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Originally published as JCO Early Release 10.1200/JCO.2008.20.4875 on May 4 2009 © 2009 American Society of Clinical Oncology.
Toward Noninvasive Genomic Screening of Lung Cancer PatientsMassachusetts General Hospital Cancer Center, Boston, MA Understanding the molecular profile of non–small-cell lung cancer (NSCLC) is becoming ever more relevant to patient care and treatment decision making. The development of molecularly targeted therapies against the epidermal growth factor receptor (EGFR) tyrosine kinase has led to the discovery of mutations and other genomic alterations that affect the efficacy of these agents. A set of activating mutations in EGFR, primarily overlapping deletion mutations in exon 19 and the single L858R point mutation in exon 21, are associated with increased response and survival after tyrosine kinase inhibitor (TKI) therapy, whereas the T790M point mutation or insertion mutations in exon 20 of EGFR, amplification of MET, and mutations in K-ras are associated with failure to respond.1–11 As a result, the field is moving toward acceptance of molecular testing as an integral component of clinical care. However, several practical obstacles to this paradigm shift remain. These include the acquisition and availability of appropriate tissue samples and the time required for analysis. The lung cancer diagnostic procedure of choice is frequently a fine needle aspiration, either via bronchoscopy or using the computed tomography–guided percutaneous approach, which often yields insufficient material for performing one or multiple molecular analyses. When considering EGFR mutation screening, the problem of limited tissue is confounded by reliance on the direct sequencing method, which is not highly sensitive and often results in falsely negative or noninformative results when performed on cytology specimens.12–14 Direct sequencing can often take up to 3 to 4 weeks to yield results, which is not clinically optimal.14,15 Consequently, there is great interest in establishing alternate methods of EGFR mutation screening other than direct sequencing, as well as developing blood-based methods of analyzing these somatic mutations. In this issue of the Journal of Clinical Oncology, Bai and colleagues16 present the results from a prospective clinical trial designed to assess the value of noninvasive EGFR mutation analysis via denaturing high-performance liquid chromatography (DHPLC) analysis of peripheral blood specimens. DHPLC has previously been used to analyze tumor tissue biopsies for EGFR mutation with promising results. Jänne et al demonstrated that by predigesting samples with a DNA endonuclease and then performing DHPLC, they could detect EGFR mutations with 100% sensitivity and 87% specificity compared to standard direct sequencing.17 Similarly, both Cohen and Chin found that DHPLC was more sensitive, more time efficient, and less costly than direct sequencing of tumor tissue.18,19 In contrast to standard direct sequencing, DHPLC involves denaturing polymerase chain reaction products with heat followed by cooling to permit renaturation and the formation of duplexes. The heteroduplexes formed in the presence of a heterozygous EGFR mutation differ in HPLC column retention time compared with the homoduplexes formed with wild-type EGFR, and are therefore detectable as an aberrant peak on the data readout. Although homozygous EGFR mutations are not detectable with this method, they are extraordinarily rare.17 Noting that a variation in elution time, or an aberrant peak, exists on DHPLC analysis does not define the specific mutation present, and direct sequencing of that peak is still necessary to confirm the exact mutation. In this case, Bai and colleagues16 first established standard readout patterns observed on DHPLC with the most common activating EGFR mutations, and then classified their subsequent results on the basis of these patterns. The study team enrolled 230 consecutive patients with advanced NSCLC from the Beijing Cancer Hospital over a 3-year period. All patients had primary tumor samples available for DHPLC analysis and comparison to the plasma DHPLC results. The primary aim of the study was to determine if plasma DHPLC analysis was a valid surrogate for tumor tissue EGFR mutation analysis. They detected EGFR mutations in 34% of the plasma samples and 33% of the tumor tissue samples, although this did not represent the same cohort of patients using each method. There was 80% concordance between DHPLC analysis of tumor tissue and of plasma samples, and six of the 30 discordant samples were analyzed by direct sequencing to confirm that the mismatched results were accurate. The investigators then assessed the patient population for clinical outcome and confirmed that EGFR mutations identified in the plasma were more frequent among patients with adenocarcinoma and low-smoking histories, and predicted for a significantly higher response and progression-free survival among patients treated with second-line gefitinib, consistent with results from prior studies analyzing tumor tissue.3,4,20,21 To our knowledge, this study represents the first prospective clinical trial examining the ability to detect EGFR mutations from a blood-based assay and signifies an important advance for the field. As noted earlier, tumor biopsy tissue from NSCLC patients is often too limited to perform molecular analyses. Furthermore, though mechanisms of resistance to EGFR-targeted therapies are well described and identification of these could inform choice of the most appropriate salvage therapies or clinical trials,9,12,22 serial molecular analyses from repeat tumor biopsies are simply not feasible outside the setting of a tertiary research hospital. Consequently, a reliable blood-based EGFR mutation assay would be immediately useful in clinical research and patient care. One notable shortcoming of the study design is that although tumor tissue was available for all patients, the current gold standard method for testing for EGFR mutations, direct sequencing, was not performed except in a small subset of the cases in which tumor DHPLC results did not match serum results. Consequently, there is no external validation of the DHPLC results. This could present an additional problem in the interpretation of the frequency of the resistance mutation T790M, which is located close to the common polymorphism Q787Q and may not be distinguishable on DHPLC. Fortunately, response to second-line gefitinib was assessed