REVIEWS. Personalized medicine in lung cancer: what we need to know. Tony S. K. Mok

Personalized medicine in lung cancer: what we need to know Tony S. K. Mok Abstract Lung cancer is a complex and often fatal disease. The recent discovery of activating mutations in EGFR and fusion genes involving ALK has set the stage for personalized medicine for lung cancer. Patients selected using biomarkers have benefited from the development of EGFR tyrosine kinase inhibitors and ALK inhibitors with considerable improvement in tumor control and survival. Four key areas of knowledge that are essential to the development of targeted therapy are discussed in this Review: knowing the target, knowing the biomarker, knowing the end point and knowing the mechanisms of resistance. Mok, T. S. K. Nat. Rev. Clin. Oncol. 8, 661 668 (2011); published online 23 August 2011; doi:10.1038/nrclinonc.2011.126 Introduction Historically, non-small-cell lung cancer (NSCLC) was considered to be a single disease entity because the different histologic subtypes into which it can be classified seemed to share the same cause, clinical characteristics and treatment outcomes. For this reason, the same treatment has been used for all patients with NSCLC for the past few decades, and the median overall survival was persistently less than 12 months. 1,2 Only when NSCLC was recognized to be a collection of diseases that are identifiable by molecular abnormalities could treatment be personalized and improved. The first breakthrough towards personalized treatment took place in 2004 when Lynch et al. 3 and Paez et al. 4 reported the presence of activating mutations in the tyrosine kinase domain of EGFR in patients who had a dramatic response to the EGFR tyrosine kinase inhibitor (TKI), gefitinib. Since then, personalized medicine for lung cancer has become a reality. Personalized medicine is not a novel concept. Since the development of traditional Chinese herbal medicine one of the oldest schools of medical therapies treatment has been based on the personalized approach of diag nosis that takes into account the patient s symptoms and the healer s observations. 5,6 The modern approach to personalized cancer medicine is founded on the identification of molecular abnormalities that could account for the patient s disease and, ideally, the identification of a single molecular event that is highly specific to malignant cells. However, hundreds of genomic abnormalities occur in a cancer cell and not all of them are suitable targets for personalized therapy. 7 This is underscored by the fact that the number of successful clinical trials of molecular-targeted therapies for lung cancer is far less than the number of unsuccessful trials. 8 To improve the chances of success in Competing interests T. S. K. Mok declares associations with the following companies: AstraZeneca, Boehringer Ingelheim, Merck Serono, Pfizer and Roche. See the article online for full details of the relationships. the development of personalized medicine for lung cancer we need to know the target, the biomarker, the therapeutic end point, and the mechanisms of resistance. This Review discusses the development and use of drugs targeting activating mutations in EGFR and the oncogenic fusion gene EML4 ALK as outstanding examples of how personalized medicine is a reality for patients with NSCLC. Know the target Multiple mutations occur in NSCLC, and the spectrum of mutations is very different between smokers and nonsmokers, and between squamous-cell carcinoma (SCC) and adenocarcinoma. 7,9 A candidate target for personalized therapy should be specific to the cancer cell and should drive cell proliferation in such a way that inhibit ion of the mutated protein would result in cessation of cancer growth. This information should, ideally, be established using preclinical studies that demonstrate differential growth rates in tumor cells with and without the mutation in the target protein. Clinical characteristics of patients with mutations in the target gene should also be known, so that the incidence of a given target mutat ion is established in a specific population. Lastly, and most importantly, clinical studies of a specific molecular-target ed inhibitor must show a high tumor response rate in patients with the target mutation, a phenomenon that would not be expected in patients without the target mutation. Activating mutations in EGFR are the best-known target in lung cancer. However, when gefitinib was first introduced as a drug targeting EGFR, tumor response rates in unselected patients were persistently below 20%, which was inferior to standard first-line chemotherapy. 10 Preclinical studies testing EGFR TKIs were only performed retrospectively after the discovery of activating mutations in EGFR in 2004. Cell growth of lung cancer cell lines transfected with mutant EGFR was inhibited by treatment with EGFR TKIs at much lower concentration than cell lines expressing wild-type EFGR. 11 Multiple Department of Clinical Oncology, The Chinese University of Hong Kong, State Key Laboratory of South China, Sha Tin, Hong Kong, China. tony@clo.cuhk.edu.hk NATURE REVIEWS CLINICAL ONCOLOGY VOLUME 8 NOVEMBER 2011 661

