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High T790M Detection Rate in TKI-Naive NSCLC with EGFR Sensitive Mutation: Truth or Artifact?

2013; Elsevier BV; Volume: 8; Issue: 9 Linguagem: Inglês

10.1097/jto.0b013e31829f691f

1556-1380

Xin Ye, Zhongzheng Zhu, Lei Zhong, Yachao Lu, Yun Sun, Xiaolu Yin, Zhenfan Yang, Guanshan Zhu, Qunsheng Ji,

Peptidase Inhibition and Analysis

T790M in epidermal growth factor receptor (EGFR) accounts for about 50% of the acquired resistance to EGFR tyrosine kinase inhibitor (TKI) in patients with non–small-cell lung cancer (NSCLC) carrying sensitive EGFR mutations. Earlier studies suggested that T790M mutation was also detected in about 2% of TKI-naive NSCLCs. Recently, three groups reported that, by using highly sensitive assays, T790M mutation was detected in about 40% of TKI-naive NSCLCs with sensitive EGFR mutations. When we carefully studied these reports, we realized that all of those data were generated from formalin-fixed paraffin-embedded (FFPE) tumor tissue samples, which raised a concern that the high mutation positivity could be consequence of formalin fixation. To address this, we assessed the T790M mutation in 36 pairs of frozen and FFPE tumor tissues of TKI-naive NSCLCs with sensitive EGFR mutations, using an enzyme-based mutant-enriched polymerase chain reaction assay (ME-PCR) with 0.1% sensitivity. In addition, frozen and FFPE adjacent normal tissues from the same patients were also assessed for a further comparison. Although 41.7% (15 of 36) of the T790M-positive rate was detected in the tumor FFPE samples, which was consistent with previous reports, only one of the 36 frozen counterparts (2.8%) was T790M positive. As suspected, 48.5% (16 of 33) of T790M mutation rates was also identified in FFPE adjacent normal tissues but none of the 35 frozen adjacent normal tissues was T790M positive. Our results indicate that the high T790M positivity detected in TKI-naive NSCLC, using some highly sensitive methods, may in some cases be FFPE-derived artifacts. Activating mutations in EGFR gene confer dramatic sensitivity to EGFR TKIs.1Gridelli C De Marinis F Di Maio M Cortinovis D Cappuzzo F Mok T Gefitinib as first-line treatment for patients with advanced non-small-cell lung cancer with activating epidermal growth factor receptor mutation: Review of the evidence.Lung Cancer. 2011; 71: 249-257Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar However, most patients underwent relapse within 1 year after the initiation of TKI treatment.2Sequist LV Bell DW Lynch TJ Haber DA Molecular predictors of response to epidermal growth factor receptor antagonists in non-small-cell lung cancer.J Clin Oncol. 2007; 25: 587-595Crossref PubMed Scopus (561) Google Scholar T790M, a secondary mutation in EGFR gene, has been identified as the major mechanism of the acquired EGFR-TKI resistance, accounting for about 50% of TKI-relapsed NSCLCs.3Inukai M Toyooka S Ito S et al.Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer.Cancer Res. 2006; 66: 7854-7858Crossref PubMed Scopus (407) Google Scholar, 4Engelman JA Settleman J Acquired resistance to tyrosine kinase inhibitors during cancer therapy.Curr Opin Genet Dev. 2008; 18: 73-79Crossref PubMed Scopus (249) Google Scholar, 5Pao W Miller VA Politi KA et al.Acquired resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the EGFR kinase domain.PLoS Med. 2005; 2: e73Crossref PubMed Scopus (3101) Google Scholar, 6Kobayashi S Boggon TJ Dayaram T et al.EGFR mutation and resistance of non-small-cell lung cancer to gefitinib.N Engl J Med. 2005; 352: 786-792Crossref PubMed Scopus (3493) Google Scholar, 7Kosaka T Yatabe Y Endoh H et al.Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib.Clin Cancer Res. 2006; 12: 5764-5769Crossref PubMed Scopus (551) Google Scholar, 8Sequist LV Waltman BA Dias-Santagata D et al.Genotypic and histological evolution of lung cancers acquiring resistance to EGFR inhibitors.Sci Transl Med. 2011; 3: 75ra26Crossref PubMed Scopus (2710) Google Scholar However, it is unknown how T790M arises in tumor cells. Conventional DNA sequencing revealed that about 2% T790M preexists with sensitive EGFR mutations in TKI-naive NSCLCs.9Sequist LV Martins RG Spigel D et al.First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations.J Clin Oncol. 