Oncogene (2009) 28, S14–S23 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc REVIEW Overview of molecular testing in non-small-cell lung cancer: mutational analysis, gene copy number, protein expression and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors

T John, G Liu and M-S Tsao

University Health Network, Princess Margaret Hospital Site, Ontario Cancer Institute, University of Toronto, Toronto, Ontario, Canada

Most patients with non-small-cell lung cancer (NSCLC) and seem to be more reliable predictors of response than present with advanced disease. Current treatment para- clinical features. This review discusses candidate mole- digms are shifting from cytotoxic chemotherapies alone to cular predictive markers of anti-EGFR therapy and the single-agent and combination biological and targeted different methodologies used to assess them. therapies. As patient responses to these therapies vary, predictive biomarkers will be an important facet of a patient’s diagnostic workup in personalized medicine, as there is accumulating evidence that they may enable the Molecular predictors of response to EGFR antagonists prognostication and prediction of therapeutic response. Potential biomarkers for the selection of patients with Protein expression by immunohistochemistry NSCLC most likely to benefit from epidermal growth There are several ways to detect EGFR protein and factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), measure its levels. These include radioactive-labeled ligand such as gefitinib and erlotinib, include mutations, gene binding, competitive immunoassay, western blotting and copy number increase and single-nucleotide polymorph- immunohistochemistry. The first three methods require isms of the EGFR gene, EGFR protein expression and protein extraction from fresh or snap-frozen samples. oncogenic mutation on the KRAS gene. Many techniques Although these methods are quantitative, they require are available to assay for these biomarkers. In this review, special laboratory setups that are more suitable for basic we present the current weight of evidence for using these research. In addition, as protein is extracted from whole methods as biomarkers for anti-EGFR therapy in patients tissue, normalization of results for tumor cell content can with NSCLC. be problematic. Immunohistochemistry has several ad- Oncogene (2009) 28, S14–S23; doi:10.1038/onc.2009.197 vantages over the above assays: (1) protein expression level can be evaluated directly on tumor cells or on other Keywords: erlotinib; gefitinib; polymerase chain reaction; specific cells of interest; (2) the technique can be performed biomarker; survival on formalin-fixed, paraffin-embedded (FFPE) tumor tissues, which represent the largest archival bank of human tissue samples; and (3) immunohistochemistry is a technique that is routinely used in clinical pathology Introduction practice. Countering these advantages, however, is the fact that quantification of expression levels by visual inspection Targeting the epidermal growth factor receptor (EGFR) canberegardedassemiquantitativeatbestandachallenge using tyrosine kinase inhibitors (TKIs) has proven to be to standardize. an effective treatment strategy for advanced non-small- Most laboratories use the indirect immunolabeling cell lung cancer (NSCLC). However, not all patients method for immunohistochemistry. The factors that are benefit from TKI treatment, and several investigators most likely to influence immunoreactivity and staining have sought to identify a priori markers that will predict include types of tissue fixative and condition of fixation, a response to, and benefit from treatment. Certain as well as the reactivity and specificity of the primary clinical characteristics have been shown to be associated antibody (Atkins et al., 2004; Dei Tos and Ellis, 2005; with an increased likelihood of responsiveness to EGFR Penault-Llorca et al., 2006; Buckley and Kakar, 2007). TKIs (female gender, nonsmoking history, adenocarci- Several commercial antibodies for EGFR are available. noma histology and East Asian race) (Miller et al., The two most commonly used are the DakoCytomation 2004). In addition, several molecular predictors are (Carpentaria, CA, USA) clone 18C9, which is used in the associated with responsiveness to EGFR TKI treatment, pharmDx kit, and the Zymed Laboratories Inc. (South San Francisco, CA, USA) clone 31G7 (Bhargava et al., 2006; Penault-Llorca et al., 2006). To obtain consistent Correspondence: Dr M-S Tsao, Room 7-611, 610 University Avenue, Toronto, Ontario, Canada M5G 2M9. results, immunohistochemistry studies require carefully E-mail: [email protected] controlled and validated standard operating procedures. Molecular