Key points Personalized medicine should be based on a molecular target that drives cancer cell proliferation, the inhibition of which would cease tumor growth Predictive biomarkers are crucial to identify patients with molecular targets, who are likely to respond better to targeted therapies than to standard therapy Progression-free survival could be an appropriate primary end point in randomized comparative studies with considerable crossover, as overall survival of the two groups would be similar It is likely that patients who benefit from molecular-targeted therapy will eventually become resistant to the drug; understanding the mechanisms of resistance is essential to prevent or defer this process Table 1 Tumor response rate to EGFR TKIs in patients with EGFR mutations Study n Patients with mutations in EGFR (n) EGFR TKI RR (%) Inoue et al. (2009) 12 99 16 Gefitinib 75 9.7 Rosell et al. (2009) 13 2,105 350 Erlotinib 71 14 Tamura et al. (2008) 14 118 32 Gefitinib 75 NA Sutani et al. (2006) 15 100 38 Gefitinib 78 9.4 TTP (months) Sequist et al. (2007) 10 98 31 Gefitinib 55 11.4 Abbreviations: NA, not available; RR, response rate; TKI, tyrosine kinase inhibitor; TTP, time to progression. single-arm clinical studies were then carried out, from which we learnt that the tumor response rates to EGFR TKIs in patients with EGFR-mutant tumors ranged from 55% to 78% (Table 1). 10,12 15 The incidence of EGFR mutations in different populations of patients with NSCLC was first assessed by Shigematsu et al. 16 The researchers screened 671 tumor samples and found the incidence of EGFR mutations to be higher in Japanese (26%) and Taiwanese (36%) patients than in American (10%) and Australian patients (12%). 16 In addition, multiple population-based studies were conducted and the data from these studies were summarized in the Review article by Yatabe and Mitsudomi. 17 Among the different studies assessed in the Review, a total of 2,880 patients with lung cancer were included, and the investi gators reported on the incidence of EGFR mutations in Asian versus non-asian patients (32% versus 7%); women versus men (38% versus 10%); never-smokers versus smokers (47% versus 7%); and adenocarcinoma versus non-adenocarcinoma (30% versus 2%). 17 The high response rates to treatment with EGFR TKIs in patients with EGFR-mutant tumors (Table 1) confirmed that the correct target had been identified. 18 However, the drugs still had to be compared against chemotherapy in randomized phase III studies before TKIs were accepted as a standard therapy. The Iressa TM Pan-Asia Study (IPASS) was the first comparative study to confirm that gefitinib is superior to stand ard chemotherapy. 19 Patients with Asian ancestry and adenocarcinoma NSCLC who were light or never-smokers were randomly assigned to receive either gefitinib (250 mg daily) alone or paclitaxel (200 mg/m 2 ) plus carboplatin (5 times AUC). From a total of 1,217 patients, 437 tumor samples were tested for mutations in EGFR and 261 of them (59.7%) were positive. Response rates in patients with mutation-positive tumors were 71.2% and 47.3% in the gefitinib and chemo therapy arms, respectively. Gefitinib significantly improved progression-free survival (PFS) in patients with EGFR-mutant tumors (hazard ratio [HR] = 0.48, P <0.0001), although the drug was associated with adverse outcomes in patients without mutations in EGFR (HR = 2.85, P <0.001). 