2008; 26: 2442-2449Crossref PubMed Scopus (763) Google Scholar,10Kosaka T Yatabe Y Endoh H Kuwano H Takahashi T Mitsudomi T Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications.Cancer Res. 2004; 64: 8919-8923Crossref PubMed Scopus (1129) Google Scholar It is not clear whether the majority of the T790M mutation in TKI-relapsed NSCLCs is acquired during disease progression or emerges from the preexisting T790M in TKI-naive patients as a minor population but undetectable by conventional sequencing. More sensitive assays capable of detecting low frequency of T790M are emerging to address this question. With recent progress made in the development of second-generation EGFR TKIs targeting tumors harboring T790M mutation,11Kwak EL Sordella R Bell DW et al.Irreversible inhibitors of the EGF receptor may circumvent acquired resistance to gefitinib.Proc Natl Acad Sci U S A. 2005; 102: 7665-7670Crossref PubMed Scopus (864) Google Scholar it is critical to accurately identify the T790M-positive NSCLC patients, which will help guide the patient selection in clinical trials and treatments. Although rebiopsied tumor tissues are ideal for detection of TKI-resistant T790M, which is challenging in clinical practice, it would be valuable to understand T790M baseline in TKI-naive patients and whether it could be used to predict the T790M status in TKI-relapsed patients. With the emerging highly sensitive techniques, about 40% of T790M has been identified in TKI-naïve NSCLCs in some studies,12Rosell R Molina MA Costa C et al.Pretreatment EGFR T790M mutation and BRCA1 mRNA expression in erlotinib-treated advanced non-small-cell lung cancer patients with EGFR mutations.Clin Cancer Res. 2011; 17: 1160-1168Crossref PubMed Scopus (278) Google Scholar, 13Su KY Chen HY Li KC et al.Pretreatment epidermal growth factor receptor (EGFR) T790M mutation predicts shorter EGFR tyrosine kinase inhibitor response duration in patients with non-small-cell lung cancer.J Clin Oncol. 2012; 30: 433-440Crossref PubMed Scopus (454) Google Scholar, 14Maheswaran S Sequist LV Nagrath S et al.Detection of mutations in EGFR in circulating lung-cancer cells.N Engl J Med. 2008; 359: 366-377Crossref PubMed Scopus (1477) Google Scholar which is much higher than the T790M rate detected by conventional methods.9Sequist LV Martins RG Spigel D et al.First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations.J Clin Oncol. 2008; 26: 2442-2449Crossref PubMed Scopus (763) Google Scholar,10Kosaka T Yatabe Y Endoh H Kuwano H Takahashi T Mitsudomi T Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications.Cancer Res. 2004; 64: 8919-8923Crossref PubMed Scopus (1129) Google Scholar The study by Su et al.13Su KY Chen HY Li KC et al.Pretreatment epidermal growth factor receptor (EGFR) T790M mutation predicts shorter EGFR tyrosine kinase inhibitor response duration in patients with non-small-cell lung cancer.J Clin Oncol. 2012; 30: 433-440Crossref PubMed Scopus (454) Google Scholar recently published in J Clin Oncol used matrix-assisted laser desorption ionization–time of flight mass spectrometry (1.5% sensitivity) to evaluate T790M mutation in NSCLCs and used next-generation sequencing (3.7% sensitivity) to attempt to validate the results. Matrix-assisted laser desorption ionization–time of flight mass spectrometry identified 41% (23 of 56) of T790M rate in pre-TKI NSCLCs carrying sensitive EGFR mutations. In addition, Rosell et al12Rosell R Molina MA Costa C et al.Pretreatment EGFR T790M mutation and BRCA1 mRNA expression in erlotinib-treated advanced non-small-cell lung cancer patients with EGFR mutations.Clin Cancer Res. 2011; 17: 1160-1168Crossref PubMed Scopus (278) Google Scholar. reported similar findings in 2011; they developed TaqMan assay (Applied Biosystems, Carlsbad, CA) with peptide nucleic acid (PNA) clamping (0.02% sensitivity) to assess T790M mutation in two cohorts of NSCLCs with sensitive EGFR mutations and found the T790M mutation rates to be 35% (45 of 129) and 38% (30 of 78), respectively. No validation of the results using a second sensitive method was included. Moreover, in 2008, Maheswaran's14Maheswaran S Sequist LV Nagrath S et al.Detection of mutations in EGFR in circulating lung-cancer cells.N Engl J Med. 2008; 359: 366-377Crossref PubMed Scopus (1477) Google Scholar group reported that 10 of 26 NSCLCs (38%) with sensitive EGFR mutations contained low levels of T790M mutation in the pre-TKI specimens. They used Scorpion Amplification Refractory Mutation System (DxS, Qiagen, Manchester, UK) for T790M detection and identified as few as one T790M mutation in 500 EGFR alleles (using cloning and sequencing; 0.2% sensitivity). However, importantly, other groups analyzing similar sample sets using the same method have not reported these frequencies of T790M mutations.15Fukuoka M Wu YL Thongprasert S et al.Biomarker analyses and final overall survival results from a phase III, randomized, open-label, first-line study of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non-small-cell lung cancer in Asia (IPASS).J Clin Oncol. 