| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
From the Department of Pathology
*
and the Department of Medicine,
Program in Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, New York
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
90% of mutated cases: short in-frame deletions in exon 19 and a specific point mutation in exon 21 at codon 858 (L858R). Screening for these mutations has been based mainly on direct sequencing. We report here the development and validation of polymerase chain reaction-based assays for these two predominant types of EGFR mutations. The assay for exon 19 mutations is based on length analysis of fluorescently labeled polymerase chain reaction products, and the assay for the exon 21 L858R mutation is based on a new Sau96I restriction site created by this mutation. Using serial dilutions of DNAs from lung cancer cell lines harboring either exon 19 or 21 mutations, we detected these mutations in the presence of up to 90% normal DNA. In a test set of 39 lung cancer samples, direct sequencing detected mutations in 25 cases whereas our assays were positive in 29 cases, including 4 cases in which mutations were not apparent by sequencing. These assays offer higher sensitivity and ease of scoring and eliminate the need for sequencing, providing a robust and accessible approach to the rapid identification of most lung cancer patients likely to respond to EGFR inhibitors. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
70,000 in the US, the volume of molecular diagnostic assays for EGFR mutations could rival that of ERBB2 amplification assays in breast cancers, if EGFR testing becomes part of standard lung cancer management.
Two mutations account for 90% of EGFR mutations reported to date in lung adenocarcinomas (Table 1)
. The most common mutation type, seen in 46% of cases with EGFR mutations, is a short in-frame deletion of 9, 12, 15, 18, or 24 nucleotides in exon 19. The second most common mutation, seen in 43% of cases with EGFR mutations, is a point mutation (CTG to CGG) in exon 21 at nucleotide 2573, that results in substitution of leucine by arginine at codon 858 (L858R). Other much less common mutations have been described in exons 18, 20, and 21 (Table 1)
. Combining data from four studies,1, 2, 3, 4
it appears that 80% of tumors that respond to gefitinib or erlotinib contain missense mutations or in-frame deletions in the EGFR tyrosine kinase domain, compared to none of 36 drug-refractory tumors (P < 0.05). These studies show that these EGFR mutations correlate strongly with sensitivity to specific EGFR inhibitors and that their detection could be used to predict which patients will respond to these drugs.
|
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
EGFR Exon 19 Deletion Assay
Because 99% of exon 19 mutations reported to date have been short in-frame deletions (Table 1)
, we designed an assay based on length analysis of fluorescently labeled PCR products. A 207-bp genomic fragment including all of exon 19 was amplified using primers EGFR-Ex19-FWD1 and EGFR-Ex19-REV1 (Table 2)
. The reverse primer was labeled with the 6-FAM fluorophore (6-FAM emits fluorescence with a peak wavelength of 522 nm). The PCR reaction mix was made up as follows: HotStarTaq DNA polymerase and 10 x buffer (Qiagen, Valencia, CA), EGFR-Ex19-FWD1 and EGFR-Ex19-REV1 primers (15 pmol each), genomic DNA template (100 ng), PCR Carry-Over prevention kit reagents (N-glycosylase + dUTP) (Applied Biosystems, Foster City, CA), remaining dNTPs, MgCl2 (0.5 mmol/L and sterile distilled water (to 50 µl). The PCR was performed as follows: 50°C x 2 minutes (to complete the PCR Carry-Over prevention procedure), 95°C x 15 minutes (to inactivate N-glycosylase and activate TaqDNA polymerase), followed by 40 cycles of 95°C x 0.5 minutes, 60°C x 1 minute, 72°C x 1 minute, and a final extension of 72°C x 10 minutes. For DNA extracted from frozen tissue, 35 cycles was sufficient. PCR product intensity was checked on a 2% agarose gel. If PCR product intensity was strong (equal or stronger than size marker), a 1 in 50 dilution was made of which 1 µl was added into 20 µl of formamide plus 1 µl of Genescan 400HD size standard (Applied Biosystems). If the PCR product intensity was weak (band fainter than size marker), up to 1 µl of undiluted PCR product was added to 20 µl or formamide and 1 µl of Genescan 400HD size standard. The samples were denatured at 95°C for 5 minutes and cooled on ice for 5 minutes. They were then subjected to capillary electrophoresis using POP4 polymer with an excitation wavelength of 494 nm and a detection wavelength of 522 nm on an ABI 3100 Avant genetic analyzer (Applied Biosystems).