19 A similar study, FIRST-SIGNAL (FIRST-line Single agent Iressa TM versus Gemcitabine and cisplatin trial in Never-smokers with Adenocarcinoma of the Lung) selected patients by clinical parameters never-smokers and positive for adenocarcinoma and used overall survival as the primary end point. Results were presented at the World Conference in Lung Cancer in 2009 but to date they are only available in abstract form. 20 There was no significant difference in overall survival, but in the small subset of patients (n = 42 out of 96 patients tested) who harbored EGFRactivating mutations, PFS was higher in the gefitinib arm (8.4 months versus 6.7 months, P = 0.08). By contrast, in patients without EGFR-mutant tumors (n = 54) PFS was higher in the chemotherapy arm (2.1 months versus 6.4 months, P = 0.07). These two studies confirmed that patient selection should be based on EGFR-mutation status and not on clinical parameters. The importance of biomarker selection is further supported by four other randomized studies that selected patients with EGFR mutations, who were randomized to receive either EGFR TKIs or chemotherapy (Table 2). 21 24 The consistently higher response rates and longer PFS have confirmed EGFR TKIs to be a standard therapy for patients with activating mutations in EGFR. Development of inhibitors of the EML4 ALK fusion gene product took a different path. It was previously established that chromosomal translocation of the ALK gene that fuses with nucleophosmin (NPM) results in the expression of an oncogene that drives tumor growth in lymphoma. 25 Soda et al. 26 reported a similar type of translocation in NSCLC that resulted in aberrant fusion of ALK with EML4 and encoded a cytoplasmic chimeric protein with kinase activity. 26 In an in vivo study, the researchers showed that nude mice injected subcutaneously with 3T3 fibroblasts transformed with either the EML4 ALK or NPM ALK fusion gene had substantial tumor growth, whereas tumor growth was not observed with transfection of the ALK or EML4 genes alone. 26 This finding suggested that the EML4 ALK fusion gene was a driver gene of tumorigenesis. However, this is a relatively rare genomic abnormality, and only five of every 75 Japanese patients (6.7%) who were tested harbored the fusion gene. 26 Following this report, the incidence rates in studies in different countries were shown to range from 1.6% to 11.6%. 27 30 The major clinical characteristics of patients with the EML4 ALK fusion gene are similar to those of patients with EGFR mutations, namely nonsmoker and adenocarcinoma. Almost all patients with the EML4 ALK fusion gene do not have mutations in EGFR. 30 The incidence of EML4 ALK fusion seems to be higher in the Chinese population, 27 but this has not been validated and the testing methods are different to other comparable studies. Kwak et al. 31 reported the 662 NOVEMBER 2011 VOLUME 8 www.nature.com/nrclinonc

Table 2 Treatment outcome in patients with EGFR mutations after treatment with a EGFR TKI or chemotherapy Study n Treatment Tumor response rate* (%) Median PFS* (months) IPASS 19 261 Gefitinib versus paclitaxel + carboplatin 71.2 versus 47.3 9.8 versus 6.4 FIRST-SIGNAL 20 42 Gefitinib versus gemcitabine + cisplatin 84.6 versus 37.5 8.4 versus 6.7 WJTOG 3405 21 86 Gefitinib versus docetaxel + cisplatin 62.1 versus 32.2 9.2 versus 6.3 NEJSG 002 22 114 Gefitinib versus paclitaxel + carboplatin 73.7 versus 30.7 10.8 versus 5.4 OPTIMAL 23 154 Erlotinib versus gemcitabine + carboplatin 83.0 versus 36.0 13.1 versus 4.6 EURTAC 24 175 Erlotinib versus platinum-based doublets 54.5 versus 10.5 9.4 versus 5.2 *EGFR TKI versus chemotherapy. Abbreviations: PFS, progression-free survival; TKI, tyrosine kinase inhibitor. first phase II study of crizotinib, an ALK inhibitor, in patients with the EML4 ALK fusion gene. After screening approximately 1,500 patients for ALK rearrangements, the researchers found 82 ALK-positive patients who were eligible for the study, 96% of whom had adenocarcinoma and 76% of whom were non-smokers and also had adenocarcinoma. The tumor response rate was 57%, and 33% of patients achieved stable disease; PFS was approximately 9.2 months. 31 Similar to EGFR mutations, EML4 ALK has been established as an important molecular target for the management of lung cancer. Two randomized studies comparing crizotinib with standard chemotherapy are ongoing. 32,33 Know the biomarker Targeted therapy implies the use of a specific drug in a selected patient population that expresses the target; thus, the therapy is unlikely to be clinically applicable unless a validated biomarker that identifies the right patients is available (Box 1). 