testing of EGFR in NSCLC T John et al S15 Immunohistochemistry results can be evaluated qua- chromosome corresponding to the gene, with a specific litatively for the absence or presence of staining of the fluorescent dye. As multiple fragments can be labeled studied cells, and semiquantitatively for the intensity of using fluorophores with different emission wavelengths, staining, percentage of stained cells or both. In addition, multiple FISH probes can be co-hybridized to detect by multiplying the staining intensity by the percentage different genes in a single assay. The fluorescent labels of cells stained, an H-score can be derived. A simple enable the visualization of the number of copies and H-score may be based on a single predominant staining location of the target genes in metaphase as well as intensity, whereas more complex H-scores can include interphase nuclei. The technique for the EGFR FISH the sum of individual H-scores for each intensity level assay is well described (Varella-Garcia, 2006) and observed. The sample can then be scored as positive or performed routinely using B4-mm-thick FFPE tissue negative on the basis of specific adopted cutoff points. sections. The most commonly used probe is the LSI The method for determining such a cutoff is one of the EGFR SpectrumOrange/CEP 7 SpectrumGreen probe controversial issues in immunohistochemistry scoring. set (Vysis Inc., Downers Grove, IL, USA). This probe set includes directly labeled DNA FISH probes for the EGFR immunohistochemistry as a prognostic and EGFR gene and the centromere of chromosome 7. There predictive biomarker are several technical considerations that could compro- Studies using different antibody sources have reported a mise the FISH assay, including lengths of proteinase K high prevalence of EGFR immunostaining in NSCLCs digestion and probe hybridization duration and condi- (Rusch et al., 1997; Fontanini et al., 1998; Hsieh et al., tion (Varella-Garcia, 2006). 2000; Hirsch et al., 2003). The rate is consistently higher in The currently used EGFR FISH scoring system was squamous-cell carcinomas (55–100%) than in adenocarci- developed by the Colorado group (Cappuzzo et al., nomas (35–60%) or large-cell carcinomas (20–60%). 2005) and requires the use of an epifluorescence Several studies have shown an association between EGFR microscope equipped with a genetic workstation. The protein expression and response to EGFR TKIs; however, cutoffs used for this scoring system were determined in a there have been conflicting results with regard to the value retrospective study of gefitinib-treated patients with of EGFR protein expression in predicting survival benefit advanced NSCLC (Cappuzzo et al., 2005). Unlike the with EGFR TKIs (see Hirsh et al., 2009). FISH assay for the HER2 gene in breast cancer, the scoring system for the EGFR gene copy changes in NSCLC is more complex, perhaps reflecting the EGFR immunohistochemistry as a clinical test different biological impact of the two genes in the two Several major issues with technical aspects of immuno- diseases. The system stratifies tumors into six categories histochemistry require standardization, although the on the basis of the quantity of tumor cells with a specific most important variables include the type and source of EGFR gene copy number compared with the chromo- antibody and the scoring system used (Dei Tos and Ellis, some 7 copy number, which is scored using the CEP7 2005; Eberhard et al., 2008; Hirsch et al., 2008a). probe. Tumors are considered FISH positive if they Discordance between antibodies may be significant, show amplification (the presence of tight gene clusters ranging from 12 to 24% (Buckley and Kakar, 2007; and a ratio of X2 genes/chromosomes per cell or X15 Hirsch et al., 2008a). EGFR immunohistochemistry is gene copies per cell in X10% of analysed cells) or high therefore not yet optimal in determining a patient’s polysomy (X4 copies of the gene in X40% of cells). eligibility to receive EGFR TKI therapy (Ciardiello and Tumors considered FISH negative are those with no Tortora, 2008), despite results indicating that EGFR amplification as defined above or with 40% of cells immunohistochemistry-negative patients demonstrate o showing X4 copies of the EGFR gene per cell (Varella- reduced responses and clinical benefit. Garcia, 2006). EGFR gene copy or dosage The EGFR gene is located on the short arm of EGFR FISH as a prognostic and predictive biomarker chromosome 7 (7p21), which is commonly overrepre- The association between the high EGFR gene copy by sented or amplified in NSCLC. There are several FISH (FISH-positive), as defined by the Colorado methods for detecting and determining EGFR gene group (Cappuzzo et al., 2005), and the