Droplet Digital PCR™: Comparison of a Novel Method of HER2 Testing to Immunohistochemistry and Fluorescence In Situ Hybridization
Stephanie C Tanner, Jesus Monico, Phil Belgrader, Jack Regan, Ryan Koehler, Alexandra S Brown. The University of Mississippi Medical Center, Jackson, MS; Bio-Rad Laboratories, Hercules, CA
Background: Recently, laboratories have begun offering HER2 testing using conventional quantitative PCR (qPCR). Although sensitive, qPCR has limitations in distinguishing and accurately measuring small changes in template copy number. In this feasibility study, we examined detection and quantification of HER2 DNA amplification by droplet digital PCR (ddPCR), a highly precise method for absolute DNA quantitation.
Design: The surgical pathology archives at our institution were searched for 50 cases of invasive breast carcinoma that had IHC and/or FISH results, available formalin-fixed paraffin-embedded (FFPE) tissue, and at least 5 mm of invasive tumor. For each case, DNA was extracted from FFPE tissue and a PCR reaction mixture was produced. Each PCR reaction mixture was divided into an emulsion of ∼20,000 1 nL mono-sized droplets. Each droplet served as an independent PCR reaction and contained either one or zero molecules of template. Droplets were thermal cycled and analyzed using an automated reader. Software counted the fraction of positive droplets for each sample, then calculated the concentration of HER2 and CEP17 genes in each sample. The copy number ratio of HER2 to CEP17 was used to determine whether the sample was positive or negative for HER2. We compared these data with HER2 status previously defined by IHC (27 cases), FISH (3 cases) and IHC and FISH (20 cases).
Results: Initial HER2 status was negative or positive with the following frequencies: IHC only 19(38%), 7(14%); FISH only 2(4%), 1(2%); combined IHC and FISH 19(38%), 2(4%). For ddPCR, results were negative in 41(82%), positive in 9(18%), showing 100% concordance with IHC and FISH results. Of the 11 cases that were equivocal by IHC, ddPCR converted HER2 status to positive in 9 and negative in 2. There was no change in HER2 status with ddPCR for patients evaluated by FISH or by both IHC and FISH.
Conclusions: ddPCR detected HER2 amplification in all cases having previous positive results while all negative cases were correctly assigned as negative (100% concordance). In eleven cases with equivocal IHC, ddPCR was able to detect presence or absence of HER2 amplification when compared to FISH. In conclusion, ddPCR is a highly precise method for measuring DNA copy number and these preliminary results demonstrate the feasibility of measuring HER2 amplification in breast carcinoma.
Wednesday, March 21, 2012 1:00 PM
Poster Session VI # 10, Wednesday Afternoon