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Journal ArticleDOI

EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples

01 Feb 2013-Journal of Clinical Pathology (BMJ Publishing Group)-Vol. 66, Iss: 2, pp 79-89
TL;DR: Cytology samples including fine needle aspirate and pleural effusion can be used successfully to determine EGFR mutation status provided that sensitive testing methods are employed, and several different testing methods offer a more sensitive alternative to direct sequencing for the detection of common EGFR mutations.
Abstract: Aims Activating mutations in the gene encoding epidermal growth factor receptor (EGFR) can confer sensitivity to EGFR tyrosine kinase inhibitors such as gefitinib in patients with advanced non-small-cell lung cancer. Testing for mutations in EGFR is therefore an important step in the treatment-decision pathway. We reviewed reported methods for EGFR mutation testing in patients with lung cancer, initially focusing on studies involving standard tumour tissue samples. We also evaluated data on the use of cytology samples in order to determine their suitability for EGFR mutation analysis. Methods We searched the MEDLINE database for studies reporting on EGFR mutation testing methods in patients with lung cancer. Results Various methods have been investigated as potential alternatives to the historical standard for EGFR mutation testing, direct DNA sequencing. Many of these are targeted methods that specifically detect the most common EGFR mutations. The development of targeted mutation testing methods and commercially available test kits has enabled sensitive, rapid and robust analysis of clinical samples. The use of screening methods, subsequent to sample micro dissection, has also ensured that identification of more rare, uncommon mutations is now feasible. Cytology samples including fine needle aspirate and pleural effusion can be used successfully to determine EGFR mutation status provided that sensitive testing methods are employed. Conclusions Several different testing methods offer a more sensitive alternative to direct sequencing for the detection of common EGFR mutations. Evidence published to date suggests cytology samples are viable alternatives for mutation testing when tumour tissue samples are not available.
Citations
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Journal ArticleDOI
TL;DR: The 2013 guideline was largely reaffirmed with updated recommendations to allow testing of cytology samples, require improved assay sensitivity, and recommend against the use of immunohistochemistry for EGFR testing.
Abstract: Context.— In 2013, an evidence-based guideline was published by the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular P...

491 citations

Journal Article
TL;DR: Co-amplification at Lower Denaturation temperature (COLD-PCR) as mentioned in this paper is a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence.
Abstract: 1796 Introduction: The Polymerase Chain Reaction (PCR) has become the cornerstone of molecular diagnosis, with almost every genetic test aiming to identify DNA sequence variation incorporating PCR. A commonly encountered problem is that variant DNA sequences exist in the presence of a large majority of wild type alleles such as when DNA is obtained from heterogeneous cancer biopsies or when fetal alleles are sought in maternal blood. As a result, downstream assays are severely limited in their ability to identify subtle genetic changes that can have profound impact in clinical decision-making and outcome. We describe Co-amplification at Lower Denaturation temperature (COLD-PCR), a novel form of PCR that amplifies minority alleles selectively from mixtures of wild-type and mutation-containing sequences irrespective of the mutation type or position on the sequence (Nature Medicine, In Press). Methods: In COLD-PCR, an intermediate annealing temperature is used during PCR-cycling to allow cross-hybridization of mutant and wild type alleles; hetero-duplexes, which melt at lower temperatures than homo-duplexes, are then selectively denatured and amplified at Critical Denaturation Temperature (Tc), while homo-duplexes remain double-stranded and do not amplify efficiently. By fixing the denaturation temperature to Tc, mutations at any position along the sequence are enriched during COLD-PCR amplification. To validate COLD-PCR, we used serial dilutions of DNA from tumor-derived cell lines containing point mutations or micro-deletions at different positions along p53 exon 8 and Kras exon 1 (codons 12-13), or samples with micro-deletions in EGFR exon 19. In addition, genomic DNA from a series of colon and lung cancer surgical specimens, and plasma-circulating DNA collected under IRB approval were utilized for validation on clinical specimens. Results: By replacing regular PCR with COLD-PCR prior to application of a range of assays (Sanger sequencing; Pyrosequencing; MALDI-TOF; dHPLC; RFLP; and Taqman) we improved mutation detection sensitivity up to 100-fold and identified several additional p53/Kras/EGFR mutations in heterogeneous cancer samples. 4 of 43 surgical samples and 3 of 10 plasma samples tested contained clinically important mutations that were not detected by any of the methods tested when preceded by regular-PCR, but they were detectable following COLD-PCR. Conclusion: Replacement of regular PCR with COLD-PCR provides a universal boost to most mutation detection technologies and enables them to be used with the required confidence in routine screening of cancer specimens for somatic mutations, including low-level mutation screening of surgical/FFPE tumor samples or bodily fluids. COLD-PCR is also anticipated to find application in pre-natal diagnosis, in infectious diseases and in epigenetics.