clinical end copy number and dosage, including fluorescence in situ points of EGFR TKI therapies has been investigated in hybridization (FISH), chromogenic in situ hybridization a number of studies. In the Italian Expanded Access (CISH) and real-time quantitative polymerase chain Study of gefitinib, the BR.21 and the ISEL studies, an reaction (qPCR). association was observed between EGFR FISH positiv- ity and either increased response rates or an improved FISH survival benefit (Hirsch et al., 2003, 2006; Tsao et al., The development of fluorescent-labeling techniques has 2005; see Hirsch et al., 2009). The high EGFR gene copy enabled multiple targets on the same or separate number detected by FISH may also be predictive of chromosomes to be analysed simultaneously, thereby benefit from chemotherapy combined with cetuximab allowing control probes to be hybridized and used as a (Hirsch et al., 2008b). reference (Trask et al., 1985). This involves labeling the Compared with reports in Caucasian patients, only a DNA fragment of a gene, or the bacterial artificial limited number of FISH studies have been conducted in

Oncogene Molecular testing of EGFR in NSCLC TJohnet al S16 East Asian patients with NSCLC treated with EGFR scoring system proposed for FISH (Cappuzzo et al., TKIs (Ichihara et al., 2007; Sone et al., 2007). In the 2005) may not be extrapolated automatically to CISH. studies by Ichihara et al. (2007) and Sone et al. (2007), Nevertheless, with a scoring system that depends only 41% of patients were FISH positive, but FISH positivity on an EGFR gene signal, CISH is an alternative method was not associated with survival benefit, suggesting a for assessing EGFR gene copy status. discrepancy between East Asian and Caucasian ethni- In a recent study comparing the performance of FISH cities. It is hoped that the FISH analysis from the and CISH for the detection of increased EGFR copy recently presented I-PASS study will further clarify this number in 77 patients with NSCLC treated by surgery issue (Mok et al., 2008). alone (Figure 1), a significant correlation was found between the number of signals detected by both methods (Spearman’s r ¼ 0.81; Po0.0001) (Sholl et al., 2007). CISH Discrepancies between the two tests were seen in only CISH is an alternative assay to FISH for determining 7% of cases. At a signal of 4.5, CISH had very high gene copy number and amplification in tissue sections. sensitivity (89%) and specificity (89%) for the discrimi- The major difference between the two methods is that nation of low and high EGFR polysomy (Po0.0001). At FISH requires an epifluorescent system, whereas CISH a signal of 7.1, CISH was also effective at distinguishing can be evaluated by the pathologist using bright-field between high polysomy and amplification (P ¼ 0.0003). microscopy and correlated directly with histopathology. Another study comparing CISH and FISH in 58 Furthermore, as with immunohistochemistry, CISH- NSCLC samples showed similar results, with a high stained slides can be archived and stored permanently. concordance between EGFR copy number determined The DNA probes are labeled with digoxigenin or by the two tests (Gallegos Ruiz et al., 2007). Overall, biotin (Gallegos Ruiz et al., 2007; Sholl et al., 2007; these data support the feasibility of using CISH to Chang et al., 2008; Li et al., 2008) and then detected evaluate the function of EGFR gene copy as a predictive using a CISH Detection Kit (Zymed Laboratories, marker for EGFR TKI therapy, although further South San Francisco, CA, USA). Gene/chromosome evaluation and validation in clinical trial samples is probes are visualized as dark brown dots, which are needed before this technique can be adopted clinically. counted in parallel sections using standard light micro- scopy and are converted into the number of nuclear signals per cell. CISH may be useful in samples that are qPCR difficult to interpret by fluorescence microscopy because Several different methods of performing qPCR mea- of sample heterogeneity, high autofluorescent back- surements can be used to detect small amounts of RNA ground signal or both (Daniele et al., 2007). The main or DNA (Bustin, 2002). Using probes that are labeled potential drawback of CISH compared with FISH is with both a fluorophore and fluorescence-quenching that most CISH studies have been performed on frozen molecule, amplicon generation can be detected and sections and only a single color can be developed per quantified in real time during a PCR. An alternative and section, meaning that one is not able to evaluate a less expensive method uses the double-stranded DNA control probe for aneusomy. Therefore, the complex intercalating agent, SYBR Green, as the signal emitter.

