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M. Windsor

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    P1.06 - Poster Session 1 - Prognostic and Predictive Biomarkers (ID 161)

    • Event: WCLC 2013
    • Type: Poster Session
    • Track: Biology
    • Presentations: 1
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      P1.06-003 - Comparative effectiveness of ddPCR for the detection of EGFR mutations. (ID 357)

      09:30 - 16:30  |  Author(s): M. Windsor

      • Abstract

      Selection of EGFR TKIs for lung cancer requires accurate detection of activating mutations. Traditional techniques are limited by small biopsy sizes. We compared droplet digital PCR (ddPCR, Bio-Rad) to Sanger sequencing, mutant-enriched PCR (ME-PCR) and high resolution melt (HRM) PCR.

      A comparative effectiveness study was performed on 317 resected NSCLCs with salt extracted gDNA. EGFR exons 19 & 21 Sanger sequencing and (Shigematsu et al., 2005) and HRM/ME-PCR mutation detection (Sriram et al., 2011) was previously reported. ddPCR (Bio-Rad) was performed with competitive allele specific Taqman PCR (CastPCR; Life Technologies); EGFR L858R assay for exon 21 c.2573T>G and c.2572_2572CT>AG; exon 19 deletion assay for 20 common deletions (EGFR_ex19dels_mu, EGFR_6224_mu). 8ng gDNA was tested in 20uL reactions partitioned into 20000 droplets; valid reads contained ≥ 10000 droplets. Controls were 8ng gDNA from mutation positive cell lines (AJCC: H1975, H1650), human female DNA (Promega) and no template controls. QuantaLife (Bio-Rad) calculated Poisson statistics determined allele copy/uL; samples with minimum estimated mutant copy number ≥ 0.15/uL (3 copies/8ng) were “called” positive.

      Serial dilution of control assay demonstrated detection of template to 40pg input gDNA. 209 (66%) men and 108 (34%) women of mean age 63 years (range 36 to 83) were included. 295 (93%) had smoked and 18 (6%) were never smokers; 4 (1%) were unknown. 1 subject (0.3%) was Asian. pTNM stages were I (47%), II (31%) and III (22%) respectively (6[th] Ed). 171 (54%) were adenocarcinomas, 109 (34%) squamous cell carcinomas, and 37 (12%) other histologies. Figure 1 Exon 19 and 21 ddPCR assays yielded valid results for 300 and 301 samples respectively. ddPCR detected all mutations previously demonstrated by Sanger sequencing (13) and HRM (13) but not ME-PCR (13/15). Mean droplet counts were lower in ddPCR only called (30 droplets/ng) than those samples also called by other methods (3600 droplets/ng; p=0.039). Median percentage tumour and necrosis content of mutation positive samples only by ddPCR were 30% and 0% respectively, identical to those called by other methods.

      ddPCR identifies mutations detected by Sanger sequencing and HRM. Limitations include nanodrop quantitation of input DNA and data replication is required. This technique demonstrates high sensitivity but limited specificity and requires further validation to examine the significance of low droplet number positive calls. The authors acknowledge the assistance of R Harrison and A Beckhouse, Bio-Rad. Financial support gratefully received from: NHMRC (MD PhD Scholarship), CCQ (MD PhD Scholarship), Cancer Australia, TPCH Foundation, Queensland Health.