|
that can be used as the basis for a PCR restriction fragment length polymorphism (PCR-RFLP) assay design. Another Sau96I restriction site upstream in exon 21 provides an internal restriction enzyme digestion control. The digested fluorescently labeled PCR products are analyzed by capillary electrophoresis. The precise product sizing possible by this approach allows unambiguous and sensitive identification of the 173-bp digested wild-type product and 87-bp digested mutant PCR product. Specifically, a 222-bp genomic fragment including all of exon 21 was amplified using primers EGFR-Ex21-FWD1 and EGFR-Ex21-REV1 (Table 2)
. Again, the reverse primer was labeled with the 6-FAM fluorophore. The PCR reaction mix was made up as above, but with EGFR-Ex21-FWD1 and EGFR-Ex21-REV1 primers (15 pmol each). The PCR was performed as follows: 50°C x 2 minutes (to complete PCR Carry-Over prevention procedure), 95°C x 15 minutes (to inactivate N-glycosylase and activate TaqDNA polymerase), followed by 95°C x 0.5 minutes, 60°C x 1 minute, 72°C x 1 minute, and a final extension of 72°C x 10 minutes for 40 cycles if the DNA was from paraffin blocks, 35 cycles if the DNA was from frozen tissue. PCR product intensity was checked on a 2% agarose gel. The PCR products were then purified using PCR Kleen Spin column (Bio-Rad, Hercules, CA) at 735 x g for 2 minutes. This step was not included in the procedure for the exon 19 deletion analysis, but was found to be necessary in the exon 21 assay to reduce baseline noise and increase sensitivity after restriction enzyme digestion. The Sau96I digestion reaction was then performed at 37°C for 2 hours and consisted of the following: 5 µl of PCR product, 2 µl of 10x NEBuffer 4 (New England Biolabs, Beverly, MA), 2 µl (10 U) of Sau96I restriction enzyme (New England Biolabs), and 11 µl of sterile distilled water. After digestion, 1 µl of undiluted Sau96I-digested PCR product was added to 20 µl of formamide and 1 µl of Genescan 400HD size standard. The samples were denatured at 95°C for 5 minutes, cooled on ice for 5 minutes, and subjected to capillary electrophoresis as described above.
|
|
Results |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. All cases with deletions also showed a germline product of 207 bp. Exon 19 deletion with loss of the remaining normal allele appears rare but preferential PCR amplification of the shorter deleted product could result in rare cases with a very small germline product of 207 bp. Furthermore, the nonneoplastic cells present in lung cancer samples should always provide a template for PCR amplification of the germline allele. Negative cases showed the expected germline product of 207 bp and the absence of any peak above background noise at 9, 12, 15, 18, 21, or 24 bp below the normal product in the electrophoretogram. Negative controls (including placenta and 21 lymphoma samples) consistently showed the germline product only.
|
. In comparison, detection of the exon 19 deletion by direct sequencing was only readily possible down to a dilution of 12.5% H1650 DNA (Supplemental Figure 1
at http://jmd.amjpathol.org/). We should note that because of ploidy differences between cancer cell lines and nonneoplastic cells, sensitivities based on dilutions of DNA should be viewed only as approximations of the absolute sensitivity based on dilutions of cells, however this should not affect relative comparisons of the analytical sensitivities of different techniques.
|
EGFR Exon 21 L858R Mutation Assay
The same cases were also screened for the exon 21 L858R mutation by a PCR-RFLP assay based on a new Sau96I restriction site created by the L858R mutation (2573T>G). The Sau96I-digested fluorescently labeled PCR products were analyzed by capillary electrophoresis. Examples of undigested, digested germline, and digested mutant PCR products are shown in Figure 4
. All cases showed a germline product of 173 bp. Digestion of both Sau96I sites was complete in all runs as shown by the absence of a 222-bp undigested peak. Any case in which the peak corresponding to the mutated allele was less than five times the local background noise in the electrophoretogram (for example, see peak at 1.6% tumor cells in Figure 5
) was considered at risk for a false-positive result and was repeated at least once using two to five times more template. Negative controls showed the expected germline digested product of 173 bp and the absence of any peak above background noise at 87 bp. Negative controls (including placenta and 21 lymphoma samples) consistently showed the germline product only.
|
|
. In comparison, detection of the exon 21 L858R mutation by direct sequencing was only possible down to a dilution of 6.25% H1975 DNA (Supplemental Figure 2
at http://jmd.amjpathol.org/).