Figure 1 Fluorescent in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) for EGFR gene copy in NSCLC. EGFR FISH probe (green) and 7q control probe (red) show EGFR gene high polysomy (a) and amplification (c). The corresponding slides stained with the EGFR gene probe by the CISH method are shown in (b and d). Adapted with permission from Sholl et al. (2007).

Oncogene Molecular testing of EGFR in NSCLC T John et al S17 Using multiple primers labeled with different fluoro- anced PCR amplification and the relative amount of phores, current qPCR technology can quantitate multi- contaminating wild-type allele of non-tumor cells can ple genes, including control genes, simultaneously in one influence the sensitivity of mutant detection by direct reaction. sequencing (Engelman et al., 2006). In addition, DNA The greatest pitfall for using qPCR to measure gene extracted from FFPE tissues could give rise to sequen- dosage is the inability to control for contamination from cing artifacts, thus requiring a confirmation by the normal host cells that result in the dilution of tumor cell sequencing of independent PCR products. gene copy. Performing laser-capture microdissection can Owing to concern regarding the sensitivity of the reduce this problem, but this is laborious and clinically direct-sequencing method, a variety of other methods impractical. The other issue with qPCR-based gene have been investigated to detect low abundance muta- dosage estimation is normalization. Normalization tions and increase the sensitivity of the mutation assay. using another region of the same chromosome as the These include high-resolution melting analysis (Nomoto studied gene, such as centromeric sequences, will detect et al., 2006; Willmore-Payne et al., 2006; Fukui et al., true amplification, but will not detect increased gene 2008), single-stranded conformational polymorphism copy number due to polysomy. Normalization using and denaturing high-performance liquid chromatogra- centromeric sequences of multiple chromosomes may phy analysis of PCR amplicons after digestion by CEL1 allow the assessment of the degree of aneusomy. endonuclease (Ja¨nne et al., 2006). These methods allow There have been two studies on EGFR gene dosage by the rapid mutation screening of large numbers of qPCR to predict treatment benefit in patients with samples with increased sensitivity but still require direct NSCLC receiving TKI therapy. Both studies failed to sequencing to identify or confirm mutations. The demonstrate its clinical applicability (Bell et al., 2005; amplification refractory mutation system (ARMS) Dziadziuszko et al., 2006). EGFR qPCR was not (Newton et al., 1989; Whitcombe et al., 1999), combined predictive of response to treatment, disease control, with the scorpion method (Kimura et al., 2006) and the progression-free survival or overall survival, nor did it peptide nucleic acid-locked clamping method (Nagai correlate with FISH, immunohistochemistry or mRNA et al., 2005), are alternative sensitive methods that may expression (Bell et al., 2005; Takano et al., 2005; be used to detect rapidly specific EGFR mutations using Dziadziuszko et al., 2006, 2007). real-time PCR-based technology.

