QClamp® XNA-PCRA highly sensitive, rapid, cost effective technology

for

low frequency & cell free circulating mutant DNA

detection

Mike Powell, PhD

CSO, DiaCarta, Inc

© 2014 DiaCarta, Inc.

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The Targeted Therapy Dilemma

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Need for assays with 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

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Low Frequency Mutations - Clinical Significance

Emergence of Resistance Mutations in CRC and NSCLC Therapy

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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

Allelic Frequency vs Survival NSCLC EGFR T790M

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PFS OS

Wang et. Al., PLoS One 2014, 9(11) e110780

QClamp®: How it works

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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

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

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QClamp® EGFR Assay

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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

QClamp® Assays detect mutations missed by ddPCR

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• 35 DNA samples from melanoma patients were analyzed for BRAF mutations byNGS 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

QClamp Sensitivity

Makes Liquid Biopsy 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

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QClamp JAK2 V617F mutant detection directly from whole blood

QClamp® Sequencing Enhancements

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Sanger Sequencing misses BRAF V600 mutations in Melanoma FFPE samples

ValineGTGCAC

Glutamic AcidGAGCTC

Sanger Sequencing of 5% BRAF V600Emisses mutation

QClamp XNA PCR followed by Sanger - 5% BRAF V600Emutation detected

Conclusions and Thank You

• 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.

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