Biomarkers, Prediction, and Prognosis in Non–Small-Cell Lung Cancer: A Platform for Personalized Treatment

Original Study

Biomarkers, Prediction, and Prognosis in Non–Small-Cell Lung Cancer: A Platform for Personalized Treatment Akshay Sudhindra, Roberto Ochoa, Edgardo S. Santos Abstract In lung cancer, the introduction of targeted agents in those patients who carry a genetic abnormality has resulted in better clinical outcomes with better quality of life. These molecular abnormalities have also become predictive biomarkers. It is imperative that we continue searching for these biomarkers in different tumorigenesis pathways, so we can provide the most appropriate therapy to each individual in the near future. Since the 1980s, chemotherapy for patients with advanced non–small-cell lung cancer has been shown to provide a small improvement in survival. In the early 1990s, platinum-based regimens became the backbone of treatment for this disease. In 2002, the Eastern Cooperative Oncology Group 1594 clinical trial showed that there was no overall survival difference among four common chemotherapy regimens used in non–small-cell lung cancer. It was not until 2006 when the introduction of biologic agents into the field of lung cancer improved, for the first time ever, median overall survival beyond 1 year. To date, we recognize that there are differences between all histologic subtypes of non–small-cell lung cancer in terms of their response to specific agents. All these plus the introduction of molecular medicine have resulted in the identification of markers for prognosis and prediction in lung cancer. In this review, we describe the actual and ongoing clinical efforts to validate the prognostic and predictive value of these potential markers in lung cancer. We hope that the clinical use of biomarkers will help us to deliver personalized medicine to our lung cancer patients by improving their quality of response which may translate into further survival advantage. Clinical Lung Cancer, Vol. 12, No. 6, 360-8 © 2011 Elsevier Inc. All rights reserved. Keywords: BRCA-1, EML4/ALK, Epidermal growth factor receptor (EGFR), Excision repair cross-complementing enzyme 1 (ERCC1), Ribonucleotide reductase M-1 protein (RRM1), Thymidilate synthase (TS), ␤-tubulin

Introduction Lung cancer remains the number one cause of cancer-related deaths in the United States in both women and men.1 For many decades, clinicians have treated all sub-types of non–small-cell lung cancer (NSCLC) in the same way, with the same chemotherapeutic regimens which included, for the most part, platinum-based therapies for those patients who have good performance status. In 2002, the Eastern Cooperative Oncology Group 1594 published its results

Sylvester Comprehensive Cancer Center/University of Miami Leonard M. Miller School of Medicine, Miami, FL Submitted: Nov 19, 2010; Revised: Dec 29, 2010; Accepted: Feb 22, 2011 Address for correspondence: Edgardo S. Santos, MD, FACP, Associate Professor of Medicine, Associate Director, Hematology/Oncology Fellowship Program, Thoracic Oncology Program, University of Miami Leonard M. Miller School of Medicine, Sylvester Comprehensive Cancer Center, 1475 NW 12th Avenue, D8-4, Miami, FL 33136 Tel: 305-243-3286; Fax: 305-243-3289; e-mail contact: [email protected]

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in which four of the most commonly used chemotherapy regimens were used to treat an unselected population of NSCLC2; the study revealed that there was no significant difference between response rate (RR) and overall survival (OS) among the four groups. However, in the last 5 years, we have seen improvement in OS of NSCLC patients because of the use of novel biologic agents (eg, bevacizumab, cetuximab) in combination with conventional chemotherapy, as well as the use of other targeted agents against certain mutations [eg, epidermal growth factor receptor (EGFR) and echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase (EML4ALK)].3-6 The latter truly represent targets for novel agents such as tyrosine kinase inhibitors (TKI) which induces response rates higher than those seen with the use of conventional cytotoxic agents. Molecular medicine has changed the paradigm of many oncologic diseases, and lung cancer has not been the exception. However, the survival in early and advanced NSCLC stages continues to be poor. As molecular medicine has identified specific tumorigenesis pathways to be targeted, it has also been able to identify certain proteins

1525-7304/$ - see frontmatter © 2011 Elsevier Inc. All rights reserved. doi: 10.1016/j.cllc.2011.02.003

Table 1 Potential Biomarkers Being Studied for Validation in NSCLC Biomarker

Prognostic

Predictive

Excision Repair Cross-complementing Group 1 (ERCC1)

Early stage (resected): High ERCC1 ¡ good Low ERCC1 ¡ poor

Platinum-based therapy: High ERCC1¡ predicts resistance Low ERCC1 ¡ predicts sensitivity

Ribonucleotide Reductase M1 Protein

Early stage (resected): High RRM1 ¡ good Low RRM1 ¡ poor

Gemcitabine-based therapy: High RRM1 ¡ predicts resistance Low RRM1 ¡ predicts sensitivity

Epidermal Growth Factor Receptor (EGFR)

Advanced disease: Mutant EGFR ¡ good

Erlotinib/Gefitinib-based therapy: Mutant EGFR ¡ good response

Breast Cancer Susceptibility Gene 1 (BRCA1)

?

Platinum-base therapy: High BRCA1 ¡ predicts resistance Taxane-based therapy: Low BRCA1 ¡ predicts sensitivity

Thymidilate Synthase (TS)

?

