A novel liquidchip platform for simultaneous detection of 70 alleles of DNA somatic mutations on EGFR, KRAS, BRAF and PIK3CA from formalin-fixed and paraffin-embedded slides containing tumor tissue.
BACKGROUND
DNA somatic mutations of EGFR, KRAS, BRAF and PIK3CA in the epidermal growth factor receptor (EGFR) signaling pathway play critical roles in the response or resistance of tumors to targeted therapy with tyrosine kinase inhibitors (EGFR-TKIs). To provide a high-throughput (HTP) clinical testing service for detecting these mutations, we developed a novel platform, SurPlex®-xTAG70plex-EGFR liquidchip.


METHODS
This platform was developed based on a universal 100-tag system. The procedures for multiplex PCR, allele specific primer extension (ASPE) and hybridization were optimized and standardized.


RESULTS
A total of 70 alleles of somatic mutations of EGFR, KRAS, BRAF and PIK3CA can be detected simultaneously in one reaction from one formalin-fixed and paraffin-embedded (FFPE) slide within one day. Cross-reaction was < 8% between individual amplimers and 70 different ASPE primers. The sensitivity for detecting mutants in the wild-type DNA was 1%-5%. Seventy-three FFPE samples with somatic mutations were used to validate the 70plex. Seventy-one showed a complete match, while two were not detected.


CONCLUSIONS
A simple, accurate, sensitive HTP technology was developed and standardized for detecting simultaneously 70 different alleles of EGFR, KRAS, BRAF and PIK3CA gene mutations from FFPE tumor slides.
SurExam Bio-Tech Co. Ltd., Guangzhou Technology Innovation Base, Science City, Guangzhou, PR China.
Guoqiang
G
Li
Xiaodi
X
Luo
Jiaying
J
He
Zeyao
Z
Zhu
Gang
G
Yu
Huijuan
H
Qin
Tao
T
Zeng
Zhiming
Z
Liu
Shiyang
S
Wu
Jiasen
J
Xu
Lifen
L
Ren-Heidenreich
D000483:Alleles; D058534:Class I Phosphatidylinositol 3-Kinases; D004252:DNA Mutational Analysis / Q000191:economics / Q000379:methods*; D066246:ErbB Receptors / Q000235:genetics; D005557:Formaldehyde / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D009369:Neoplasms / Q000235:genetics / Q000473:pathology*; D016612:Paraffin Embedding; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics; D048493:Proto-Oncogene Proteins B-raf / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D013997:Time Factors; D016707:Tissue Fixation; D018631:ras Proteins / Q000235:genetics
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D005557:Formaldehyde; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C484760:PIK3CA protein, human; C512478:EGFR protein, human; D066246:ErbB Receptors; C482119:BRAF protein, human; D048493:Proto-Oncogene Proteins B-raf; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins