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Tri-Con_2015
1) The document describes QClamp XNA-PCR, a technology from DiaCarta that uses molecular "clamps" to highly sensitively detect low frequency and cell-free circulating mutant DNA. 2) It needs assays that can detect biomarkers present at low frequency in samples containing a high amount of normal DNA. Existing technologies are inadequate for this need. 3) QClamp assays can reliably detect all possible mutations in a gene region using a single reaction. They can achieve ultra-high sensitivity down to 0.01% from minimal sample input.
Tri-Con_2015
- 1. QClamp® XNA-PCR A highlysensitive, rapid, cost effective technology for low frequency & cell free circulating mutant DNA detection Mike Powell, PhD CSO, DiaCarta, Inc © 2014 DiaCarta, Inc. All Rights Reserved
- 2. The Targeted TherapyDilemma © DiaCarta, Inc. 2014 All Rights Reserved
- 3. Need for assayswith high analytical sensitivity to detect biomarkers present at low frequency • Low frequency tumor ‘driver’ and ‘resistance’ mutations exist in well-known tumor oncogenes and growth factor receptors • Minimally invasive biomarker assays for monitoring and diagnosing disease should be able to detect biomarkers present at low frequency in a high abundance of normal DNA background present in the sample • Existing technologies are not adequate to address the need © 2014 DiaCarta, Inc. All Rights Reserved
- 4. Low Frequency Mutations- Clinical Significance Emergence of Resistance Mutations in CRC and NSCLC Therapy © 2014 DiaCarta, Inc. All Rights Reserved CRC KRAS c12(Panitumumab Amgen) Nature 2012, 486 (7404), 537-540 NSCLC EGFR T790M (CO1685 Clovis) AACR 2014 *Early detection of resistance mutations critical to enable therapy decisions to be made
- 5. Allelic Frequency vsSurvival NSCLC EGFR T790M © 2014 DiaCarta, Inc. All Rights Reserved PFS OS Wang et. Al., PLoS One 2014, 9(11) e110780
- 6. QClamp®: How itworks © 2014 DiaCarta, Inc. All Rights Reserved GET RID OF THE HAYSTACK! MAKE MORE NEEDLES!! Molecular “Clamps” bind to wild-type DNA preventing PCR amplification of WT DNA; Only mutant DNAs are amplified
- 7. QClamp® qPCR Assays •Target actionable genetic alterations – EGFR, KRAS, NRAS, BRAF, PIK3CA, JAK2 etc. • Reliable detection of ALL possible mutations within a particular region of a target gene in a single reaction • Complete suppression of PCR amplification of wild-type DNA • Achieve ultra-high sensitivity – up to 0.01% with minimal sample input • Easily genotype enriched mutations by Sanger sequencing or follow-on QClamp genotyping assays with mutant specific primers © 2014 DiaCarta, Inc. All Rights Reserved
- 8. QClamp® EGFR Assay ©2014 DiaCarta, Inc. All Rights Reserved G719 MIX_0.1% G719S (5ng) 32.6 G719 MIX_WT 40.0 Ex 19del MIX_0.1% Ex 19 del (5ng) 36.5 Ex 19del MIX_WT 40.0 T790 MIX_0.1% T790 (5ng) 35.5 T790 MIX_WT 40.0 L858 Mix_0.1% T790 (5ng) 34.5 L858 MIX_WT 40.0
- 9. QClamp® Assays detectmutations missed by ddPCR © 2014 DiaCarta, Inc. All Rights Reserved • 35 DNA samples from melanoma patients were analyzed for BRAF mutations by NGS and ddPCR • NGS and ddPCR detected mutations in 31 samples • QClamp BRAF Mutation Test (CE) detected mutations in all 35 samples • Sanger sequencing of the QClamp PCR product confirmed that these mutations were BRAF V600K mutations
- 10. QClamp Sensitivity Makes LiquidBiopsy Possible • Cell-free circulating tumor DNA (ctDNA) • Dying tumor cells release small pieces of their DNA into the bloodstream • Minimally invasive monitoring of patients tumor genomic landscape © 2014 DiaCarta, Inc. All Rights Reserved QClamp JAK2 V617F mutant detection directly from whole blood
- 11. QClamp® Sequencing Enhancements ©2014 DiaCarta, Inc. All Rights Reserved Sanger Sequencing misses BRAF V600 mutations in Melanoma FFPE samples Valine GTG CAC Glutamic Acid GAG CTC Sanger Sequencing of 5% BRAF V600E misses mutation QClamp XNA PCR followed by Sanger - 5% BRAF V600E mutation detected
- 12. Conclusions and ThankYou • QClamp XNA PCR is a rapid, convenient and highly sensitive method for detection of somatic mutations in clinical samples on existing real-time PCR instruments • XNA clamp probes have wide applicability in improving the sensitivity of detection of low frequency genetic variations in nucleic acid target amplification assays © 2014 DiaCarta, Inc. All Rights Reserved