231 citations

Journal ArticleDOI
TL;DR: The 2019 edition of the World Health Organization (WHO) Classification of Thoracic Tumours was published earlier this year, with classification of lung tumors being one of the chapters as discussed by the authors .

230 citations

Journal ArticleDOI
TL;DR: Adenocarcinomas are further classified based on architectural pattern to delineate tissue types of prognostic significance in resection specimens and in small biopsy or cytology specimens to effectively select tumors for targeted molecular testing.

222 citations


Cites methods from "EGFR mutation testing in lung cance..."

  • ...Targeted methods include polymerase chain reaction (PCR) -based targeted methods, such as ARMS (Amplification Refractory Mutation SystemARMS) and SmartAMP (Smart Amplification Process).(85,86) Gefitinib, erlotinib, and afatinib are currently...

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References
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Journal ArticleDOI
TL;DR: The results for 20 world regions are presented, summarizing the global patterns for the eight most common cancers, and striking differences in the patterns of cancer from region to region are observed.
Abstract: Estimates of the worldwide incidence and mortality from 27 cancers in 2008 have been prepared for 182 countries as part of the GLOBOCAN series published by the International Agency for Research on Cancer. In this article, we present the results for 20 world regions, summarizing the global patterns for the eight most common cancers. Overall, an estimated 12.7 million new cancer cases and 7.6 million cancer deaths occur in 2008, with 56% of new cancer cases and 63% of the cancer deaths occurring in the less developed regions of the world. The most commonly diagnosed cancers worldwide are lung (1.61 million, 12.7% of the total), breast (1.38 million, 10.9%) and colorectal cancers (1.23 million, 9.7%). The most common causes of cancer death are lung cancer (1.38 million, 18.2% of the total), stomach cancer (738,000 deaths, 9.7%) and liver cancer (696,000 deaths, 9.2%). Cancer is neither rare anywhere in the world, nor mainly confined to high-resource countries. Striking differences in the patterns of cancer from region to region are observed.

21,040 citations

Journal ArticleDOI
TL;DR: A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib, and these mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor.
Abstract: BACKGROUND Most patients with non-small-cell lung cancer have no response to the tyrosine kinase inhibitor gefitinib, which targets the epidermal growth factor receptor (EGFR). However, about 10 percent of patients have a rapid and often dramatic clinical response. The molecular mechanisms underlying sensitivity to gefitinib are unknown. METHODS We searched for mutations in the EGFR gene in primary tumors from patients with non-small-cell lung cancer who had a response to gefitinib, those who did not have a response, and those who had not been exposed to gefitinib. The functional consequences of identified mutations were evaluated after the mutant proteins were expressed in cultured cells. RESULTS Somatic mutations were identified in the tyrosine kinase domain of the EGFR gene in eight of nine patients with gefitinib-responsive lung cancer, as compared with none of the seven patients with no response (P<0.001). Mutations were either small, in-frame deletions or amino acid substitutions clustered around the ATP-binding pocket of the tyrosine kinase domain. Similar mutations were detected in tumors from 2 of 25 patients with primary non-small-cell lung cancer who had not been exposed to gefitinib (8 percent). All mutations were heterozygous, and identical mutations were observed in multiple patients, suggesting an additive specific gain of function. In vitro, EGFR mutants demonstrated enhanced tyrosine kinase activity in response to epidermal growth factor and increased sensitivity to inhibition by gefitinib. CONCLUSIONS A subgroup of patients with non-small-cell lung cancer have specific mutations in the EGFR gene, which correlate with clinical responsiveness to the tyrosine kinase inhibitor gefitinib. These mutations lead to increased growth factor signaling and confer susceptibility to the inhibitor. Screening for such mutations in lung cancers may identify patients who will have a response to gefitinib.

10,879 citations


"EGFR mutation testing in lung cance..." refers background in this paper

  • ...While fragment length analysis is used widely in practice, it can only detect insertions or deletions and does not allow detection of point mutations in EGFR....

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  • ...4 Two types of mutation—short in-frame deletions in exon 19, clustered around the amino-acid residues 747–750 and a specific exon 21 point mutation (L858R)—have been reported to comprise up to 90% of all activating EGFR mutations.(3) 4 13 Other activating mutations include point mutations in exon 18 (including mutations in codon 719) and point mutations and in-frame insertions in exon 20 (including T790M)....

