Correspondence
Assessing EGFR Mutations
N Engl J Med 2006; 354:526-528February 2, 2006
- Article
To the Editor:
Recent reports have noted the presence of novel mutations of the epidermal growth factor receptor (EGFR) gene in small samples of DNA extracted from paraffin-embedded sections or laser-microdissected specimens.1,2 Nagahara et al.1 reported EGFR mutations in colon carcinomas. These mutations were novel G→A or A→G transitions. Similarly, Tsao et al. (July 14 issue)2 report that 24 (53 percent) of the mutations they found in specimens of non–small-cell lung cancers were novel variant mutations; 22 (92 percent) of these mutations were C→T/G→A or A→G/T→C transitions. These mutations had not been reported previously in more than 2000 analyses for EGFR mutations performed on DNA extracted from pieces of frozen tumors.
By sequencing multiple polymerase-chain-reaction (PCR) amplification products (10 per sample) from 70 samples of lung-tumor DNA extracted from a single (5 μm) paraffin-embedded section, we found 45 uncommon mutations in exons 18 through 21 of the EGFR gene, including the 22 transitions reported by Tsao et al. and the 4 mutations reported by Nagahara et al. All the uncommon mutations detected were found to be artifacts. Indeed, the mutations were also found in multiple amplifications of paraffin-embedded DNA samples from 50 normal tissues (lymph nodes) obtained from 50 patients who did not have neoplasms; the mutations were identified in a minority of the amplifications from the same sample; and the C→T/G→A mutations were prevented by adding uracil-N-glycosylase to the DNA template before PCR amplification.
The artifacts we observed can be ascribed to postmortem deamination of cytosine or adenine, resulting in uracil or hypoxanthine residues, respectively.3 When DNA containing such lesions is used as a template for PCR, C→T/G→A or A→G/T→C transitions will result. C→T/G→A mutations can be prevented by treating the template with uracil-N-glycosylase, which removes uracil from DNA, creating a strand break. Artifactual mutations have been described by several authors,3-5 and alternative explanations have also been reported.4,5
These artifacts can easily be observed by carrying out multiple PCR amplifications of very small amounts of DNA, particularly if the DNA is isolated from paraffin-embedded tissues3-5 (Figure 1Figure 1
Sequences of Multiple PCR Amplifications of Small Amounts (5 ng) of DNA Extracted from Paraffin-Embedded Tissues.). With the use of a low DNA copy number, PCR amplification starts from single or few templates, making possible the detection of alterations present in subsets of DNA molecules that are otherwise undetectable.4 Since EGFR mutations may be used for guidance in the clinical management of lung cancer, extreme care must be taken when working with small amounts of DNA, especially if the samples were extracted from paraffin. The occurrence of artifacts can be prevented with the use of larger amounts of template DNA (at least 1 μg of DNA recovered from paraffin). If very few copies of template molecules must be used, as in the case of DNA obtained from microdissected sections, the examination of multiple amplifications is imperative.
5 ReferencesAntonio Marchetti, M.D.
Lara Felicioni, M.D.
Fiamma Buttitta, M.D.
Center for Excellence on Aging, 66013 Chieti, Italy
amarchetti@unich.it 1
Nagahara H ,Mimori K ,Ohta M , et al. Somatic mutations of epidermal growth factor receptor in colorectal carcinoma. Clin Cancer Res 2005;11:1368-1371
CrossRef | Web of Science | Medline2
Tsao M-S ,Sakurada A ,Cutz J-C , et al. Erlotinib in lung cancer -- molecular and clinical predictors of outcome. N Engl J Med 2005;353:133-144
Full Text | Web of Science | Medline3
