Sponsored Paper ApplicationForum
Evaluating EGFR Genotyping using HDx™ Reference Standards Dr. Marsha Speevak, Discipline Lead Molecular Genetics and Cytogenetics, Credit Valley Site, Trillium Health Partners.
Chantal Murray, Senior Technologist Department of Laboratory Medicine and Genetics, Credit Valley Site, Trillium Health Partners.
“People are always begging friends and colleagues for reference material - It is precious” Introduction
Dr. Marsha Speevak
Excessive activation of EGFR has been shown to be associated with advanced stages of cancer and a poor prognosis found in several types of non-small cell lung cancer. Detection and identification of these mutations has become a critical factor in stratifying patients who will benefit from anti-EGFR targeted therapies and those who are less likely to respond.
In particular, non-specific and inefficient probe binding in mutations can lead to late crossing points creating these false- negatives as identified in the proficiency testing schemes. EGFR G719A/S and T790M are particular mutations that have been highlighted as areas of high error.
HDx™ Reference Standards from Horizon Diagnostics are provided as Research Use Only reference materials. Trillium Health Partners have adopted HDx Reference Standards to evaluate performance characteristics and have provided the data within this report. No financial transactions have taken place between parties for the provision of data within this report.
Methodology Kit: Entrogen EGFR Kit (EGFR exons 18, 19, 20 and 21) Sequencer: Roche LC480 Real Time PCR
About: Allele discriminating assays rely upon the ability of the probes to bind correctly and the software setting to correctly set a crossing point baseline. HDx Reference Standards were used to evaluate the assay and establish the detection limits for each mutation.
EGFR HDx Reference Standards as DNA were used at allelic frequencies of 50%, and matched Wild Type was used to dilute mutations down to a 2.5% allelic frequency.
Results
HDx Reference Standards were used for four applications: assay evaluation and determination of the limit of detection for each mutation; highlighting of problematic probe bindings; comparison of an assay’s performance between mutations; and for the identification of muta- tions that are true positives but have low percentage tumor content.
Assay evaluation Trillium Health Partners were able to quickly deter- mine the limit of detection and assay specific cross- ing point cut-offs for each available mutation. The different allelic frequencies used in the assay evalua-
tion for EGFR L861Q are highlighted in Figure 1.
Figure 1 – Assay evaluation specific crossing points cut-offs for EGFR L861Q
cut-offs for EGFR L861Q Vol. 58 | No. 5 | 2015 Vol. 58 | No. 5 | 2015 268
www.BioTechniques.com www.BioTechniques.com
Assay evaluation specific crossing points
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56 |
Page 57 |
Page 58 |
Page 59 |
Page 60 |
Page 61 |
Page 62 |
Page 63 |
Page 64 |
Page 65 |
Page 66 |
Page 67 |
Page 68