Comparison to Direct Sequencing
We then tested 39 lung cancer samples with both assays and compared the results to direct sequencing. To provide an adequate test of the sensitivity of our assays, this series of cases was enriched for mutated cases based on previous sequencing data (and is therefore not representative of mutation prevalence). The exon 19 deletion assay was positive in 15 of 39 cases and the exon 21 L858R assay was positive in 14 of 39 cases. No cases were positive for both assays. In four cases (two exon 19, two exon 21), the PCR-based assays detected mutations not apparent by direct sequencing. In an additional two cases positive by our exon 21 assay, direct sequencing was only equivocally positive (results not shown). The results are summarized in Table 3
.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
. It has been previously shown that this assay design is as accurate as and more sensitive than direct sequencing for the detection of in-frame deletion (or insertion) mutations9
(in the context of exon 11 KIT mutations in gastrointestinal stromal tumors). Although there is a theoretical risk that an in-frame deletion could create a novel stop codon (with opposite biological consequences), this has not been reported in more than 100 exon 19 deletions sequenced to date. Interestingly, the only other type of mutation so far reported in exon 19, an 18-bp insertion found in a single tumor,8
should also be detected by the present assay. Indeed, we have recently observed another such case in our clinical testing (unpublished data).
Using this assay on 200 clinical samples throughout a 6-month period, we have observed 9-, 12-, 15-, 18-, and 24-bp deletions in exon 19 of EGFR. By far the most common deletion size is 15 bp. A peculiar observation is that 21-bp deletions have so far not been reported and 12-bp deletions are rare. Novel deletions (eg, 6 bp, 21 bp, or 27 bp) not previously characterized in terms of function or association with therapy response should be considered of uncertain significance although their functional impact may be similar if they affect the LREA motif that forms the core of all presently described deletions. It should also be noted that, although a minority of tumors have been reported to carry two mutations (Table 1)
, the two hotspot mutations in exons 19 and 21 have never been found together.
Our assays found 10% (4 of 39) more mutations than were detected by direct sequencing. Another two cases were only equivocally positive by direct sequencing (Table 3)
. Thus, the overall potential false-negative rate for direct sequencing compared to our assays is 15% (6 of 39), and this may be an underestimate because our study group was enriched for cases with mutations based on previous sequencing data. We attribute this to the greater analytical sensitivity of our assays. That this should be significant in the setting of lung cancer samples is not surprising given the often considerable admixture of nonneoplastic elements in these tumors.
Based on the aggregate data from four studies,1, 2, 3, 5
10% of lung adenocarcinomas from American or European patients who are unselected for response to EGFR inhibitors contain EGFR mutations. This percentage also matches the response rate of 10% seen in single agent trials of gefitinib or erlotinib performed before the discovery of EGFR mutations.10, 11
However, there appear to be striking ethnic differences in the prevalence of EGFR mutations. It had been previously observed that responses to gefitinib were significantly more frequent in Japanese non-small cell lung cancer patients than in non-Japanese patients.11
That unusual clinical observation is now explained by the finding of a fourfold higher prevalence of EGFR mutations in Asian non-small cell lung cancer patients, ie, 40%.4, 8
It is important to note that the use of the clinical term non-small cell lung cancer may result in unnecessary testing. This is because the vast majority of EGFR mutations are found in moderately to well differentiated adenocarcinomas, in particular those with partial or complete bronchioloalveolar features. The same histological features had been shown to correlate with response to EGFR inhibitors before the discovery of these mutations.12 In contrast, mutations appear exceedingly rare in large cell carcinomas2, 5, 7 and adenosquamous carcinomas.4, 8 Lastly, numerous pure squamous carcinomas (>500), including one with apparent response to gefitinib,3 have been tested but were uniformly negative.4, 5, 8
Other strong correlates of EGFR mutations (and response to EGFR inhibitors) include female sex and never smoker status.3, 5, 8, 12 It is presently estimated that females are approximately three times as likely as males to have mutation-positive tumors and never smokers are at least five times as likely to have mutation-positive tumors as past or present smokers.13 However, the interrelationships between these different factors have not yet been fully explored by multivariate analyses.