34 Gefitinib was originally developed to target overexpression of EGFR; therefore, high EGFR protein expression levels was initially thought to be a potential predictive biomarker. Only when mutations in EGFR were identified, were both EGFR mutations and EGFR gene-copy number (measured by fluorescence in situ hybridization [FISH]) used as biomarkers in multiple clinical trials. The ISEL 35 and BR 21 36 studies compared gefitinib and erlotinib, respectively, with placebo in patients who had failed to respond to one or more chemotherapy regimens. Data on the predictive value of EGFR protein expression and EGFR gene-copy number were not consistent. Both biomarkers seemed to be marginally predictive in the ISEL study (interaction test P = 0.049 and 0.045, respectively) but the two biomarkers were not predictive in the BR 21 study (interaction test P = 0.33 and 0.12, respectively). However, the number of patients who harbored an EGFR mutation was too small (26 and 15 patients for ISEL and BR 21, respectively) for meaningful analysis in these studies; thus, the negative interaction test result should not exclude EGFR mutations as a biomarker. The INTEREST study compared gefitinib with docetaxel as second-line and third-line therapy, and none of the three biomarkers (EGFR protein expression, EGFR gene-copy number, EGFR mutations) were predictive of survival. 37 As before, the small number of patients with EGFR mutations (44 patients) may explain this observation. Conversely, in the IPASS study, EGFR mutations were a robust predictive biomarker for both objective tumor response to gefinitib and PFS (P <0.001), but not for overall survival (P = 0.99). 38 EGFR gene-copy number seemed to be predictive of PFS in patients with high EGFR copy number (HR = 0.66; 95% CI 0.50 0.88; P = 0.005). However, there was no signifi cant difference between gefitinib and chemotherapy in patients with low EGFR gene-copy number (HR = 1.24; 95% CI 0.87 1.76; P = 0.237). The predictive power of high EGFR gene-copy number can be explained by the concurrent presence of EGFR mutations (78% of all patients with high EGFR copy number) compared with patients with low EGFR gene-copy number, only 33% of whom carried mutations in EGFR. 38 Gefitinib was associated with longer PFS in patients with both high gene-copy number and mutat ions in EGFR (HR = 0.48; 95% CI 0.34 0.67); by contrast, chemo therapy was superior to gefitinib in patients with high gene-copy number without mutations in EGFR (HR = 3.85; 95% CI 2.09 7.09). 38 Therefore, EGFR mutations should be the predictive biomarker of choice for first-line EGFR TKIs in patients with advanced stage NSCLC. The prognostic or predictive value of EGFR mutations in early-stage NSCLC is less-well defined. Patients with resectable lung cancer who harbor EGFR mutations may have a better survival outcome than patients without EGFR mutations. 39 In a retrospective study of 397 Japanese patients with adenocarcinoma after surgical resection, 197 patients (49%) were found to have EGFR mutations and their overall survival was longer than patients without EGFR mutations (P = 0.0046). 39 However, multivariate analysis only confirmed smoking status and not mutations in EGFR to be an independent prognostic factor. Predictive value of EGFR mutations in early-stage disease is also controversial. The BR 19 randomized phase III study compared adjuvant gefitinib with placebo in an unselected patient population with resectable NSCLC. 40 Overall survival of the study population was similar between the two groups (HR = 1.23; P = 0.136). More speci fically, patients with EGFR mutations (n = 76) seemed to have a worse survival outcome with adjuvant gefitinib than with placebo (HR = 1.58; P = 0.18). This surprising outcome may be partly explained by the small sample size and short duration of therapy (the median exposure to gefitinib was 4 months). 40 Alternatively, tumor biology of EGFR mutations in early-stage disease may be different from advanced-stage disease. NATURE REVIEWS CLINICAL ONCOLOGY VOLUME 8 NOVEMBER 2011 663

Box 1 The definition of a biomarker The NIH Biomarkers Definition Working Group defined a biomarker as a characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacological responses to a therapeutic intervention. 31 A biomarker is usually a measurable parameter that reflects a normal or physiopathological genomic process. A biomarker can be prognostic, predictive, or both. Being prognostic, the presence of a biomarker is associated with better or worse survival compared with the absence of it. Being predictive, the presence of a biomarker is associated with a greater difference in treatment outcomes when comparing two treatments. The most definitive evidence on the predictive value of a biomarker is its positive interaction test in a randomized comparative study of two active treatments (A and B) such that, in the presence of the biomarker, treatment A attains better outcomes than treatment B, and vice versa in the absence of the biomarker. A