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Droplet Digital™ PCR: Multiplex Detection of KRAS Mutations in Formalin-Fixed, Paraffin-Embedded Colorectal Cancer Samples Wei Yang, Dawne N Shelton, Jennifer R Berman, Bin Zhang, Samantha Cooper, Svilen Tzonev, Eli Hefner, and John F Regan
Digital Biology Center, Bio-Rad Laboratories, Inc., 5731 W Las Positas Blvd, Pleasanton, CA 94588 Abstract
Targeted therapies in many cancers have allowed unprecedented progress in the treatment of disease. However, routine implementation of genomic testing is constrained due to: 1) limited amounts of sample (pg–ng range) per biological specimen, 2) diagnostic turnaround time and workflow, 3) cost, and 4) difficulties in detection of mutational loads below 5%. KRAS is mutated in approximately 40% of colorectal cancers (CRCs). The majority of mutations affect codons 12, 13, and 61 and indicate a negative response to anti–epidermal growth factor receptor (EGFR) therapy. To optimize therapy strategies for personalized care, it is critical to rapidly screen patient samples for the presence of multiple KRAS mutations.
We have developed a multiplexing strategy to screen seven action- able KRAS mutations in colorectal cancer sam- ples using digital PCR. This panel includes KRAS point mutations with indi- vidual frequencies higher than 1% and covers 98% of KRAS mutant colorec- tal cancers (Faulkner et al. 2010, unpublished data). No preamplification step is required. This KRAS screening assay was used to quantify KRAS mutational load in a panel of formalin-fixed, paraf- fin-embedded
(FFPE)
samples from patients with advanced meta- static colorectal cancer. KRAS mutations present at <1% fractional abun- dance were detected in multiple samples. This sensitive and inexpensive method reduces the risk of contamination and can be easily implemented for rapid, routine screening of cancer samples.
Materials and
Methods • 16 mCRC (7 female, 9 male, average age 64
Vol. 58 | No. 5 | 2015 Vol. 58 | No. 5 | 2015 270
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