Hofreiter M ,Jaenicke V ,Serre D ,Haeseler Av A ,Paabo S . DNA sequences from multiple amplifications reveal artifacts induced by cytosine deamination in ancient DNA. Nucleic Acids Res 2001;29:4793-4799
CrossRef | Web of Science | Medline4
Akbari M ,Hansen MD ,Halgunset J ,Skorpen F ,Krokan HE . Low copy number DNA template can render polymerase chain reaction error prone in a sequence-dependent manner. J Mol Diagn 2005;7:36-39
CrossRef | Web of Science | Medline5
Williams C ,Ponten F ,Moberg C , et al. A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am J Pathol 1999;155:1467-1471
CrossRef | Web of Science | Medline
Author/Editor Response
Marchetti et al. have highlighted the difficulties in performing an EGFR mutation analysis on small, formalin-fixed, paraffin-embedded biopsy specimens, which commonly are the only samples available from patients with advanced lung cancer and are insufficient to permit repeated assays. We recently completed mutational assays on 45 additional samples from the BR.21 trial and successfully obtained results for exons 19 and 21 in 25 samples (success rate, 56 percent). Among the results, we identified four new cases with exon 19 deletions or exon 21 L858R mutations. Given the uncertainty surrounding the identity or functional significance of novel mutations, we have reanalyzed our data, focusing only on exon 19 deletions and exon 21 L858R mutations for which the functional significance is clearer. Including the new samples, 24 cases that could be evaluated had these classic mutations, among 201 patients, for a prevalence rate of 11.9 percent, which is similar to mutation rates reported by other investigators. Among patients with classic mutations who were treated with erlotinib, the response rate was 30.0 percent (3 of 10 patients), as compared with 7.5 percent (8 of 106) for the remaining 106 patients (P=0.05). The hazard ratio for death associated with erlotinib, as compared with placebo, was 0.52 (95 percent confidence interval, 0.21 to 1.31; P=0.16) among those with classic mutations (Figure 1AFigure 1
Kaplan–Meier Estimates of Survival.), whereas it was 0.75 (95 percent confidence interval, 0.53 to 1.07; P=0.11) among those with wild-type EGFR or other variant mutations of indeterminate significance (Figure 1B). The interaction P value for treatment by classic-mutation status was 0.45. These findings, although based on small numbers, are consistent with our previous results showing no significant difference in the survival benefit achieved with erlotinib between patients with classic mutations and patients with wild-type EGFR. We therefore continue to believe that testing for EGFR mutations is not necessary to identify patients for treatment with erlotinib, since patients with wild-type EGFR and those with mutant genotypes both have the potential to benefit from therapy.Ming-Sound Tsao, M.D.
Suzanne Kamel-Reid, Ph.D.
Frances A. Shepherd, M.D.
Princess Margaret Hospital, Toronto, ON M56 2M9, Canada- Citing Articles (44)
Citing Articles
1
Sinchita Roy Chowdhuri, Liqiang Xi, Trinh Hoc-Tran Pham, Jeffrey Hanson, Jaime Rodriguez-Canales, Arlene Berman, Arun Rajan, Giuseppe Giaccone, Michael Emmert-Buck, Mark Raffeld, Armando C Filie. (2011) EGFR and KRAS mutation analysis in cytologic samples of lung adenocarcinoma enabled by laser capture microdissection. Modern Pathology
CrossRef2
Tommaso De Pas, Francesca Toffalorio, Michela Manzotti, Caterina Fumagalli, Gianluca Spitaleri, Chiara Catania, Angelo Delmonte, Monica Giovannini, Lorenzo Spaggiari, Filippo de Braud, Massimo Barberis. (2011) Activity of Epidermal Growth Factor Receptor-Tyrosine Kinase Inhibitors in Patients with Non-small Cell Lung Cancer Harboring Rare Epidermal Growth Factor Receptor Mutations. Journal of Thoracic Oncology 6:11, 1895-1901
