G12D NVD Input DNA (ng); Gen Equiv (G.E.); Calc Starting Mut % from ICP Outcome Amt: 102 ng G.E. 31,400 Mut % calc: 9.5% Stable Disease Enriches for ANY & ALL Mutations from Low Amounts of Starting DNA Amt: 67 ng G.E.: 20,800 Mut % calc: 0.8% Stable Disease Detects Mutations from Liquid Biopsy Samples Amt: 18 ng G.E.: 5,700 Mut % calc: 0.3% Partial Response Amt: 22 ng G.E.: 6,800 Mut % calc: 0% Partial Response Helps Predict Therapeutic Options Improves Clinical Outcomes 20 MX-ICP Enables Quantification of Amount of Mutation in Starting Sample: LoD for EGFR S492R (3 reps) 10 Average LOD Amount of Alteration Prior to MX-ICP ICP (%) y= 0.0199e0.0557x Digitally verified Sample 4.1% before MX-ICP 90% after MX-ICP R² = 0.986 1 Sample Dilution (Rep 4) 0.04 before MX-ICP 15% after MX-ICP CRC: Sample ?? before MX-ICP 40% after MX-ICP Calculated 0.2% Before MX-ICP 0.1 0.01 0 20 40 60 80 100 CRC: Sample ?? before MX-ICP 10% after MX-ICP Calculated 0.03% Before MX-ICP Amount of Alteration After MX-ICP (%) 21 New MX-ICP Assay for C797S Mutation in EGFR Exon 20 – no additional MX-PCR EGFR Exon 20 C797S – G to C 20%→80% 5→65% 1→50% 0.5→35% 0.25→20% 0.1→10% 0.5→20% 0.25→10% 0.1→5% EGFR Exon 20 C797S – T to A 20%→95% 5→55% 1→35% 1 MX-PCR including EGFR Exon 20 ICP assays for T790M and C797S 1 RS-oligo for T790M Region 1 RS-oligo for C797S Region 2 different mutations for C797S 22 Questions/Challenges What levels of mutations are relevant for the patient? In Tissues - tumor heterogeneity? In FFPE what does it mean for the Pathologist/Oncologist/Patient? In Liquid Biopsies – amounts of sample needed? Clinical Trials have limited amounts IRBs allow only so much blood drawn from a patient Patients not on Clinical Trials can have more blood drawn Are these levels the same for all mutations? Will different mutations have different cut points? Will cut points be both mutation and patient dependent? Are these levels the same for all cancers? Suspect different cancers will have same mutation but different cut point Maybe cancer and patient dependent 23 Solutions? Inclusion of ICP in research projects will help provide answers! Use of ICP for mutational analysis of nucleic acids isolated from Liquid Biopsies will help provide answers! Inclusion of ICP in Clinical Trial Protocols will help provide answers! For all of the above, Correlation to Patient Treatment Outcomes will be required! 24 Summary and Products Biopsy Learn more Ultra-sensitive mutation detection in circulating free DNA (cfDNA) Transgenomic® Advancing Personalized Medicine 25 Summary of the Advantages of MX ICP Pharma/biotech “Plug-and Play” into existing workflow for in-house use or preferred service provider Detection of low level mutations that correlate to response Patient Stratification: drug sensitivity, resistance markers Clinical Diagnostic Development of companion diagnostic Ease of technology transfer with Pharma/Biotech Partner Real-time monitoring of patient condition, improving clinical outcome Clinical Oncologists Real-time monitoring of patient condition and to improve patient care. Rapid identification of all relevant DNA alterations using currently validated platforms. Implementation of plasma/serum oncology assays 26 Products Released Biomarker Services: Launched new MX-ICP services at AACR CLIA Testing: First Test launched at ASCO EGFR T790M Mutation, 6 more targeted tests/panels this year NSCLC, CRC & Melanoma Kits Research Use Only (ICEme): multiplex and customizable kits launched globally Licensing: Commercial arm of University of Melbourne, others 27 Thank You 28