genomic biomarker commonly refers to either a single genetic abnormality or a panel of genetic abnormalities. Single abnormalities may be studied at the genetic level using PCR sequencing and fluorescence in situ hybridization, or at the protein level using immunohistochemistry. Multiple genetic abnormalities may be studied using microarrays. A predictive biomarker for targeted therapy should be specific to a single genetic abnormality, otherwise the selection of testing methods and the clinical application of the biomarker could be confusing. Similar to the detection of mutations in EGFR, the EML4 ALK fusion gene can be detected by PCR sequencing, FISH, or immunohistochemical (IHC) staining. However, unlike the development of EGFR mutations as biomarkers for EGFR TKIs, the identification of biomarkers for response to crizotinib is being developed prospectively. Kwak et al. 31 selected all 82 patients for their phase I II study by FISH using an ALK breakpoint probe. A tumor sample was considered ALK positive when 15% or more of the scored cells had either a split 5' and 3' probe or a single 3' signal indicating that the translocation had occurred. Reverse transcription (RT)-PCR and IHC were carried out in a subgroup of 31 patients with available tissue for biomarker study. Only 20 of 29 patients who were ALK-positive as detected by FISH had the EML4 ALK fusion breakpoint confirmed by RT PCR; 19 of 25 ALKpositive patients had ALK expression levels that scored 2+ or 3+ by IHC. Of the four patients who had progressive or stable disease after treatment with crizotinib, two did not have confirmed fusion breakpoint by RT PCR but all four had ALK-positive tumors scoring 2+ by IHC. On the basis of these findings, the investigators decided to use FISH as the standard method of identifying biomarkers in patients enrolled in two subsequent randomized phase III studies. 32,33 The first study enrolled patients who had not responded to first-line chemo therapy and were ALK-positive by FISH, and who were randomly assigned to receive either crizotinib or single-agent chemo therapy (docetaxel or pemetrexed). The second study had a similar design for treatment-naive patients assigned to receive either crizotinib or platinum-based doublet chemo therapy. Both studies are ongoing and may potentially establish the role of crizotinib as firstline, second-line or third-line therapy in ALK-positive patients determined by FISH. 32,33 However, these studies may not confirm the predictive power of FISH as a biomarker; by definition, a positive interaction test is essential for confirmation of the predictive power. As patients without the EML4 ALK fusion gene are not eligible for the study, treatment outcomes to crizotinib in patients not harboring this fusion gene will not be known, either prospectively or retrospectively. Know the end point Overall survival is the most important primary end point for regulatory approval of cytotoxic chemotherapy. This is understandable, given the short treatment duration and limited improvement in survival in either the firstline, second-line or third-line setting in patients with advanced-stage NSCLC. 41,42 The treatment benefit of second-line or third-line chemotherapy is also much less than the benefit of first-line chemotherapy. 43 Development of molecular-targeted therapies poses a new challenge both for investi gators and regulators. Patients with a specific molecular target should respond to targeted therapies independently of the line of therapy. The Spanish Lung Cancer Study Group enrolled 217 patients with EGFR mutations in a single-arm study of erlotinib. 13 Tumor response, PFS and overall survival were similar between patients who received erlotinib as first-line, secondline or third-line therapy. 13 In any randomized study comparing one molecular-targeted therapy with standard chemo therapy, it would be unethi cal not to allow patients in the control arm to cross over to the experimental molecular-targeted drug group upon disease progression. If a significant portion of patients cross over to the study group, overall survival will likely be similar between the two arms. Therefore, PFS is probably the only rational end point for an effective targeted therapy. Figure 1 illustrates the concept of treatment crossover between moleculartargeted therapy and chemotherapy. This phenomenon is exemplified in the IPASS study; 38 despite the dramatic difference in PFS in patients with mutations in EGFR, there was no difference in overall survival between the gefitinib and chemotherapy arm (21.6 months versus 21.9 months; HR = 1.00; 95% CI 0.76 1.33; P = 0.990). About 64% of patients who received the combination of paclitaxel and carboplatin as first-line therapy, received EGFR TKIs as post-study therapy. This was also observed in the study by Maemondo et al., 22 in which the median overall survival for the gefinitib and paclitaxel carboplatin arms was 30.5 months and 23.6 months, respectively (P = 0.31). In this study, 94.8% of the patients in the chemotherapy arm crossed over to receive gefitinib and the tumor-response rate in this group was 58.8%. 