Quantifying EGFR alterations in the lung cancer genome with nanofluidic digital PCR arrays.
BACKGROUND
The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable.


METHODS
We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array.


RESULTS
The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors.


CONCLUSIONS
The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors.
Wang
Jun
J
Fluidigm Corporation, South San Francisco, CA, USA.
Ramakrishnan
Ramesh
R
Tang
Zhe
Z
Fan
Weiwen
W
Kluge
Amy
A
Dowlati
Afshin
A
Jones
Robert C
RC
Ma
Patrick C
PC
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000483:Alleles; D004252:DNA Mutational Analysis; D004273:DNA, Neoplasm / Q000235:genetics*; D018450:Disease Progression; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000235:genetics*; D005260:Female; D015894:Genome, Human / Q000235:genetics; D005838:Genotype; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D009154:Mutation; D009367:Neoplasm Staging; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; C512478:EGFR protein, human; D066246:ErbB Receptors