in 102 patients who received this therapy and is a reasonable surrogate marker of true EGFR mutation status. The response rate to gefitinib among EGFR mutants by plasma analysis was 60%, on the lower end of what is typically seen among mutation-positive patients, and for the plasma wild-type patients the response rate was 23%, on the higher end of the expected range for this cohort. This raises some concern that serum DHPLC testing may miss a portion of both activating and resistance EGFR mutations of interest, though it is not outside the range of realistic response rates for each genotype, especially in the second-line setting.4,23 Other groups have pursued different assays for blood-based EGFR mutation analysis. Kimura and colleagues tested serum samples with an ultra-sensitive allele-specific PCR-based technique that identifies a predefined set of EGFR mutations (the Scorpion Amplification Refractory Mutation System [SARMS]) and found that the serum analysis matched tumor tissue direct sequencing results in 72% and 93% of cases in two separate studies.24,25 Moran et al also examined serum samples using both direct sequencing of free DNA and allele-specific assays and found that blood-based testing was concordant with tumor results in 69%.26 Our group investigated the ability to determine EGFR mutation status from peripheral blood samples by examining captured circulating tumor cells (CTCs) using the SARMS technique along with a microfluidic CTC isolation platform that can capture high numbers of viable CTCs in an array of antiepithelial antibody-coated posts on the surface of a chip.27,28 We found that NSCLC patients known to harbor EGFR mutations had a high number of captured CTCs and that SARMS analysis of genomic DNA from these cells identified the expected mutation in 92% of cases. The CTC methodology also allowed for serial analysis of patients, akin to serial "noninvasive biopsies," which confirmed shifts in the dominant genotype over time including documenting the emergence of the T790M resistance mutation in correlation with clinical progression on EGFR TKI therapy. To date, all of the reported blood-based EGFR mutation detection methods hold promise, with a high concordance between blood results and tumor tissue results, and all have the general advantages of being noninvasive, highly sensitive, and performed more rapidly than standard direct sequencing of tumor biopsy tissue. It is not clear that any particular one of the techniques studied thus far will or should become the universal standard technique, as there are benefits and disadvantages with each method. For example, the serum DHPLC method used by Bai et al16 appears useful for widespread screening of populations since negative results require no further testing and the cost is low, making it widely applicable, even in rural settings or in cases of limited resources, as discussed by the authors. In contrast, the CTC-based method of EGFR mutation analysis may have selective advantages in serial analysis of patients known to harbor mutations and undergoing treatment with sequential targeted therapies as the test provides both dynamic enumeration of CTCs that correlates with response to therapy and snapshots of the shifting genotype in response to therapies.27,28 Bai and colleagues16 have made an important contribution with this prospective clinical trial demonstrating that blood-based results correlate with tissue-based results. Given the results of recently reported clinical trials we now have strong evidence that molecular profiling of lung cancer can be of enormous clinical significance. Several studies have demonstrated that first-line EGFR TKI therapy in a genotype-defined EGFR mutation-positive cohort leads to response rates of 55% to 75% and median progression-free survivals of about 9 months.22,29–31 Importantly, this strategy has recently been validated in a large prospective randomized clinical trial. Mok and colleagues reported at the European Society for Medical Oncology meeting in Stockholm 2008 that when Asian patients with adenocarcinoma and a low or never-smoking history were randomly assigned to receive either first-line gefitinib or first-line carboplatin/paclitaxel chemotherapy, progression-free survival was markedly improved among EGFR mutation-positive patients randomly assigned to gefitinib (hazard ratio [HR], 0.48; 95% CI, 0.36 to 0.64) and was markedly decreased among EGFR wild-type patients randomly assigned to gefitinib (HR, 2.85; 95% CI, 2.05 to 3.98).3 This study tells us that EGFR genotype trumps the phenotype of never-smoking adenocarcinoma patients when considering first-line EGFR TKIs, and that outcomes can be improved using genetic information to make first-line therapy decisions. In summary, Bai and colleagues16 have performed a timely and clinically relevant study that demonstrates that a blood-based assay of EGFR mutation status is feasible, reliable as a surrogate for tumor tissue analysis, and affordable. And given what we now know about EGFR mutations and their impact on patient outcome when personalized therapy is administered, we can no longer afford not to offer a mechanism of EGFR testing to our patients. AUTHORS' DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST Although all authors completed the disclosure declaration, the following author(s) indicated a financial or other interest that is relevant to the subject matter under consideration in this article. Certain relationships marked with a "U" are those for which no compensation was received; those relationships marked with a "C" were compensated. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment or Leadership Position: None Consultant or Advisory Role: Jeffrey A. Engelman, Hoffman-LaRoche (C), Bristol-Myers Squibb (C), Schering-Plough (C), Aveo (C), Novartis (C); Thomas J. Lynch, Merck Serono (C), Bristol-Myers Squibb (C), ImClone Systems (C), Genentech (C), OSI Pharmaceuticals (C), AstraZeneca (C), Roche (C), Boehringer Ingelheim (C) Stock Ownership: None Honoraria: None Research Funding: None Expert Testimony: None Other Remuneration: Thomas J. Lynch, Genzyme AUTHOR CONTRIBUTIONS Conception and design: Lecia V. Sequist, Jeffrey A. Engelman, Thomas J. Lynch Manuscript writing: Lecia V. Sequist, Jeffrey A. Engelman, Thomas J. Lynch Final approval of manuscript: Lecia V. Sequist, Jeffrey A. 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Copyright © 2009 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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