CrossRef3
Sapna P. Patel, Kevin B. Kim, Nicholas E. Papadopoulos, Wen-Jen Hwu, Patrick Hwu, Victor G. Prieto, Menashe Bar-Eli, Maya Zigler, Andrey Dobroff, Yulia Bronstein, Roland L. Bassett, Anna G. Vardeleon, Agop Y. Bedikian. (2011) A phase II study of gefitinib in patients with metastatic melanoma. Melanoma Research 21:4, 357-363
CrossRef4
Aude Lamy, France Blanchard, Florence Le Pessot, Richard Sesboüé, Frédéric Di Fiore, Jessie Bossut, Elodie Fiant, Thierry Frébourg, Jean-Christophe Sabourin. (2011) Metastatic colorectal cancer KRAS genotyping in routine practice: results and pitfalls. Modern Pathology 24:8, 1090-1100
CrossRef5
Michèle Beau-Faller, Armelle Degeorges, Estelle Rolland, Mounia Mounawar, Martine Antoine, Virginie Poulot, Audrey Mauguen, Véronique Barbu, Florence Coulet, Jean-Luc Prétet, Ivan Bièche, Hélène Blons, Jean-Christophe Boyer, Marie-Pierre Buisine, Florence de Fraipont, Sarab Lizard, Sylviane Olschwang, Patrick Saulnier, Delphine Prunier-Mirebeau, Nicolas Richard, Claire Danel, Elisabeth Brambilla, Christos Chouaid, Gérard Zalcman, Pierre Hainaut, Stefan Michiels, Jacques Cadranel. (2011) Cross-Validation Study for Epidermal Growth Factor Receptor and KRAS Mutation Detection in 74 Blinded Non-small Cell Lung Carcinoma Samples. Journal of Thoracic Oncology 6:6, 1006-1015
CrossRef6
Gilda da Cunha Santos, Frances A. Shepherd, Ming Sound Tsao. (2011) EGFR Mutations and Lung Cancer. Annual Review of Pathology: Mechanisms of Disease 6:1, 49-69
CrossRef7
S. Merkelbach-Bruse, E. Wardelmann, H. Künstlinger, R. Büttner, H.-U. Schildhaus. (2011) Molekulare Methoden in der Sarkomdiagnostik. Der Pathologe 32:1, 24-31
CrossRef8
Roland Penzel, Christine Sers, Yuan Chen, Ulrich Lehmann-Mühlenhoff, Sabine Merkelbach-Bruse, Andreas Jung, Thomas Kirchner, Reinhard Büttner, Hans H. Kreipe, Iver Petersen, Manfred Dietel, Peter Schirmacher. (2011) EGFR mutation detection in NSCLC—assessment of diagnostic application and recommendations of the German Panel for Mutation Testing in NSCLC. Virchows Archiv 458:1, 95-98
CrossRef9
Patrick Adam, Stefanie Hahner, Martina Hartmann, Barbara Heinrich, Marcus Quinkler, Holger S Willenberg, Wolfgang Saeger, Silviu Sbiera, Sebastian Schmull, Hans-Ullrich Voelker, Philipp Ströbel, Bruno Allolio, Martin Fassnacht. (2010) Epidermal growth factor receptor in adrenocortical tumors: analysis of gene sequence, protein expression and correlation with clinical outcome. Modern Pathology 23:12, 1596-1604
CrossRef10
Matthew Meyerson, Stacey Gabriel, Gad Getz. (2010) Advances in understanding cancer genomes through second-generation sequencing. Nature Reviews Genetics 11:10, 685-696
CrossRef11
Akito Hata, Hiroshige Yoshioka, Shiro Fujita, Kei Kunimasa, Reiko Kaji, Yukihiro Imai, Keisuke Tomii, Masahiro Iwasaku, Akihiro Nishiyama, Tadashi Ishida, Nobuyuki Katakami. (2010) Complex Mutations in the Epidermal Growth Factor Receptor Gene in Non-small Cell Lung Cancer. Journal of Thoracic Oncology 5:10, 1524-1528
CrossRef12
Grégory Verhoest, Jean-Jacques Patard, Patricia Fergelot, Florence Jouan, Salim Zerrouki, Stéphane Dreano, Stéphanie Mottier, Nathalie Rioux-Leclercq, Marc G. Denis. (2010) Paraffin-embedded tissue is less accurate than frozen section analysis for determining VHL mutational status in sporadic renal cell carcinoma. Urologic Oncology: Seminars and Original Investigations
CrossRef13
Tomoya Yokota, Noriko Shibata, Takashi Ura, Daisuke Takahari, Kohei Shitara, Kei Muro, Yasushi Yatabe. (2010) Cycleave polymerase chain reaction method is practically applicable for V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS)/V-raf murine sarcoma viral oncogene homolog B1 (BRAF) genotyping in colorectal cancer. Translational Research 156:2, 98-105