2011; 29: 2866-2874Crossref PubMed Scopus (1202) Google Scholar,16Chen HJ Mok TS Chen ZH et al.Clinicopathologic and molecular features of epidermal growth factor receptor T790M mutation and c-MET amplification in tyrosine kinase inhibitor-resistant Chinese non-small cell lung cancer.Pathol Oncol Res. 2009; 15: 651-658Crossref PubMed Scopus (96) Google Scholar In summary, all three groups, by using highly sensitive assays (3.71%–0.02% sensitivity), identified approximately 40% T790M-positive rate in TKI-naive NSCLCs with sensitive EGFR mutations. If this is true, with T790M being detected in NSCLCs before TKI, it would help tremendously in the trial design and in the optimization of treatment strategies to maximize their efficacious benefits. In addition, the presence of high frequency of T790M in pre-TKI NSCLCs would prompt the first-line application of second-generation TKIs, which might benefit in delaying or preventing the TKI resistance. However, when we looked into the sample information of these reports, we realized that all three groups had used formalin-fixed samples for T790M detection and in some cases the developed assays had not been validated on the FFPE material they were being used on, but rather unfixed cell lines. This raised a concern that the high T790M positivity could be a consequence of formalin fixation, as there were reports showing that artificial gene mutations could be detected from formalin-fixed sample when using sensitive methods and insufficient assay validation.17Williams C Pontén F Moberg C et al.A high frequency of sequence alterations is due to formalin fixation of archival specimens.Am J Pathol. 1999; 155: 1467-1471Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar, 18Quach N Goodman MF Shibata D In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR.BMC Clin Pathol. 2004; 4: 1Crossref PubMed Scopus (111) Google Scholar, 19Wong C DiCioccio RA Allen HJ Werness BA Piver MS Mutations in BRCA1 from fixed, paraffin-embedded tissue can be artifacts of preservation.Cancer Genet Cytogenet. 1998; 107: 21-27Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar To address this concern, we collected 36 TKI-naive NSCLC tumor tissue samples carrying sensitive EGFR mutations, including 19-Del (13 of 36; 36%), L858R (18 of 36; 50%) and other mutations (5 of 36; 14%), and their adjacent normal lung tissue samples when available, in both frozen and FFPE formats. Deoxyribonucleic acids (DNA) from frozen and FFPE specimens were extracted by Puregene DNA extraction kit (Cat. #: 158388; Qiagen, Germantown, MD) and QIAamp DNA FFPE Tissue Kit (Cat. #: 56404; Qiagen, Hilden, Germany), respectively. T790M was detected using an enzyme-based ME-PCR assay with 0.1% sensitivity according to a previous report with some modifications.3Inukai M Toyooka S Ito S et al.Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer.Cancer Res. 2006; 66: 7854-7858Crossref PubMed Scopus (407) Google Scholar All samples were tested in duplicate with 50 ng DNA input per reaction. When the cases positive in both singleton and duplicate were counted, a T790M-positive rate of 41.7% (15 of 36) in the tumor FFPE samples was detected, which was similar to that in previous reports. However, only one of the 36 frozen counterparts (2.8%) was T790M positive, which showed positive in duplicate and was validated by standard Sanger sequencing (the tumor FFPE sample from this patient was also positive in duplicate, although the adjacent normal FFPE sample was positive in singleton). Surprisingly, 48.5% (16 of 33) of T790M-positive rate was identified in the adjacent normal FFPE tissues as well whereas none of the 35 adjacent normal frozen tissues was T790M positive (3 normal FFPE samples and 1 normal frozen sample were not available for detection). If only the cases positive in duplicate were taken into consideration, about 20% of T790M-positive rate would have been detected in both tumor and adjacent normal FFPE samples. Our results (summarized in Table 1), for the first time, suggested that the high T790M positivity detected in TKI-naive NSCLCs using highly sensitive methods may actually be the FFPE-derived artifacts.TABLE 1EGFR-T790M Mutation Detection by Mutant-Enriched Polymerase Chain ReactionFFPE SamplesPaired Frozen SamplesTumorAdjacent NormalTumorAdjacent NormalTotal number of cases.36333635T790MPositive in singleton/duplicateaIncluding the cases positive in only one singleton reaction and the cases positive in duplicate reactions.15 (41.7%)16 (48.5%)1 (2.8%)0Positive in duplicatebIncluding only the cases positive in duplicate reactions.7 (19.4%)7 (21.2%)1 (2.8%)0EGFR, epidermal growth factor receptor; FFPE, formalin-fixed paraffin-embedded.a Including the cases positive in only one singleton reaction and the cases positive in duplicate reactions.b Including only the cases positive in duplicate reactions. Open table in a new tab EGFR, epidermal growth factor receptor; FFPE, formalin-fixed paraffin-embedded. It was reported that formalin fixation could lead to false-positive somatic mutations although most, if not all of these artifacts can be distinguished from true mutations by performing duplicate sequencing from the original DNA sample.20Ellison G Donald E McWalter G et al.A comparison of ARMS and DNA sequencing for mutation analysis in clinical biopsy samples.J Exp Clin Cancer Res. 2010; 29: 132Crossref PubMed Scopus (135) Google Scholar In this study, however, duplicate analysis would not have resolved the issue because of the frequency of the artifacts in T790M locus generated by this assay on FFPE material. Williams et al.17Williams C Pontén F Moberg C et al.A high frequency of sequence alterations is due to formalin fixation of archival specimens.Am J Pathol. 