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  • ...The conformational change seen in the TK domain of mutated EGFRs increases the activation of the domain and its affinity for ATP (and EGFR TKIs) compared with wild-type EGFR.(3)The resulting increase in binding of EGFR TKIs produces greater inhibition of the domain and blocking of signal transduction pathways implicated in the proliferation and survival of cancer cells....

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  • ...Gefitinib also improved PFS versus chemotherapy in two Phase III trials performed solely in patients with EGFR mutationpositive advanced NSCLC.6 7 In addition, in two Phase III erlotinib trials that recruited EGFR mutation-positive patients, PFS was significantly increased with first-line erlotinib relative to chemotherapy.9 10 As a result of these data, the accurate identification of patients who might benefit from EGFR TKI therapy has become an important step in the treatment-decision pathway for advanced NSCLC.9 12 Mutations associated with enhanced sensitivity to EGFR TKIs are found in exons 18–21 of the TK domain of EGFR.3 4 Two types of mutation—short in-frame deletions in exon 19, clustered around the amino-acid residues 747–750 and a specific exon 21 point mutation (L858R)—have been reported to comprise up to 90% of all activating EGFR mutations.3 4 13 Other activating mutations include point mutations in exon 18 (including mutations in codon 719) and point mutations and in-frame insertions in exon 20 (including T790M)....

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  • ...In patients with wild-type EGFR, carboplatin/paclitaxel was associated with significantly longer PFS than gefitinib.8 The conformational change seen in the TK domain of mutated EGFRs increases the activation of the domain and its affinity for ATP (and EGFR TKIs) compared with wild-type EGFR.3The resulting increase in binding of EGFR TKIs produces greater inhibition of the domain and blocking of signal transduction pathways implicated in the proliferation and survival of cancer cells....

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Journal ArticleDOI
04 Jun 2004-Science
TL;DR: Results suggest that EGFR mutations may predict sensitivity to gefitinib, and treatment with the EGFR kinase inhibitor gefitsinib causes tumor regression in some patients with NSCLC, more frequently in Japan.
Abstract: Receptor tyrosine kinase genes were sequenced in nonsmall cell lung cancer (NSCLC) and matched normal tissue. Somatic mutations of the epidermal growth factor receptor gene EGFR were found in 15 of 58 unselected tumors from Japan and 1 of 61 from the United States. Treatment with the EGFR kinase inhibitor gefitinib (Iressa) causes tumor regression in some patients with NSCLC, more frequently in Japan. EGFR mutations were found in additional lung cancer samples from U.S. patients who responded to gefitinib therapy and in a lung adenocarcinoma cell line that was hypersensitive to growth inhibition by gefitinib, but not in gefitinibinsensitive tumors or cell lines. These results suggest that EGFR mutations may predict sensitivity to gefitinib. Protein kinase activation by somatic mutation or

9,265 citations

Journal ArticleDOI
TL;DR: Gefit inib is superior to carboplatin-paclitaxel as an initial treatment for pulmonary adenocarcinoma among nonsmokers or former light smokers in East Asia and the presence in the tumor of a mutation of the EGFR gene is a strong predictor of a better outcome with gefitinib.
Abstract: METHODS In this phase 3, open-label study, we randomly assigned previously untreated patients in East Asia who had advanced pulmonary adenocarcinoma and who were nonsmokers or former light smokers to receive gefitinib (250 mg per day) (609 patients) or carboplatin (at a dose calculated to produce an area under the curve of 5 or 6 mg per milliliter per minute) plus paclitaxel (200 mg per square meter of body-surface area) (608 patients). The primary end point was progression-free survival. RESULTS The 12-month rates of progression-free survival were 24.9% with gefitinib and 6.7% with carboplatin–paclitaxel. The study met its primary objective of showing the noninferiority of gefitinib and also showed its superiority, as compared with carboplatin– paclitaxel, with respect to progression-free survival in the intention-to-treat population (hazard ratio for progression or death, 0.74; 95% confidence interval [CI], 0.65 to 0.85; P<0.001). In the subgroup of 261 patients who were positive for the epidermal growth factor receptor gene (EGFR) mutation, progression-free survival was significantly longer among those who received gefitinib than among those who received carboplatin–paclitaxel (hazard ratio for progression or death, 0.48; 95% CI, 0.36 to 0.64; P<0.001), whereas in the subgroup of 176 patients who were negative for the mutation, progression-free survival was significantly longer among those who received carboplatin–paclitaxel (hazard ratio for progression or death with gefitinib, 2.85; 95% CI, 2.05 to 3.98; P<0.001). The most common adverse events were rash or acne (in 66.2% of patients) and diarrhea (46.6%) in the gefitinib group and neurotoxic effects (69.9%), neutropenia (67.1%), and alopecia (58.4%) in the carboplatin–paclitaxel group. CONCLUSIONS Gefitinib is superior to carboplatin–paclitaxel as an initial treatment for pulmonary adenocarcinoma among nonsmokers or former light smokers in East Asia. The presence in the tumor of a mutation of the EGFR gene is a strong predictor of a better outcome with gefitinib. (ClinicalTrials.gov number, NCT00322452.)

