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Detection of epidermal growth factor receptor mutation in lung cancer by droplet digital polymerase chain reaction.

Xu Q, Zhu Y, Bai Y, Wei X, Zheng X, Mao M, Zheng G - Onco Targets Ther (2015)

Bottom Line: A resistance mutation in exon 20 (T790M) has been found to accompany drug treatment when patients relapse.The ddPCR assay for T790M could detect patient samples that the qPCR method failed to detect.Two patients with the ex19del mutation also had a naïve T790M mutation.

View Article: PubMed Central - PubMed

Affiliation: Translational Bioscience and Diagnostics, WuXi AppTec, Shanghai, People's Republic of China.

ABSTRACT

Background: Two types of epidermal growth factor receptor (EGFR) mutations in exon 19 and exon 21 (ex19del and L858R) are prevalent in lung cancer patients and sensitive to targeted EGFR inhibition. A resistance mutation in exon 20 (T790M) has been found to accompany drug treatment when patients relapse. These three mutations are valuable companion diagnostic biomarkers for guiding personalized treatment. Quantitative polymerase chain reaction (qPCR)-based methods have been widely used in the clinic by physicians to guide treatment decisions. The aim of this study was to evaluate the technical and clinical sensitivity and specificity of the droplet digital polymerase chain reaction (ddPCR) method in detecting the three EGFR mutations in patients with lung cancer.

Methods: Genomic DNA from H1975 and PC-9 cells, as well as 92 normal human blood specimens, was used to determine the technical sensitivity and specificity of the ddPCR assays. Genomic DNA of formalin-fixed, paraffin-embedded specimens from 78 Chinese patients with lung adenocarcinoma were assayed using both qPCR and ddPCR.

Results: The three ddPCR assays had a limit of detection of 0.02% and a wide dynamic range from 1 to 20,000 copies measurement. The L858R and ex19del assays had a 0% background level in the technical and clinical settings. The T790M assay appeared to have a 0.03% technical background. The ddPCR assays were robust for correct determination of EGFR mutation status in patients, and the dynamic range appeared to be better than qPCR methods. The ddPCR assay for T790M could detect patient samples that the qPCR method failed to detect. About 49% of this patient cohort had EGFR mutations (L858R, 15.4%; ex19del, 29.5%; T790M, 6.4%). Two patients with the ex19del mutation also had a naïve T790M mutation.

Conclusion: These data suggest that the ddPCR method could be useful in the personalized treatment of patients with lung cancer.

No MeSH data available.


Related in: MedlinePlus

Background of L858R and ex19del assays in formalin-fixed, paraffin-embedded genomic DNA.Notes: (A) Twenty-three samples positive in ex19del showed no copies of the L858R allele. (B) Twelve samples positive in L858R showed no copies of the ex19del allele.
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f2-ott-8-1533: Background of L858R and ex19del assays in formalin-fixed, paraffin-embedded genomic DNA.Notes: (A) Twenty-three samples positive in ex19del showed no copies of the L858R allele. (B) Twelve samples positive in L858R showed no copies of the ex19del allele.

Mentions: To determine the background level of ddPCR assays in the clinical samples, we compared samples with known L858R and ex19del mutation status based on their mutual exclusivity. For the L858R assay, no copies of mutant alleles were detected in any of the 23 ex19del-positive samples (Figure 2A); and for the ex19del assay, no copies of mutant alleles were detected in any of the 12 L858R-positive samples (Figure 2B). This result is consistent with the high specificity result obtained with 20,000 copies of normal human blood genomic DNA described above, suggesting that the two assays were highly specific in the FFPE specimens.


Detection of epidermal growth factor receptor mutation in lung cancer by droplet digital polymerase chain reaction.

Xu Q, Zhu Y, Bai Y, Wei X, Zheng X, Mao M, Zheng G - Onco Targets Ther (2015)

Background of L858R and ex19del assays in formalin-fixed, paraffin-embedded genomic DNA.Notes: (A) Twenty-three samples positive in ex19del showed no copies of the L858R allele. (B) Twelve samples positive in L858R showed no copies of the ex19del allele.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4482376&req=5

f2-ott-8-1533: Background of L858R and ex19del assays in formalin-fixed, paraffin-embedded genomic DNA.Notes: (A) Twenty-three samples positive in ex19del showed no copies of the L858R allele. (B) Twelve samples positive in L858R showed no copies of the ex19del allele.
Mentions: To determine the background level of ddPCR assays in the clinical samples, we compared samples with known L858R and ex19del mutation status based on their mutual exclusivity. For the L858R assay, no copies of mutant alleles were detected in any of the 23 ex19del-positive samples (Figure 2A); and for the ex19del assay, no copies of mutant alleles were detected in any of the 12 L858R-positive samples (Figure 2B). This result is consistent with the high specificity result obtained with 20,000 copies of normal human blood genomic DNA described above, suggesting that the two assays were highly specific in the FFPE specimens.

Bottom Line: A resistance mutation in exon 20 (T790M) has been found to accompany drug treatment when patients relapse.The ddPCR assay for T790M could detect patient samples that the qPCR method failed to detect.Two patients with the ex19del mutation also had a naïve T790M mutation.

View Article: PubMed Central - PubMed

Affiliation: Translational Bioscience and Diagnostics, WuXi AppTec, Shanghai, People's Republic of China.

ABSTRACT

Background: Two types of epidermal growth factor receptor (EGFR) mutations in exon 19 and exon 21 (ex19del and L858R) are prevalent in lung cancer patients and sensitive to targeted EGFR inhibition. A resistance mutation in exon 20 (T790M) has been found to accompany drug treatment when patients relapse. These three mutations are valuable companion diagnostic biomarkers for guiding personalized treatment. Quantitative polymerase chain reaction (qPCR)-based methods have been widely used in the clinic by physicians to guide treatment decisions. The aim of this study was to evaluate the technical and clinical sensitivity and specificity of the droplet digital polymerase chain reaction (ddPCR) method in detecting the three EGFR mutations in patients with lung cancer.

Methods: Genomic DNA from H1975 and PC-9 cells, as well as 92 normal human blood specimens, was used to determine the technical sensitivity and specificity of the ddPCR assays. Genomic DNA of formalin-fixed, paraffin-embedded specimens from 78 Chinese patients with lung adenocarcinoma were assayed using both qPCR and ddPCR.

Results: The three ddPCR assays had a limit of detection of 0.02% and a wide dynamic range from 1 to 20,000 copies measurement. The L858R and ex19del assays had a 0% background level in the technical and clinical settings. The T790M assay appeared to have a 0.03% technical background. The ddPCR assays were robust for correct determination of EGFR mutation status in patients, and the dynamic range appeared to be better than qPCR methods. The ddPCR assay for T790M could detect patient samples that the qPCR method failed to detect. About 49% of this patient cohort had EGFR mutations (L858R, 15.4%; ex19del, 29.5%; T790M, 6.4%). Two patients with the ex19del mutation also had a naïve T790M mutation.

Conclusion: These data suggest that the ddPCR method could be useful in the personalized treatment of patients with lung cancer.

No MeSH data available.


Related in: MedlinePlus