22 The overall survival outcome of another trial (WJTOG 3405) is immature but preliminary data indicate similar survival outcomes between the gefitinib and chemotherapy arms. 21 Gefitinib has been approved in 75 countries, including in the EU, as first-line therapy for patients with mutations in EGFR. PFS was the primary end point in all four random ized studies that supported the registration. 19 22 The ongoing registration studies of crizotinib in patients with the EML4 ALK fusion gene have also adopted PFS as the primary end point. This paradigm shift will help investigators to design future studies. However, potential shortfalls should be noted. Tumor progression must be rigorously monitored with diagnostic imaging. Unlike cytotoxic chemotherapy, patients may stay on the 664 NOVEMBER 2011 VOLUME 8 www.nature.com/nrclinonc

Diagnosis Chemotherapy Gefitinib DP DP Chemotherapy Gefitinib DP 2 nd or 3 rd line therapy Death 0 20 24 months Figure 1 Overall survival in patients with mutations in EGFR receiving TKIs and chemotherapy. Most patients with activating EGFR mutations will benefit from EGFR TKIs irrespective of the line of therapy. Patients who receive a first-line EGFR TKI will have a prolonged PFS and, upon DP*, have a relatively shorter duration of disease controlled by chemotherapy. Patients who receive first-line chemotherapy will have a relatively shorter PFS and, upon DP, have prolonged PFS from second-line EGFR TKI. Patients will have similar overall survival as long as an EGFR TKI is administered during the course of illness. Therefore, PFS is a more valid indicator of treatment effectiveness than overall survival. *DP defined by RECIST criteria. Abbreviations: DP, disease progression; PFS, progression-free survival; TKI, tyrosine kinase inhibitor. targeted drug for many months, and long-term frequent imaging could be a laborious process. Furthermore, most of the past studies did not routinely monitor the central nervous system if the brain was normal at enrollment. As the central nervous system is one of the most common sites of progression for patients receiving TKIs, and many patients may not be symptomatic until significant tumor progression occurs, diagnosis of disease progression could be delayed by this omission. Accurate assessment of PFS should, therefore, include regular imaging of the central nervous system. Know the mechanisms of resistance Molecular-targeted therapies can be effective, but almost all treated tumors will eventually become resistant and progress. Knowing the mechanisms of resistance will help to manage patients at disease progression, and may prevent or defer the process. The main reasons for primary resistance to EGFR TKIs include the absence of EGFR mutations and presence of KRAS mutations, and the two mutations are essentially mutually exclusive. 44 The two major mechanisms of acquired resistance are the EGFR point mutation T790M in exon 20 and overexpression of the components of the hepatocyte growth factor receptor (HGFR, also known as MET) pathway. 45 48 Two independent groups reported the single amino acid change of T790M in EGFR in patients with clinical resistance to gefitinib. 44,45 This substitution alters the conformation of the protein, interfering with the binding of EGFR TKIs and thus restoring ATP binding to the levels of wild-type EGFR. 45 Over 50% of acquired resistance is explained by this mechanism. 46 48 However, whether the T790M mutation pre-exists in a small percentage of cells in treatment-naive tumors or is acquired from exposure to EGFR TKIs remains controversial. Maheswaran et al. 49 reported the presence of the T790M mutation in 28% of tumor samples with no previous exposure to TKIs and, in the IPASS study, 19 11 of 261 patients with EGFR mutations had the T790M mutation at baseline. The prognostic value of the T790M mutation is also debatable. Using a highly sensitive method, Rosell et al. 50 identified the T790M mutation in 35% of patients enrolled in the Spanish Lung Cancer Study Group trial. Patients with this mutation had a worse PFS when treated with erlotinib than patients without the mutation. However, patients who were found to have the T790M mutation at the time of progression seemed to have slower progressing disease. In a study of 93 patients with a tumor re-biopsy taken at the time of resistance to EGFR TKIs, 58 patients (62%) were found to have the T790M mutation. 