Somatic EGFR gene mutations EGFR mutations as biomarkers In May 2004, two independent groups of investigators Exon 19 deletion and exon 21 L858R substitution reported the discovery of somatic mutations in the TK account for 85–90% of the drug-sensitive EGFR domain (exons 18–23) of EGFR (Lynch et al., 2004; mutations seen in NSCLC (Riely et al., 2006; Sakurada Paez et al., 2004). Practically all mutations that have et al., 2006). The mutations are more common in been reported are on exons 18 through 21. These adenocarcinomas, in persons of East Asian ethnicity, mutations affect the adenosine triphosphate (ATP)- women and in never smokers. These phenotypic features binding cleft of EGFR, which is also where TKIs bind. had already been marked as clinical predictors of In vitro studies have shown that EGFR mutants have response to TKIs before the discovery of the mutations constitutive TK activity and, therefore, a greater (Fukuoka et al., 2003; Kris et al., 2003). The prevalence sensitivity to anti-EGFR inhibition. Among all muta- of EGFR mutations varies by ethnicity with ranges from tions, four predominantly result in TKI drug sensitivity 20–40% in Asian populations (Huang et al., 2004; by in vitro and in vivo studies. These include point Kosaka et al., 2004; Han et al., 2005; Shigematsu et al., mutations in exons 18 (G719A/C) and 21 (L858R and 2005; Tokumo et al., 2005) to 5–20% among Caucasians L861Q) and in-frame deletions in exon 19, which (Cappuzzo et al., 2005; Marchetti et al., 2005). eliminate four amino acids (LREA) downstream of the The association between the presence of activating lysine residue at position 745; other mutations appear to EGFR mutations and high response rates to TKIs was be associated with variable or less sensitivity (Eberhard demonstrated in a number of phase II studies in which et al., 2005; Chen et al., 2006; Sharma et al., 2007; see all patients received EGFR TKI therapy. The overall Gazdar, 2009). response rates were as high as 80% in patients harboring The most commonly used method to detect mutations mutations compared with p10% in patients with wild- is direct sequencing of the PCR-amplified exon se- type EGFR (Huang et al., 2004; Lynch et al., 2004; Mu quences. Tumor sections are initially stained with et al., 2004; Paez et al., 2004; Pao et al., 2004; Bell et al., hematoxylin and eosin (H&E) or methylene blue to 2005; Cappuzzo et al., 2005; Chou et al., 2005; Cortes- determine tumor cell percentage and to account for all Funes et al., 2005; Eberhard et al., 2005; Han et al., cellular components present. For the direct sequencing 2005; Kim et al., 2005; Kondo et al., 2005; Mitsudomi method, samples with tumor cellularity o40% should et al., 2005; Takano et al., 2005; Taron et al., 2005; be enriched by microdissection. DNA is extracted from Tomizawa et al., 2005; Tsao et al., 2005; Zhang et al., the selected tumor tissue using standard methods or a 2005; Shih et al., 2006; Uramoto et al., 2006). In commercially available kit. After PCR amplification of addition, the presence of mutations has also been each exon of the EGFR TK domain, amplicons are associated with improved progression-free survival and sequenced. The copy number of mutant allele, imbal- overall survival with EGFR TKI treatment compared

Oncogene Molecular testing of EGFR in NSCLC TJohnet al S18 with patients with wild-type EGFR (Cappuzzo et al., American study (Liu et al., 2008) found associations 2005; Cortes-Funes et al., 2005; Han et al., 2005; with lung cancer survival or toxicity with gefitinib, Mitsudomi et al., 2005; Takano et al., 2005; Tsao whereas others found no significant association (Ichi- et al., 2005); however, several studies have since reported hara et al., 2007; Gregorc et al., 2008). Shorter CA that non-TKI-treated patients with NSCLC and EGFR repeat lengths were associated with poorer survival in mutations have a more favorable prognosis than do the absence of therapy with an EGFR TKI, which is a patients with wild-type EGFR (Eberhard et al., 2005; reversal of expectations in TKI-treated patients (Dubey Riely et al., 2006; Sasaki et al., 2006; Kosaka et al., 2007; et al., 2006). Yoshida et al., 2007; see Gazdar, 2009). The multidrug transporter ABCG2 was shown to be Mutations in the kinase domain of EGFR have also active in removing gefitinib from cells (Elkind et al., been associated with an acquired resistance to EGFR 2005). In