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The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

ABSTRACT: Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected based on the specific EGFR mutations. Therefore, the reliable detection of mutations is critical for the application of appropriate therapy. In this study, we tested a two-tiered mutation detection strategy using real-time PCR assays as a well-validated high-sensitivity method and multiplex ligation-dependent probe amplification (MLPA)-based EGFRmut+ assay as a second-tier standard-sensitivity method. One additional advantage of the applied MLPA method is that it allows the simultaneous detection of EGFR mutations and copy-number alterations (i.e., amplifications) in EGFR, MET and ERBB2. Our analysis showed high concordance between these two methods. With the use of this two-tier strategy, we reliably determined the frequency of EGFR mutations and EGFR, MET and ERBB2 amplifications in over 200 lung cancer samples. Additionally, taking advantage of simultaneous copy number and small mutation analyses, we showed a very strong correlation between EGFR mutations and EGFR amplifications and a mutual exclusiveness of EGFR mutations/amplifications with MET and ERBB2 amplifications. Our results proved the reliability and usefulness of the two-tiered EGFR testing strategy.

SUBMITTER: Lewandowska MA

PROVIDER: S-EPMC4342230 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

Lewandowska Marzena Anna MA Czubak Karol K Klonowska Katarzyna K Jozwicki Wojciech W Kowalewski Janusz J Kozlowski Piotr P

PloS one 20150226 2

Lung cancer is the leading cause of cancer-related death worldwide. Recent progress in lung cancer diagnosis and treatment has been achieved due to a better understanding the molecular mechanisms of the disease and the identification of biomarkers that allow more specific cancer treatments. One of the best known examples of personalized therapy is the use of tyrosine kinase inhibitors, such as gefitinib and erlotinib, for the successful treatment of non-small-cell lung cancer patients selected b ...[more]

PMID: 25719557

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Project description:BackgroundThe epidermal growth factor receptor (EGFR) gene is the prototype member of the type I receptor tyrosine kinase (TK) family and plays a pivotal role in cell proliferation and differentiation. There are three well described polymorphisms that are associated with increased protein production in experimental systems: a polymorphic dinucleotide repeat (CA simple sequence repeat 1 [CA-SSR1]) in intron one (lower number of repeats) and two single nucleotide polymorphisms (SNPs) in the promoter region, -216 (G/T or T/T) and -191 (C/A or A/A). The objective of this study was to examine distributions of these three polymorphisms and their relationships to each other and to EGFR gene mutations and allelic imbalance (AI) in non-small cell lung cancers.Methods and findingsWe examined the frequencies of the three polymorphisms of EGFR in 556 resected lung cancers and corresponding non-malignant lung tissues from 336 East Asians, 213 individuals of Northern European descent, and seven of other ethnicities. We also studied the EGFR gene in 93 corresponding non-malignant lung tissue samples from European-descent patients from Italy and in peripheral blood mononuclear cells from 250 normal healthy US individuals enrolled in epidemiological studies including individuals of European descent, African-Americans, and Mexican-Americans. We sequenced the four exons (18-21) of the TK domain known to harbor activating mutations in tumors and examined the status of the CA-SSR1 alleles (presence of heterozygosity, repeat number of the alleles, and relative amplification of one allele) and allele-specific amplification of mutant tumors as determined by a standardized semiautomated method of microsatellite analysis. Variant forms of SNP -216 (G/T or T/T) and SNP -191 (C/A or A/A) (associated with higher protein production in experimental systems) were less frequent in East Asians than in individuals of other ethnicities (p < 0.001). Both alleles of CA-SSR1 were significantly longer in East Asians than in individuals of other ethnicities (p < 0.001). Expression studies using bronchial epithelial cultures demonstrated a trend towards increased mRNA expression in cultures having the variant SNP -216 G/T or T/T genotypes. Monoallelic amplification of the CA-SSR1 locus was present in 30.6% of the informative cases and occurred more often in individuals of East Asian ethnicity. AI was present in 44.4% (95% confidence interval: 34.1%-54.7%) of mutant tumors compared with 25.9% (20.6%-31.2%) of wild-type tumors (p = 0.002). The shorter allele in tumors with AI in East Asian individuals was selectively amplified (shorter allele dominant) more often in mutant tumors (75.0%, 61.6%-88.4%) than in wild-type tumors (43.5%, 31.8%-55.2%, p = 0.003). In addition, there was a strong positive association between AI ratios of CA-SSR1 alleles and AI of mutant alleles.ConclusionsThe three polymorphisms associated with increased EGFR protein production (shorter CA-SSR1 length and variant forms of SNPs -216 and -191) were found to be rare in East Asians as compared to other ethnicities, suggesting that the cells of East Asians may make relatively less intrinsic EGFR protein. Interestingly, especially in tumors from patients of East Asian ethnicity, EGFR mutations were found to favor the shorter allele of CA-SSR1, and selective amplification of the shorter allele of CA-SSR1 occurred frequently in tumors harboring a mutation. These distinct molecular events targeting the same allele would both be predicted to result in greater EGFR protein production and/or activity. Our findings may help explain to some of the ethnic differences observed in mutational frequencies and responses to TK inhibitors.