7,246 citations


"EGFR mutation testing in lung cance..." refers background in this paper

  • ...In patients with wild-type EGFR, carboplatin/paclitaxel was associated with significantly longer PFS than gefitinib.(8) The conformational change seen in the TK domain of mutated EGFRs increases the activation of the domain and its affinity for ATP (and EGFR TKIs) compared with wild-type EGFR....

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Journal ArticleDOI
TL;DR: First-line gefitinib for patients with advanced non-small-cell lung cancer who were selected on the basis of EGFR mutations improved progression-free survival, with acceptable toxicity, as compared with standard chemotherapy.
Abstract: In the planned interim analysis of data for the first 200 patients, progression-free survival was significantly longer in the gefitinib group than in the standard-chemotherapy group (hazard ratio for death or disease progression with gefitinib, 0.36; P<0.001), resulting in early termination of the study. The gefitinib group had a significantly longer median progression-free survival (10.8 months, vs. 5.4 months in the chemotherapy group; hazard ratio, 0.30; 95% confidence interval, 0.22 to 0.41; P<0.001), as well as a higher response rate (73.7% vs. 30.7%, P<0.001). The median overall survival was 30.5 months in the gefitinib group and 23.6 months in the chemotherapy group (P = 0.31). The most common adverse events in the gefitinib group were rash (71.1%) and elevated amino transferase levels (55.3%), and in the chemotherapy group, neutropenia (77.0%), anemia (64.6%), appetite loss (56.6%), and sensory neuropathy (54.9%). One patient receiving gefitinib died from interstitial lung disease. CONCLUSIONS First-line gefitinib for patients with advanced non–small-cell lung cancer who were selected on the basis of EGFR mutations improved progression-free survival, with acceptable toxicity, as compared with standard chemotherapy. (UMIN-CTR number, C000000376.)

4,829 citations


"EGFR mutation testing in lung cance..." refers background in this paper

  • ...Key messages ▸ The development of targeted EGFR mutation testing methods and commercially available test kits has enabled sensitive, rapid and robust analysis of clinical samples from patients with NSCLC. ▸ The use of screening methods, either used alone or in conjunction with targeted methods, enables the detection of more rare and novel EGFR mutations....

    [...]

  • ...Gefitinib also improved PFS versus chemotherapy in two Phase III trials performed solely in patients with EGFR mutationpositive advanced NSCLC.(6) 7 In addition, in two Phase III erlotinib trials that recruited EGFR mutation-positive patients, PFS was significantly increased with first-line erlotinib relative to chemotherapy....

    [...]

  • ...Gefitinib also improved PFS versus chemotherapy in two Phase III trials performed solely in patients with EGFR mutationpositive advanced NSCLC.6 7 In addition, in two Phase III erlotinib trials that recruited EGFR mutation-positive patients, PFS was significantly increased with first-line erlotinib relative to chemotherapy.9 10 As a result of these data, the accurate identification of patients who might benefit from EGFR TKI therapy has become an important step in the treatment-decision pathway for advanced NSCLC.9 12 Mutations associated with enhanced sensitivity to EGFR TKIs are found in exons 18–21 of the TK domain of EGFR.3 4 Two types of mutation—short in-frame deletions in exon 19, clustered around the amino-acid residues 747–750 and a specific exon 21 point mutation (L858R)—have been reported to comprise up to 90% of all activating EGFR mutations.3 4 13 Other activating mutations include point mutations in exon 18 (including mutations in codon 719) and point mutations and in-frame insertions in exon 20 (including T790M)....

    [...]

  • ...Both screening and targeted methods have been used to identify EGFR mutations in clinical trials of EGFR TKIs in patients with advanced NSCLC.6–10 14–16 84 These trials were not identified by our search due to our focus on method comparison studies....

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