The two assays described here can be used as first line assays in all cases submitted for EGFR mutation analysis. The advantages of this overall approach to EGFR mutation screening is that the two types of mutation accounting for 90% of all mutations (exon 19 deletion and exon 21 L858R mutation) are detected by techniques that are faster (ie, 1 day versus 2 days) and more sensitive than direct sequencing, allowing for prompt initiation of treatment in most of the patients likely to respond to EGFR inhibitors. If both assays are negative, these first line assays can be followed by assays based on direct sequencing of EGFR exons 18, 20, and 21 to detect the remaining 10% of EGFR mutations. The predictive value of these assays could be further enhanced by combining them with testing for KRAS exon 2 mutations. Recent data from our center and elsewhere have shown that KRAS exon 2 mutations, present in 15 to 30% of lung adenocarcinomas, rarely, if ever, co-exist with EGFR mutations.8, 14
This is biologically consistent because KRAS is downstream in the EGFR signaling pathway. It is therefore not unexpected that KRAS-mutated lung cancers fail to respond to EGFR inhibitors, a clinically important observation.14
Although there is currently still no consensus on the role of EGFR mutation screening in patient management, it is quite possible that testing of tumor tissue for EGFR mutations may soon be indicated in most or all patients with moderately to well differentiated lung adenocarcinoma,13 to aid in selecting therapy in neoadjuvant, adjuvant, and advanced/metastatic settings. In American or European centers, at least 10% of lung adenocarcinomas will harbor EGFR mutations and 90% of these will be detectable using the two EGFR mutation assays described here. Mutational analysis of EGFR is likely to become the mainstay of laboratory testing in this setting because other parameters such as EGFR immunoreactivity or EGFR gene amplification show at best only weak correlation with response to EGFR inhibitors.7, 15, 16 Immunohistochemistry with antibodies to phosphorylated EGFR remains to be systematically studied in this setting but the potential of this approach is uncertain given the observation that mutant EGFR proteins generally do not show simple constitutive autophosphorylation in vitro.1, 3 The search for EGFR mutations in isolated cases of other cancers that have responded to EGFR inhibitors is in progress in many research laboratories. A wide variety of other cancers have already been screened with negative results.13 However, it remains possible that such studies could define new indications for EGFR testing in clinical molecular pathology laboratories in the coming months or years.
|
Acknowledgments |
|---|
|
Footnotes |
|---|
Supported by the Chest and Lungevity Foundations of the American College of Chest Physicians and by an anonymous donor (to W.P.).
Accepted for publication March 16, 2005.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
 |
|
 |
|
  A. R. Li, D. Chitale, G. J. Riely, W. Pao, V. A. Miller, M. F. Zakowski, V. Rusch, M. G. Kris, and M. Ladanyi EGFR Mutations in Lung Adenocarcinomas: Clinical Testing Experience and Relationship to EGFR Gene Copy Number and Immunohistochemical Expression J. Mol. Diagn., May 1, 2008; 10(3): 242 - 248. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 V. A. Miller, G. J. Riely, M. F. Zakowski, A. R. Li, J. D. Patel, R. T. Heelan, M. G. Kris, A. B. Sandler, D. P. Carbone, A. Tsao, et al. Molecular Characteristics of Bronchioloalveolar Carcinoma and Adenocarcinoma, Bronchioloalveolar Carcinoma Subtype, Predict Response to Erlotinib J. Clin. Oncol., March 20, 2008; 26(9): 1472 - 1478. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Bean, C. Brennan, J.-Y. Shih, G. Riely, A. Viale, L. Wang, D. Chitale, N. Motoi, J. Szoke, S. Broderick, et al. MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib PNAS, December 26, 2007; 104(52): 20932 - 20937. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Takano, Y. Ohe, K. Tsuta, T. Fukui, H. Sakamoto, T. Yoshida, U. Tateishi, H. Nokihara, N. Yamamoto, I. Sekine, et al. Epidermal Growth Factor Receptor Mutation Detection Using High-Resolution Melting Analysis Predicts Outcomes in Patients with Advanced Non Small Cell Lung Cancer Treated with Gefitinib Clin. Cancer Res., September 15, 2007; 13(18): 5385 - 5390. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
W. Pao and M. Ladanyi Epidermal Growth Factor Receptor Mutation Testing in Lung Cancer: Searching for the Ideal Method Clin. Cancer Res., September 1, 2007; 13(17): 4954 - 4955. [Full Text] [PDF]