published reports, ABCG2 polymorphisms TKIs. A secondary mutation involving the substitution have been identified as being associated with increased of methionine for threonine in codon 790 (T790M) is EGFR TKI concentrations, toxicity or both, in patients present in approximately 50% of cases of acquired treated with gefitinib and erlotinib (Cusatis et al., 2006; resistance (Kobayashi et al., 2005; Pao et al., 2005a; Li et al., 2007; Rudin et al., 2008). Kosaka et al., 2006; see Gazdar, 2009). Screening for the In summary, genetic polymorphisms may be impor- emergence of the T790M mutation during treatment tant contributors to variability in response to EGFR may allow for the earlier identification of acquired TKI therapy, but evidence is thus far contradictory. resistance. A recent study showed that T790M can be Several reasons exist to explain the inconsistent data: detected before EGFR TKI treatment with a highly (1) studies have been underpowered, with sample sizes sensitive allele-specific assay (Maheswaran et al., 2008). generally lower than 200 patients; (2) different definitions The investigators found that the pretreatment presence are used for key variables, such as ‘short’ and ‘long’ of T790M did not preclude responses with EGFR TKIs, intron 1 CA repeat lengths and ‘clinical toxicity’; (3) but was associated with significantly shorter progres- confounders such as race, polymorphism allele frequen- sion-free survival compared with patients without cies, mutation rates, copy number changes and other detectable T790M before TKI treatment (7.7 vs 16.5 clinical and molecular prognostic and predictive factors months; Po0.001). These results suggest that T790M have rarely been accounted for in these polymorphism may be a useful pretreatment biomarker for identifying analyses. Comprehensive analyses are needed to deter- patients who are not likely to experience long-term mine whether genetic polymorphisms are independently benefits with reversible EGFR TKIs (that is, erlotinib predictive with EGFR TKI therapy, are simply corre- and gefitinib). lated with other molecular or clinical prognostic factors, or are prognostic but not predictive.

Genetic polymorphisms and EGFR-targeted drugs The EGFR gene contains numerous genetic polymorphic KRAS gene mutation variants. The evaluation of germ line genetic variants RAS is considered a ‘master switch’ signal transduction uses DNA extracted from any non-tumor source, protein downstream of multiple types of cell surface including leukocytes from whole blood. Several genetic receptors, including tyrosine kinase and G-protein- variants are associated with alterations in the mRNA coupled receptors (Aviel-Ronen et al., 2006; Molina gene expression (Liu et al., 2005). Both the EGFR and Adjei, 2006). A point mutation on codons 12, 13 and –216G/T and –191C/A polymorphisms are located in the 61 of all RAS family genes will lead to the inability of the transcriptional start site region of the promoter, wherein RAS–guanosine triphosphatase (RAS–GTPase)-activat- multiple nuclear regulatory affinity sites are located (Liu ing protein to hydrolyse bound GTP to guanosine et al., 2005). Two studies in Caucasian patients reported diphosphate, thus resulting in a constitutive activation that the –216G/T variant, alone or in combination with of the RAS protein. In NSCLC, >90% of RAS –191C/A, was associated with improved outcome, mutations involve the KRAS gene, with most occurring greater toxicity to gefitinib or both (Cusatis et al., in codon 12. The most direct way to detect KRAS codon 2006; Liu et al., 2008), whereas a third found no 12 and 13 mutations is by PCR amplification of exon 2 association (Gregorc et al., 2008). Among Asians, these and by direct sequencing of the amplicon. However, all variants are rare (Ichihara et al., 2007). the previously discussed caveats for the detection of One of the enhancer elements for EGFR is located in EGFR mutations using this technique on FFPE samples intron 1 (Maekawa et al., 1989; Haley and Waterfield, apply, especially the need to enrich samples to X50% 1991). Shorter alleles of the dinucleotide CA repeat tumor cellularity. The need for repeat PCR to confirm sequence polymorphism in intron 1 of EGFR are mutations is less of a concern, as any mutations detected associated with a greater EGFR expression than are outside codons 12, 13 and 61 can be ignored. the longer repeats (Gebhardt et al., 1999; Etienne- Pao et al. (2005b) first suggested that patients with Grimaldi et al., 2005). In Asian patients, a shorter CA NSCLC whose tumors harbor mutations are resistant to repeat length was associated with a greater EGFR EGFR TKI. To date, among 75 NSCLC patients expression and copy number (Zhou et al., 2006). Asian harboring KRAS mutations who were treated with patients tend to have longer CA repeat lengths. Two TKIs (Pao et al., 2005b; Hirsch et al., 2006; Massarelli Asian studies (Han et al., 2007; Nie et al., 2007) and one et al., 2007; Miller et al., 2008; Zhu et al., 2008), only