51 Median postprogression survival for patients with the T790M mutation was 19 months, which is significantly longer than the median overall survival of 12 months in patients without the T790M mutation (P = 0.036). These data are crucial to future investigation of second-generation irreversible TKIs such as afatinib and PF299084 (Pfizer, New York, USA). Both of these drugs have been shown to inhibit growth in cell lines that harbor the T790M mutation. 52 A phase III study comparing afatinib with placebo in patients with pulmonary adenocarcinoma and previous exposure to an EGFR TKI (LUX Lung 1 study) reported tumor response rates of 13.3% versus 0.5% and PFS of 3.3 months versus 1.1 month (P <0.001), but the study failed to report a significant difference in overall survival. 53 The study considered disease progression after 12 weeks or more of treatment with TKIs erlotinib and gefitinib as the key criterion for EGFR TKI resistance. Tumor re-biopsy was not required and status of exon 20 T790M was unknown at enrollment. Confirming the encouraging preclinical data would require a sufficient proportion of the enrolled patients to present with T790M-related TKI resistance. On the basis of the results from the BR 21 study, an estimated overall survival of the placebo arm of 4.7 months and an HR of 0.70 were expected for the LUX Lung 1 study. However, the median overall survival of the placebo arm in the LUX Lung 1 study was 11.96 months and, for this reason, there remains the possibility that the study could be statistically underpowered a larger sample size is needed to detect a 2 month difference from a control group with an overall survival of 11.96 months, than from a control group with an overall survival of 4.7 months. Over 60% of patients in the placebo arm were non-smokers and their median exposure to previous EGFR TKIs was about 9.7 months. In other words, a significant portion of patients in the placebo group carried activating EGFR mutations, which has in general a better prognosis. After a lack of response to placebo, 24% of patients received salvage EGFR TKIs and 43% received standard salvage chemotherapy such as pemetrexed, and this may explain the exceptionally long overall survival. Overexpression of the MET pathway accounts for about 20% of TKI resistance. 47 Tivantinib is a novel TKI targeted against MET. A randomized phase II study in NATURE REVIEWS CLINICAL ONCOLOGY VOLUME 8 NOVEMBER 2011 665

patients with non-squamous cell histology comparing a combination of tivantinib and erlotinib with erlotinib alone reported an improvement in PFS (HR = 0.61, P <0.05) and overall survival (HR = 0.58, P <0.05) favoring the combination arm. 54 Biomarkers, including MET expression by FISH and EGFR mutations by PCR, were not predictive, whereas a small number of patients with KRAS-mutant tumors seemed to survive longer with the combination therapy. A phase III study targeting patients with non-squamous cell histology with no previous exposure to EGFR TKIs is ongoing. Neither this study nor the LUX Lung 1 targeted a biomarker-selected population. Mechanisms of EGFR TKI resistance are heterogeneous and a single drug is unlikely to be effective against the diverse pathologies. Future investigation should involve biopsy of resistant tumors for biomarker analysis, and specific treatment selected according to the mechanisms of resistance if identified. Within a relatively short time of the discovery of the EML4 ALK fusion gene, investigators from Japan have already found two secondary mutations associated with resistance to the ALK inhibitor crizotinib. 55 DNA sequencing and characterization of an EML4 ALK-tumor mutant was performed in a Japanese patient who did not respond to treatment with crizotinib after only 5 months of therapy. 55 The patient harbored point mutations of the residues C1156Y and L1196M in the ALK protein. Lung cancer cell lines transfected with ALK constructs harboring these mutations were much less sensitive to crizotinib, and phosphorylation downstream of ALK was also inhibited. 