Use of Locked Nucleic Acid Probes (LNA) Greatly Increases Sensitivity of Sanger Sequencing (SS): A Comparative Experience of Genotyping of KRAS Codons 12,13
Dhananjay A Chitale, Lisa Whiteley, Linda Szymanski, Milena Cankovic. Henry Ford Hospital, Detroit, MI
Background: Targeted therapies in pursuit of personalized medicine are growing in number due to new biomarker discoveries. Emerging treatment protocols are using this approach as first-line therapies and the companion diagnostic tests are being ordered on smaller biopsy samples. This has raised the challenge on the molecular laboratories to provide rapid, reliable, accurate and sensitive methods for mutation screening of very limited samples. SS of PCR-amplified DNA, the most widely used method, has limitation due to low sensitivity (∼20-25%). Our aim was to improve the performance of lab developed SS based KRAS mutation assay by modified Sanger sequencing (MSS) with LNA and compare it with allele specific PCR (AS-PCR) and use this as a pilot project to expand on other SS based assays.
Design: To increase the sensitivity of routine SS, we modified the standard PCR assay by adding LNA probes to favor mutant DNA amplification during PCR. Mutations in KRAS codon 12, 13 using SS and MSS were evaluated. Samples were also run on Qiagen's KRAS RGQ PCR kit to detect 7 somatic mutations using real time PCR and using Scorpions and ARMS® technologies (AS-PCR). 21 clinical samples [9 primary adenocarcinomas (7 colorectal,2 lung) and 12-metastatic colorectal adenocarcinomas at various sites] and a dilution series of known positive KRAS mutant cell line were tested in parallel using the 3 above methods. All samples were either formalin-fixed, paraffin-embedded tissues or cytology slides. Manual macrodissection was performed when tumor cellularity was less than 50% of total cells(10/21 cases).
Results: MSS-LNA method detected 3 additional mutations compared to SS and 1 additional mutation compared to AS-PCR. Sensitivity of the assay was determined to be 1% by MSS-LNA & Qiagen KRAS RGQ PCR kit & 20% for SS.
Conclusions: MSS-LNA method increased the detection sensitivity of SS 20-fold, equivalent or more sensitive than AS-PCR. This is not only a cost effective method, but also offers wider coverage for discovering additional rarer mutations not detected by AS-PCR. MSS-LNA method needs much less DNA and thus is a reliable and sensitive method for detecting mutations in any sample type, especially where the sample size is limited or paucicellular.
Wednesday, March 6, 2013 9:30 AM
Poster Session V # 256, Wednesday Morning