|
|
|
|
|
|
K. Hoshi, H. Takakura, Y. Mitani, K. Tatsumi, N. Momiyama, Y. Ichikawa, S. Togo, T. Miyagi, Y. Kawai, Y. Kogo, et al. Rapid Detection of Epidermal Growth Factor Receptor Mutations in Lung Cancer by the SMart-Amplification Process Clin. Cancer Res., September 1, 2007; 13(17): 4974 - 4983. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Riely, M. G. Kris, B. Zhao, T. Akhurst, D. T. Milton, E. Moore, L. Tyson, W. Pao, N. A. Rizvi, L. H. Schwartz, et al. Prospective Assessment of Discontinuation and Reinitiation of Erlotinib or Gefitinib in Patients with Acquired Resistance to Erlotinib or Gefitinib Followed by the Addition of Everolimus Clin. Cancer Res., September 1, 2007; 13(17): 5150 - 5155. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 X. Zhang and A. Chang Somatic mutations of the epidermal growth factor receptor and non-small-cell lung cancer J. Med. Genet., March 1, 2007; 44(3): 166 - 172. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N van Zandwijk, A Mathy, L Boerrigter, H Ruijter, I Tielen, D de Jong, P Baas, S Burgers, and P Nederlof EGFR and KRAS mutations as criteria for treatment with tyrosine kinase inhibitors: retro- and prospective observations in non-small-cell lung cancer Ann. Onc., January 1, 2007; 18(1): 99 - 103. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. M. Chin, D. Anuar, R. Soo, M. Salto-Tellez, W. Q. Li, B. Ahmad, S. C. Lee, B. C. Goh, K. Kawakami, A. Segal, et al. Detection of Epidermal Growth Factor Receptor Variations by Partially Denaturing HPLC Clin. Chem., January 1, 2007; 53(1): 62 - 70. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Rosell, M. Taron, N. Reguart, D. Isla, and T. Moran Epidermal Growth Factor Receptor Activation: How Exon 19 and 21 Mutations Changed Our Understanding of the Pathway Clin. Cancer Res., December 15, 2006; 12(24): 7222 - 7231. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Riely, K. A. Politi, V. A. Miller, and W. Pao Update on Epidermal Growth Factor Receptor Mutations in Non-Small Cell Lung Cancer Clin. Cancer Res., December 15, 2006; 12(24): 7232 - 7241. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. N. Balak, Y. Gong, G. J. Riely, R. Somwar, A. R. Li, M. F. Zakowski, A. Chiang, G. Yang, O. Ouerfelli, M. G. Kris, et al. Novel D761Y and Common Secondary T790M Mutations in Epidermal Growth Factor Receptor-Mutant Lung Adenocarcinomas with Acquired Resistance to Kinase Inhibitors. Clin. Cancer Res., November 1, 2006; 12(21): 6494 - 6501. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Janne and B. E. Johnson Effect of epidermal growth factor receptor tyrosine kinase domain mutations on the outcome of patients with non-small cell lung cancer treated with epidermal growth factor receptor tyrosine kinase inhibitors. Clin. Cancer Res., July 15, 2006; 12(14): 4416s - 4420s. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. F. Zakowski, M. Ladanyi, M. G. Kris, and the Memorial Sloan-Kettering Cancer Center Lung Ca EGFR mutations in small-cell lung cancers in patients who have never smoked. N. Engl. J. Med., July 13, 2006; 355(2): 213 - 215. [Full Text] [PDF]
|
|
|
|
|
|
Y. Yatabe, T. Hida, Y. Horio, T. Kosaka, T. Takahashi, and T. Mitsudomi A Rapid, Sensitive Assay to Detect EGFR Mutation in Small Biopsy Specimens from Lung Cancer J. Mol. Diagn., July 1, 2006; 8(3): 335 - 341. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. Pham, M. G. Kris, G. J. Riely, I. S. Sarkaria, T. McDonough, S. Chuai, E. S. Venkatraman, V. A. Miller, M. Ladanyi, W. Pao, et al. Use of Cigarette-Smoking History to Estimate the Likelihood of Mutations in Epidermal Growth Factor Receptor Gene Exons 19 and 21 in Lung Adenocarcinomas J. Clin. Oncol., April 10, 2006; 24(11): 1700 - 1704. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. A. Janne, A. M. Borras, Y. Kuang, A. M. Rogers, V. A. Joshi, H. Liyanage, N. Lindeman, J. C. Lee, B. Halmos, E. A. Maher, et al. A Rapid and Sensitive Enzymatic Method for Epidermal Growth Factor Receptor Mutation Screening Clin. Cancer Res., February 1, 2006; 12(3): 751 - 758. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
G. J. Riely, W. Pao, D. Pham, A. R. Li, N. Rizvi, E. S. Venkatraman, M. F. Zakowski, M. G. Kris, M. Ladanyi, and V. A. Miller Clinical Course of Patients with Non-Small Cell Lung Cancer and Epidermal Growth Factor Receptor Exon 19 and Exon 21 Mutations Treated with Gefitinib or Erlotinib Clin. Cancer Res., February 1, 2006; 12(3): 839 - 844. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