CrossRef14
Lizette Vila, Hongyan Liu, Samer Z Al-Quran, Dominique P Coco, Hui-Jia Dong, Chen Liu. (2010) Response to Moskaluk et al. Modern Pathology 23:6, 906-907
CrossRef15
Christopher A Moskaluk, Henry F Frierson, Adel K El-Naggar, P Andrew Futreal. (2010) c-kit gene mutations in adenoid cystic carcinoma are rare. Modern Pathology 23:6, 905-906
CrossRef16
Waki Hosoda, Tadayuki Takagi, Nobumasa Mizuno, Yasuhiro Shimizu, Tsuyoshi Sano, Kenji Yamao, Yasushi Yatabe. (2010) Diagnostic approach to pancreatic tumors with the specimens of endoscopic ultrasound-guided fine needle aspiration. Pathology International 60:5, 358-364
CrossRef17
Yasushi Yatabe. (2010) EGFR mutations and the terminal respiratory unit. Cancer and Metastasis Reviews 29:1, 23-36
CrossRef18
E.-H. Tan, R. Ramlau, A. Pluzanska, H.-P. Kuo, M. Reck, J. Milanowski, J. S.-K. Au, E. Felip, P.-C. Yang, D. Damyanov, S. Orlov, M. Akimov, P. Delmar, L. Essioux, C. Hillenbach, B. Klughammer, P. McLoughlin, J. Baselga. (2010) A multicentre phase II gene expression profiling study of putative relationships between tumour biomarkers and clinical response with erlotinib in non-small-cell lung cancer. Annals of Oncology 21:2, 217-222
CrossRef19
Im Il Na, Byung Hyun Byun, Kyeong Min Kim, Gi Jeong Cheon, Du Hwan Choe, Jae Soo Koh, Duck Yong Lee, Baek-Yeol Ryoo, HeeJong Baek, Sang Moo Lim, Sung Hyun Yang, Cheol Hyeon Kim, Jae Cheol Lee. (2010) 18F-FDG uptake and EGFR mutations in patients with non-small cell lung cancer: A single-institution retrospective analysis. Lung Cancer 67:1, 76-80
CrossRef20
J-J Patard, N Rioux-Leclercq, D Masson, S Zerrouki, F Jouan, N Collet, C Dubourg, B Lobel, M Denis, P Fergelot. (2009) Absence of VHL gene alteration and high VEGF expression are associated with tumour aggressiveness and poor survival of renal-cell carcinoma. British Journal of Cancer 101:8, 1417-1424
CrossRef21
C. Tapia, S. Savic, M. Bihl, A. Rufle, I. Zlobec, L. Terracciano, L. Bubendorf. (2009) EGFR-Mutationsanalyse beim nichtkleinzelligen Lungenkarzinom. Der Pathologe 30:5, 384-392
CrossRef22
Asha Balakrishnan, Junia Y. Penachioni, Simona Lamba, Fonnet E. Bleeker, Carlo Zanon, Monica Rodolfo, Viviana Vallacchi, Aldo Scarpa, Lara Felicioni, Matthias Buck, Antonio Marchetti, Paolo M. Comoglio, Alberto Bardelli, Luca Tamagnone. (2009) Molecular profiling of the âplexinomeâ in melanoma and pancreatic cancer. Human Mutation 30:8, 1167-1174
CrossRef23
R Pérez-Soler. (2009) Individualized therapy in non-small-cell lung cancer: future versus current clinical practice. Oncogene 28, S38-S45
CrossRef24
Helena Linardou, Issa J. Dahabreh, Dimitrios Bafaloukos, Paris Kosmidis, Samuel Murray. (2009) Somatic EGFR mutations and efficacy of tyrosine kinase inhibitors in NSCLC. Nature Reviews Clinical Oncology 6:6, 352-366
CrossRef25
Giulio Metro, Federico Cappuzzo. (2009) New targeted therapies for non-small-cell lung cancer. Therapy 6:3, 335-350
CrossRef26
Hiromasa Yamamoto, Shinichi Toyooka, Tetsuya Mitsudomi. (2009) Impact of EGFR mutation analysis in non-small cell lung cancer. Lung Cancer 63:3, 315-321
CrossRef27
Suzanne WEBB, Marc THOMAS, Cecily METCALF, Amanda SEGAL, Anna K NOWAK, Jacqueline BENTEL, Michael MILLWARD. (2009) EGFR mutation testing in NSCLC: Patterns of care and outcomes in Western Australia. Asia-Pacific Journal of Clinical Oncology 5:1, 66-71
CrossRef28
Ting Liu, Carlynn Willmore-Payne, Lester J. Layfield, Joseph A. Holden. (2009) Lack of BRAF Activating Mutations in Prostate Adenocarcinoma. Applied Immunohistochemistry & Molecular Morphology 17:2, 121-125
CrossRef29
