Evaluation of Two Molecular Methods for Genotyping FCGR3a in Non Hodgkin Lymphoma Patients
Saurabh Malhotra, Paul R Burchard, Prabhjot Kaur, Gregory J Tsongalis. Geisel School of Medicine at Dartmouth, Hanover, NH; Dartmouth-Hitchcock Medical Center and Norris Cotton Cancer Center, Lebanon, NH
Background: Fc receptor III A of immunoglobulin G (FcγRIIIA, also called CD16) belongs to the Fc gamma receptor family (FCGR) which plays an important role in immunoinflammatory processes. It is a low affinity, trans membrane receptor and is mainly expressed in monocytes, NK cells and macrophages. It has been implicated in various inflammatory conditions and recently a polymorphism in this gene has been shown to influence response to rituximab (anti CD20) therapy in various disorders. We evaluated two molecular methods to genotype this polymorphism.
Design: Archived FFPE samples from 30 biopsies of Diffuse Large B Cell Lymphoma were retrieved and DNA was extracted. The samples were tested for the FCGR3a polymorphism using a real time PCR followed by melt curve analysis or by a standard Taqman allelic discrimination assay using the AB 7500 FAST real time PCR instrument. The Taqman assay consisted of the following PCR conditions: 60C for 1:00; 95C for 10:00; 40 cycles of 92C for 15 sec, 60C for 1:00; 60C for 1:00.The PCR/melt curve assay was performed using the following conditions: 95C for 5:00; 40 cycles of 95C for 20 sec, 56C for 20 sec, 72C for 30 sec followed by a continuous melt from 72C for 10 sec then 1% continuous temperature increase until 97C for 10 sec.
Results: With the Taqman allelic discrimination assay, twenty cases had the homozygous wild type F/F genotype for the FcγRIIIA receptor, while two cases had the homozygous V/V polymorphism and eight cases were heterozygous with a V/F genotype. Results with the real time PCR followed by melt curve analysis were similar for twenty five cases, however four samples did not have sufficient DNA sample for the melt curve analysis method and the result from one sample was discordant.
Conclusions: The new Taqman assay offers several advantages over previously published assays such as rapid turnaround time, ease of interpretation, less sample DNA requirement and higher specificity without compromising sensitivity. These performance characteristics make it highly suitable for use in a clinical laboratory.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 282, Wednesday Morning