APPLICATION NOTE High multiplex, ultrasensitive EGFR detection using the EGFR 6-color Crystal Digital PCR™ kit Ready-to-use | Intuitive | Rapid time to results | High multiplexing | High sensitivity | ctDNA compatible Single assay detection of more than 90% of described EGFR mutations in NSCLC A liquid biopsy is the sampling and analysis of a non-solid biological specimen, most often the case blood. Liquid biopsies are a minimally invasive and straightforward sampling approach that can overcome the heterogeneity of tumors, and thus represent a valuable source of circulating tumor DNA (ctDNA) for oncological biomarker analysis. ctDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high background of wild-type sequences. Digital PCR has emerged as a powerful technology for the next-generation analysis of liquid biopsies. Stilla® Technologies’ 6-color naica® system is an ultrasensitive digital PCR technology capable of simultaneously and precisely quantifying high numbers of biomarkers in a single sample, simplifying the detection process, minimizing detection variability, and significantly reducing the hands-on-time to results. Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Epidermal growth factor receptor (EGFR) is frequently mutated and is a well-known genetic aberration in NSCLC. The highly multiplexed, ready-to-use EGFR 6-color Crystal Digital PCR™ kit allows the detection from circulating free DNA (cfDNA) of more than 90% of EGFR mutations described in NSCLC, including 32 common, rare, activating, and resistant somatic EGFR mutations in exons 18, 19, 20 and 21 (Table 1, next page). Application Note Highlights y The EGFR 6-color Crystal Digital PCR™ kit reliably detects 32 common, rare, activating, and resistant somatic EGFR mutations in a single assay. y The EGFR 6-color Crystal Digital PCR™ kit is optimized for ultrasensitive and highly robust EGFR mutation detection on the naica ® system with LoDs ranging from 0.30 to 0.46 cp/µL, depending on the target using Sapphire chips. y The Crystal Miner software and custom EGFR 6-color analysis template enable fast and straightforward analysis of cfDNA. The EGFR 6-color Crystal Digital PCR™ kit enables highly multiplexed cfDNA analysis on the naica® system The EGFR 6-color Crystal Digital PCR™ kit uses TaqMan™ probe technology to detect eight, point mutations, as well as twenty-four Exon 19 deletion and Exon 20 insertion alterations using drop-off detection (Figure 1, next page).

Table 1 | EGFR mutations detectable with the EGFR 6-color Crystal Digital PCR™ kit Exon Base changes Exon 19 deletion Drop-off Exon 20 insertion drop-off Figure 1 | EGFR 6-color Crystal Digital PCR™ kit detection design. TaqMan™ probe positions on each exon and fluorescent color code are noted.

A major advantage of a drop-off digital PCR assay is the simplified detection of numerous proximal genetic lesions (including deletions, insertions and nucleotide substitutions) within a short genomic interval using minimal reagents. Indeed, a drop-off assay requires only two TaqMan™ probes targeting the same amplicon: a wild-type probe that spans the genetic lesion site but is uniquely complementary to the wild-type sequence, and a reference probe that hybridizes adjacent to the mutation site and is thus complementary to both the mutant and the wild-type alleles. In the presence of a wild-type...

The EGFR 6-color Crystal Digital PCR™ kit is optimized for ultrasensitive and highly specific detection on the naica® system. The EGFR 6-color Crystal Digital PCR™ kit is compatible with the 6-color naica ® system using highly sensitive Sapphire chips. To determine the sensitivity of detection of each EGFR target, the Limit of Blank (LoB) and the Limit of detection (LoD) were determined following the Clinical and Laboratory Standards Institute (CLSI) EP17-A2 standard (Protocols For Determination Of Limits Of Detection And Limits Of Quantitation; Approved Guideline). The LoB for EGFR mutation...

The EGFR 6-color Crystal Digital PCR™ kit showed robust inter-run repeatability, with concentration RSDs (relative standard deviations) ranging from 4.5 to 8.6% (N = 12), depending on the target (Figure 4). In addition, the EGFR 6-color Crystal Digital PCR™ kit showed high specificity, with no false positives obtained (Table 3). Figure 4 | Inter-run repeatability of the EGFR 6-color Crystal Digital PCR™ kit. The inter-run repeatability was measured by comparing the results obtained on one 1 positive control sample chamber in 12 different runs (N = 12) with the EGFR 6-color Crystal Digital PCR™...

The analysis template provided with the EGFR 6-color Crystal Digital PCR™ kit combined with Crystal Miner software allow for the straightforward generation of 2D-plots (Figure 5) and the rapid obtention of highly concordant results. Figure 5 | A representative example of the analysis of a L858R and T790M positive NSCLC clinical cfDNA sample using Crystal Miner software and the custom EGFR 6-color Crystal Digital PCR™ kit analysis template. The cfDNA sample showed a perfect concordance between the expected and measured results. The x- and y-axis correspond to the fluorescence units of the indicated...