M I Galleges Ruiz, K Floor, S M Steinberg, K Grünberg, F B J M Thunnissen, J A M Belien, G A Meijer, G J Peters, E F Smit, J A Rodriguez, G Giaccone. (2009) Combined assessment of EGFR pathway-related molecular markers and prognosis of NSCLC patients. British Journal of Cancer 100:1, 145-152
CrossRef30
Martin Reck, Lucio Crinò. (2009) Advances in anti-VEGF and anti-EGFR therapy for advanced non-small cell lung cancer. Lung Cancer 63:1, 1-9
CrossRef31
Laia Agell, Silvia Hernández, Silvia de Muga, José A Lorente, Núria Juanpere, Raquel Esgueva, Sergi Serrano, Antoni Gelabert, Josep Lloreta. (2008) KLF6 and TP53 mutations are a rare event in prostate cancer: distinguishing between Taq polymerase artifacts and true mutations. Modern Pathology 21:12, 1470-1478
CrossRef32
Samuel Murray, Issa J. Dahabreh, Helena Linardou, Menelaos Manoloukos, Dimitrios Bafaloukos, Paris Kosmidis. (2008) Somatic Mutations of the Tyrosine Kinase Domain of Epidermal Growth Factor Receptor and Tyrosine Kinase Inhibitor Response to TKIs in Non-small Cell Lung Cancer: An Analytical Database. Journal of Thoracic Oncology 3:8, 832-839
CrossRef33
Raffaele Califano, Fiona H. Blackhall, Giovanna Finocchiaro, Luca Toschi, Nicholas Thatcher, Federico Cappuzzo, Lucio Crinò. (2008) EGFR tyrosine kinase inhibitors in non-small cell lung cancer patients: how do we interpret the clinical and biomarker data?. Targeted Oncology 3:3, 173-186
CrossRef34
Marc Ladanyi, William Pao. (2008) Lung adenocarcinoma: guiding EGFR-targeted therapy and beyond. Modern Pathology 21, S16-S22
CrossRef35
S Savic, C Tapia, B Grilli, A Rufle, M P Bihl, A de Vito Barascud, M Herzog, L Terracciano, F Baty, L Bubendorf. (2008) Comprehensive epidermal growth factor receptor gene analysis from cytological specimens of non-small-cell lung cancers. British Journal of Cancer 98:1, 154-160
CrossRef36
Tetsuya Mitsudomi, Yasushi Yatabe. (2007) Mutations of the epidermal growth factor receptor gene and related genes as determinants of epidermal growth factor receptor tyrosine kinase inhibitors sensitivity in lung cancer. Cancer Science 98:12, 1817-1824
CrossRef37
Phillip S. Hasleton, Philip Sloan. 2007. Respiratory System. .
CrossRef38
Giovanna Finocchiaro, Luca Toschi, Isabella Garassino, Fabio De Vincenzo, Elisabetta Campagnoli, Paolo Zucali, Raffaele Cavina, Giovanni L Ceresoli, Armando Santoro, Federico Cappuzzo. (2007) EGFR tyrosine kinase inhibitors: a therapy for a few, for the majority or for all non-small cell lung cancer patients?. Expert Opinion on Medical Diagnostics 1:2, 183-191
CrossRef39
D Irmer, J O Funk, A Blaukat. (2007) EGFR kinase domain mutations – functional impact and relevance for lung cancer therapy. Oncogene 26:39, 5693-5701
CrossRef40
Elaine H. Lim, Shen-Li Zhang, Kun Yu, Min-En Nga, Dokeu A. Ahmed, Thirugananam Agasthian, Poo-Sing Wong, Gim-Chuah Chua, Daniel Wong, Lenny Tan, Kar-Yin Seto, Wee-See Yap, Seow-Ping Low, Kay-Leong Khoo, Alex Chang, Alan Ng, Patrick Tan. (2007) An Alternative Approach to Determining Therapeutic Choices in Advanced Non-small Cell Lung Carcinoma (NSCLC): Maximizing the Diagnostic Procedure and the Use of Low-Volume Lung Biopsies. Journal of Thoracic Oncology 2:5, 387-396
CrossRef41
Yasushi Yatabe, Tetsuya Mitsudomi. (2007) Epidermal growth factor receptor mutations in lung cancers. Pathology International 57:5, 233-244
CrossRef42
James Spicer, Simon Chowdhury, Peter Harper. (2007) Targeting novel and established therapies for non-small cell lung cancer. Cancer Letters 250:1, 9-16
CrossRef43
Rafal Dziadziuszko, Barbara Szostakiewicz, Fred R. Hirsch. 2007. Epidermal Growth Factor Receptor Targeted Therapy—Markers of Sensitivity and Response. , 97-122.
CrossRef44
H Uramoto, T Mitsudomi. (2007) Which biomarker predicts benefit from EGFR-TKI treatment for patients with lung cancer?. British Journal of Cancer 96:6, 857-863
CrossRef