1999; 155: 1467-1471Abstract Full Text Full Text PDF PubMed Scopus (418) Google Scholar reported that a high frequency of sequence alterations was detected in formalin-fixed samples but not in frozen samples. Moreover, 96% (27 of 28) of the artificial mutations were C-T or G-A transitions, which happened to be the mutation type of T790M. Similarly, Quach et al.18Quach N Goodman MF Shibata D In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR.BMC Clin Pathol. 2004; 4: 1Crossref PubMed Scopus (111) Google Scholar also reported significantly more mutations from formalin-fixed samples than from the corresponding frozen samples, among which 92% were transition mutations (C-T or G-A). The study by Inukai et al.3Inukai M Toyooka S Ito S et al.Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer.Cancer Res. 2006; 66: 7854-7858Crossref PubMed Scopus (407) Google Scholar,21Asano H Toyooka S Tokumo M et al.Detection of EGFR gene mutation in lung cancer by mutant-enriched polymerase chain reaction assay.Clin Cancer Res. 2006; 12: 43-48Crossref PubMed Scopus (180) Google Scholar supported the false-positive detection of T790M in FFPE samples. The authors identified 4% (4 of 95) of T790M mutation rate using the highly sensitive ME-PCR assay (0.1% sensitivity) in TKI-naive NSCLCs with sensitive EGFR mutations. These 95 samples positive for sensitive EGFR mutations were from 280 clinical specimens of different formats, with approximately 80% frozen specimens, approximately10% formalin-fixed specimens, and approximately 10% pleural fluid. Although there is no information regarding the EGFR mutation status versus sample format of individual samples, the fact that majority of samples were frozen type might explain the much lower T790M rate (4% versus ~40% reported before) detected in this cohort of samples with comparably sensitive assay applied. Previous studies9Sequist LV Martins RG Spigel D et al.First-line gefitinib in patients with advanced non-small-cell lung cancer harboring somatic EGFR mutations.J Clin Oncol. 2008; 26: 2442-2449Crossref PubMed Scopus (763) Google Scholar,10Kosaka T Yatabe Y Endoh H Kuwano H Takahashi T Mitsudomi T Mutations of the epidermal growth factor receptor gene in lung cancer: biological and clinical implications.Cancer Res. 2004; 64: 8919-8923Crossref PubMed Scopus (1129) Google Scholar suggested that direct DNA sequencing was able to detect T790M in approximately 2% of pre-TKI NSCLC patients carrying sensitive EGFR mutations. Our study here using highly sensitive assay (0.1% sensitivity) also detected 2.8% T790M rate in frozen pre-TKI NSCLC patients. It indicates that with as high as 0.1% detection sensitivity, only about 2% of pre-TKI NSCLCs carry T790M mutation. However, it remains a question whether higher authentic T790M rate could be detected from pretreatment NSCLC if a more sensitive method is applied. A recent study by Fujita et al.22Fujita Y Suda K Kimura H et al.Highly sensitive detection of EGFR T790M mutation using colony hybridization predicts favorable prognosis of patients with lung cancer harboring activating EGFR mutation.J Thorac Oncol. 2012; 7: 1640-1644Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar reported 79% positive rate of T790M among totally 38 frozen samples of pre-TKI NSCLCs carrying sensitive EGFR mutations. They used colony-hybridization technique, which was claimed to have 0.01% sensitivity. However, additional studies are needed to include more frozen tumor samples and different technical platforms are required to confirm this. In conclusion, care should be taken when analyzing FFPE material to significantly reduce or eliminate artificial errors. Tests should be validated on the material they are intended to be used on, and at least a proportion of the results should be confirmed by an alternative method. Duplicate or repeat analysis should be considered as a means of reducing the chance of error so that sensitive test results on FFPE are reliable. The authors thank Gillian Ellison (AstraZeneca) and Suzanne Jenkins (AstraZeneca) for their valuable opinion and editing assistance in the writing of the article.