Project description:BackgroundThe use of tyrosine kinase inhibitors to target the epidermal growth factor receptor gene (EGFR) in patients with non-small-cell lung cancer is effective but limited by the emergence of drug-resistance mutations. Molecular characterization of circulating tumor cells may provide a strategy for noninvasive serial monitoring of tumor genotypes during treatment.MethodsWe captured highly purified circulating tumor cells from the blood of patients with non-small-cell lung cancer using a microfluidic device containing microposts coated with antibodies against epithelial cells. We performed EGFR mutational analysis on DNA recovered from circulating tumor cells using allele-specific polymerase-chain-reaction amplification and compared the results with those from concurrently isolated free plasma DNA and from the original tumor-biopsy specimens.ResultsWe isolated circulating tumor cells from 27 patients with metastatic non-small-cell lung cancer (median number, 74 cells per milliliter). We identified the expected EGFR activating mutation in circulating tumor cells from 11 of 12 patients (92%) and in matched free plasma DNA from 4 of 12 patients (33%) (P=0.009). We detected the T790M mutation, which confers drug resistance, in circulating tumor cells collected from patients with EGFR mutations who had received tyrosine kinase inhibitors. When T790M was detectable in pretreatment tumor-biopsy specimens, the presence of the mutation correlated with reduced progression-free survival (7.7 months vs. 16.5 months, P<0.001). Serial analysis of circulating tumor cells showed that a reduction in the number of captured cells was associated with a radiographic tumor response; an increase in the number of cells was associated with tumor progression, with the emergence of additional EGFR mutations in some cases.ConclusionsMolecular analysis of circulating tumor cells from the blood of patients with lung cancer offers the possibility of monitoring changes in epithelial tumor genotypes during the course of treatment.

Project description:Lung cancer is a leading cause of cancer-related deaths in Latin America, with non-small cell lung cancer (NSCLC) being the most prevalent. The current study aimed to report real-world data on epidermal growth factor receptor (EGFR) mutational testing and treatment regimens at diagnosis and progression in patients with metastatic NSCLC across four Latin American countries (Argentina, Chile, Colombia and Uruguay). A retrospective, multicenter, observational study was conducted in patients with NSCLC using medical records from participating countries. The study population was categorized into two cohorts: Cohort 1 comprised of newly diagnosed, treatment-naïve patients with stage IV NSCLC; and cohort 2 comprised of stage IV NSCLC EGFR mutation (EGFRm)-positive patients who had progressed after first- or second-generation EGFR-tyrosine kinase inhibitor (TKI) treatment. Measures included demographic variables, health characteristics, treatment regimen, molecular testing rate and turnaround time at diagnosis and at progression for cohorts 1 and 2, respectively. Descriptive statistics were used to summarize all study measures. Of the 462 patients enrolled, 431 were newly diagnosed or treatment naïve with metastatic NSCLC. In cohort 1, the majority of patients with private health insurance (57.31%) underwent molecular diagnosis while only 41.3% of patients within the public sector had access to testing. The average molecular testing rate in cohort 1 varied across countries, with Argentina having the highest testing rate (79%) and Uruguay the lowest (27.63%). EGFRm was observed in 22% of patients. Cohort 2 comprised 31 patients who had progressed after first- or second-generation EGFR-TKI treatment and of these, only 22 (70.97%) underwent testing after progression. Access to molecular testing is still a challenge impacting the choice of first-line treatment in Latin American patients with NSCLC. These findings underline the unmet needs of ensuring early diagnosis, molecular profiling and use of correct treatment to alleviate NSCLC burden in the region.

Dataset Information

The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

Publications

The use of a two-tiered testing strategy for the simultaneous detection of small EGFR mutations and EGFR amplification in lung cancer.

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