55 The incidence of these resistant mutations remains unclear. With the ongoing phase III studies of crizotinib in patients with the EML4 ALK fusion gene, hopefully more information on the pattern and frequency of resistance will unfold. Knowing the mechanisms of resistance is not sufficient for future development of personalized medicine for lung cancer. Definition of TKI resistance dictates the duration of therapy, and when treatment is prematurely terminated, patient survival could be indirectly shortened. Jackman et al. 56 proposed four criteria for clinical definition of acquired resistance to EGFR TKIs: clinical exposure to an EGFR TKI, benefit from an EGFR TKI, evidence of disease progression by the RECIST criteria, and lack of exposure to other drugs. 56,57 Biomarkers are not included because our understanding of the biology of acquired resistance is still limited. The problem is the applicability of the RECIST criteria 58 when using moleculartargeted therapies. The criteria are well adopted for clinical investigation of cytotoxic chemotherapy. An increase of 30% in summed maximum diameter or occurrence of new metastatic lesion, are accurate reflections of chemotherapy resistance. 57 However, this principle may not be applicable to molecular-targeted therapies, which are supposed to inhibit cancer cell proliferation that is predominantly driven by a mutated pathway. A new metastatic lesion may only suggest the presence of a resistant clone and not necessarily generalized resistance of other preexisting tumors. In addition to percentage of increase in summed diameter, rate of tumor progression is equally important. Targeted therapies inhibit cancer proliferation continuously. In cases in which the tumor progresses slowly in the presence of a TKI, it is better for the patient to continue with the drug, as cessation of inhibition may lead to rapid tumor growth. 59 The concept of personalized medicine should apply to both treatment selection and treatment cessation. Conclusions Personalized medicine is now a reality for patients with lung cancer. About 10 20% of all patients with lung cancer harbor mutations in EGFR and 3% harbor mutations in EML4 ALK. 10,29 These patients will benefit from EGFR TKI and ALK inhibitors, respectively. Testing for mutations in EGFR for selection of patients for first-line EGFR TKI therapy is standard practice and, in the near future, selection of patients with EML4 ALK for crizotinib treatment will also become a new standard. As most of the patients who harbor mutations in EGFR or EML4 ALK genes are non-smokers, personalized molecular-targeted therapy may not help a large proportion of patients who are smokers. Therefore, future developments will have to identify novel molecular targets that drive cancer growth in smokers. It is likely that these targets are relatively uncommon and drug development will be difficult, but this is a challenge worth undertaking. The successful development of personalized therapy is founded on knowledge of a specific target that drives cancer growth, validation of a clinically applicable biomarker, acceptance of a rational end point, and understanding of the mechanisms of resistance. With each novel target, a standardized biomarker will be developed concurrently. Sequencing technologies will help to identify such biomarkers either in tumor tissue or plasma DNA. Our group used digital PCR to identify mutations in EGFR from plasma and reported high sensitivity and specificity. 60 A mature plasma-based biomarker analysis should make molecular profiling in patients with lung cancer universal, as an adequate tumor sample is not always available. PFS will likely become the standard end point for trials of molecular-targeted therapy in patients with lung cancer. However, the definition of progression may change. In addition to tumor size, progression may be defined according to the molecular profile of the tumor with the emergence of resistant mutations in EGFR, such as T790M or C1156Y. This implies that clinical progression needs to be validated by either re-biopsy or plasmabased biomarker analysis. With such methods, we will be able to learn more about the mechanisms of resistance and provide alternative therapies. Review criteria The relevant articles were identified by the author on the basis of his knowledge of the clinical development of EGFR and EML4 ALK inhibitors. The published data were inclusive of articles from January 2000, and abstracts from major conferences (including the Annual Meeting of ASCO and European Society of Medical Oncology from 2009 to 2011). 666 NOVEMBER 2011 VOLUME 8 www.nature.com/nrclinonc

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