Oncogene Molecular testing of EGFR in NSCLC T John et al S19 one patient has been reported to respond (Zhu et al., pretreated metastatic NSCLC and in 40–50% of East 2008). Interestingly, this patient’s tumor also had an Asian patients. The ability to predict significant clinical EGFR gene amplification. On the basis of TKI response benefits with appropriate biomarkers would be a major rates of up to 10% in patients with wild-type EGFR, one advancement in the achievement of personalized med- would expect that at least seven to eight of these 75 icine for patients with NSCLC, as it would allow the patients should have responded, suggesting that mu- tailoring of treatment with TKIs to patients who are tated KRAS results in TKI resistance. In the TRIBUTE most likely to benefit, whereas other patients would study, patients with KRAS mutant tumors, who were receive alternative therapies that may also be effective. treated with a combination of erlotinib and chemother- To date, several such biomarkers and various techno- apy, showed poorer survival than did patients treated logies to assay them have been described and studied, with chemotherapy alone or patients with wild-type but none is currently ready for clinical implementation. KRAS, regardless of treatment type (Eberhard et al., This is largely because of the fact that most biomarker 2005). In the BR.21 study, KRAS mutations were found study results have been derived from phase II trials in in 30 (15%) of 206 patients tested. The HR of erlotinib which all patients were treated, whereas very limited to placebo treatment in 30 patients with a KRAS data are available from the results of phase III trials that mutation was 1.67 (95% CI, 0.62–4.50; P ¼ 0.31), included both drug-treated and placebo-controlled whereas for 176 patients with wild-type KRAS it was patients. The latter is important when one is trying to 0.69 (95% CI, 0.49–0.97; P ¼ 0.03), with an insignificant identify significant predictive markers that truly demon- interaction P-value of 0.09. In a multivariate analysis, strate differential benefits of therapies between marker- KRAS mutation was neither prognostic for poorer negative and marker-positive patients (Shepherd and survival in untreated patients nor predictive of differ- Tsao, 2006). ential treatment effect (Zhu et al., 2008). Overall, these To maximize the therapeutic index and cost-effective- results suggest that patients with NSCLC who have ness of anti-EGFR therapy, both negative and positive KRAS mutations are unlikely to derive any benefit from predictive biomarkers should be considered. For nega- current TKI therapy, although the number of reported tive markers that may potentially exclude patients from cases remains limited. receiving therapy, the candidate markers are EGFR immunohistochemical negativity, KRAS mutation and MET amplification. Each of these has its respective MET amplification shortcomings. For immunohistochemistry, a standar- Using gefitinib-resistant NSCLC cell lines, Engelman dized assay and quantitative scoring procedures need to et al. (2007) recently found that amplification of the be established and validated prospectively. Despite MET proto-oncogene may be involved in resistance to accumulating evidence for KRAS tumors being resistant EGFR TKIs by enabling cells to activate the PI3K and to EGFR TKIs, the number of reported cases remains Akt-signaling pathway independently of EGFR through low ( 100 cases), especially considering that only 10% HER3 activation. MET is commonly overexpressed in o of tumors that are negative for both KRAS and EGFR NSCLC (Tsao et al., 1998; Ma et al., 2005) and may be a mutations still respond to TKIs. et al. et al. poor prognostic marker (Cheng , 2005; Rossi , Both EGFR mutation and a high gene copy number 2005). It has been speculated that MET amplification (FISH positivity) are promising positive selection accounts for approximately 10–20% of acquired resis- biomarkers (that is, markers to select patients who are tance to gefitinib and erlotinib (Engelman and Janne, ¨ likely to respond to TKI therapy). The current adoption 2008). In addition, preclinical data suggest that inhibi- of EGFR mutation as a selection marker would exclude tion of MET can restore sensitivity to gefitinib (Engel- EGFR TKI therapy in 80–90% of non-East Asian man et al., 2007). Although this requires further patients, whereas 50–60% would be excluded when elucidation in a clinical setting, the dual inhibition of using EGFR gene copy (FISH positivity) as a marker. EGFR and MET may be an approach to overcome However, the value of mutational analysis for predicting resistance to EGFR TKIs. response may not be uniform across various ethnicities. MET amplification can be detected either by qPCR For example, in Caucasian patients, whose overall or FISH (Engelman et al., 2007). Along with the response rate is 10%, it seems highly sensible to use general challenges that accompany these two techniques, mutational status as a marker to select the 10–20% of an added difficulty centers on the threshold level of patients who are most likely to benefit from anti-EGFR MET amplification, which is clinically relevant for therapy. Yet, many studies have consistently reported EGFR TKI treatment. Currently, this is not well 10% response rates to gefitinib and erlotinib in patients defined and will clearly be important if this molecular with wild-type EGFR, and the HR of erlotinib to factor is to be used as a biomarker for EGFR TKI placebo is 0.74 (Zhu et al., 2008), which is comparable to treatment. the overall survival benefit of erlotinib in all BR.21 patients (Shepherd et al., 2005). Therefore, it may be that one unifying predictive model does not apply to all Practical considerations patients with NSCLC, and that it is more practical to define different models for patients of various ethnicities. EGFR TKIs are associated with significant clinical Finally, the adoption of biomarker analysis into responses in 10–20% of Caucasian patients with heavily clinical practice would be significantly facilitated if such

Oncogene Molecular testing of EGFR in NSCLC TJohnet al S20 tests could be performed using blood samples, especially clinical practice are important issues that need to be considering the fact that for patients with advanced addressed. This can be achieved only by dedicated disease, the diagnosis is often made on fine-needle tumor banking and further molecular profiling of the aspirates, which are not likely to yield adequate material tumors from patients enrolled in large phase III for mutational/FISH testing. Kimura et al. (2006, 2007) randomized studies. reported the ability to detect EGFR mutations in serum samples of patients with advanced NSCLC with approximately 75% sensitivity. More recently, Mahes- Conflict of interest waran et al. (2008) reported 92% sensitivity in the detection of EGFR mutations in isolated circulating M-S Tsao has received consulting/paid advisory board tumor cells, including the detection of the T790M- fees from Roche International, AstraZeneca and Roche resistant mutants in patients undergoing TKI therapy. Canada. M-S Tsao has received lecture fees from These approaches are becoming more feasible with the AstraZeneca. The remaining authors have declared no increasing availability of automated equipment neces- conflict of interest. sary for the isolation of circulating tumor cells and may circumvent the issue of limited tissue availability. The ability to personalize small molecule-based Acknowledgements therapies on the basis of molecular markers remains This article was funded by Boehringer Ingelheim Pharmaceu- elusive in patients with NSCLC, although enormous ticals Inc. Dr John is an International Association for Study of progress has been made in a relatively short period of Lung Cancer (IASLC) Translational Fellow in Lung Cancer time. The most appropriate biomarkers for predicting Research. Dr Liu is the Alan B Brown Chair in Molecular response and the preferred gene-sequencing technology Genomics, and Dr Tsao is the M Qasim Choksi Chair in Lung for the determination of mutational status in routine Cancer Translational Research.

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