Pemetrexed-based therapy: High TS ¡ predicts resistance Low TS ¡ predicts sensitivity

␤-tubulin

?

Taxane-based therapy: High ␤-tubulin ¡ predicts resistance

?

Erlotinib/Gefitinib-based therapy: Mutant EML4/ALK ¡ predicts resistance Crizotinib (ALK inhibitor)-based therapy: Mutant EML4/ALK ¡ predicts sensitivity

Echinoderm Microtubule-associated Protein-like 4Anaplastic Lymphoma Kinase (EML4-ALK)

whose lack of expression or overexpression has been associated with either prognosis or prediction of response to some therapeutic agents. These proteins have the potential to become markers for each individual patient by analyzing the molecular phenotype of a patient’s tumor. Thus, we may deliver a “personalized” therapy, increasing the chances of obtaining a better quality in response which could translate into survival advantage. As an example, the EFGR pathway has been targeted by TKI (eg, gefitinib, erlotinib) with the presence of EGFR mutation being a biologic marker (biomarker) that is both prognostic of survival and predictive of response to these TKIs. It is important to understand the difference between prognostic and predictive biomarkers; a prognostic marker determines outcome regardless of any specific therapeutic intervention. Meanwhile, a predictive biomarker predicts the response or lack of response to a specific therapeutic agent. Many other areas, such as proteomics, genomics (eg, gene expression profile), circulating tumor cells, and others are in development for becoming prognostic or predictive biomarkers. In this review, we will focus our attention on the well-established EGFR mutation and those biomarkers which look promising but still need validation by well-designed prospective randomized clinical trials. The clinical usefulness of biomarkers is vast. Their use will not only help us to choose the right first line agent, but also will allow us to determine which patients need therapy or none at all (prognosis). Hence, by determining the biomarker profile of the patient’s tumor, we will treat the right patient with the right therapy (customizing the management of lung cancer), and ultimately avoid excess of toxicities from therapies that may not deliver any benefit to the patient. It is imperative that we validate these biomarkers in NSCLC; there is no difference between a targeted agent and conventional chemotherapy if the right biomarker is not available.

DNA Repair Genes As Predictive Biomarkers Excision Repair Cross-complementing Group 1 (ERCC1) ERCC1 is the rate-limiting protein in the nucleotide excision repair and interstrand cross-link–repair pathways which are responsible for repair of the platinum-DNA adducts formed when a patient is treated with a platinum-based chemotherapy regimen.7 ERCC1 mRNA expression [measured by quantitative reverse transcriptase (qRT)–polymerase chain reaction (PCR)] is both prognostic of survival after resection of NSCLC and predictive of benefit from treatment with platinum-based regimens.8,9 Simon et al initially showed the prognostic effects of ERCC1 on survival. In their trial of 51 patients they noted a significantly increased median survival in those with high expression of ERCC1 mRNA versus patients with low expression of ERCC1 mRNA (94.6 months versus 35.5 months (survival); P ⫽ .01) suggesting that ERCC1 over-expression confers a favorable prognosis in untreated patients with NSCLC.8 They hypothesized that this effect was due to an intact DNA repair mechanism reducing the accumulation of genetic mutations which are responsible for a tumors’ malignant potential. A study by Lord et al had similar results; in their uncontrolled trial of 56 patients with stage IIIB-IV NSCLC, patients with low ERCC1 mRNA expression had a significantly improved survival when treated with a combination cisplatin-gemcitabine versus those with high ERCC1 mRNA expression [hazard ratio (HR) 0.32, 95% confidence interval (CI) 0.140.71; P ⫽ .005].6 These findings were confirmed in the International Adjuvant Lung Cancer Trial (IALT) in which patients with tumors that were ERCC1-negative by immunohistochemistry (IHC) had a significantly better OS in the adjuvant setting when treated with cisplatin-based therapy than that were ERCC1-positive (Table 1).10 In addition, Holm et al retrospectively studied 163 patients with inop-

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362

erable NSCLC who were treated with carboplatin-gemcitabine. They found that patients who were ERCC1- negative by IHC had a significantly increased median survival when compared to patients who were ERCC1-positive by IHC (12.0 versus 8.2 months; P ⫽ .02).11 In a prospective clinical trial, the Genotypic International Lung Trial (GILT), patients were randomized to docetaxel/cisplatin (control arm) and an experimental arm in which ERCC1 mRNA levels were measured by qRT-PCR.12 Those with low ERCC1 mRNA expression were assigned to receive docetaxel/ cisplatin (selected group) whereas those with high ERCC1 mRNA expression received docetaxel/gemcitabine (selected group). The RR in the control arm and the genotypic arm (experimental) was 39.3% and 50.7 % (P ⫽ .02), respectively.12 Currently, the Tailored Post Surgical Therapy in Early Stage NSCLC (TASTE) trial is recruiting patients and customizing treatment based on EGFR and ERCC1. In this study, patients with stage II or IIIA non-squamous NSCLC are being randomized to a standard chemotherapy arm in which they will receive treatment with pemetrexed/cisplatin and an experimental arm in which EGFR mutation status and ERCC1 mRNA expression levels will be determined. Those with mutated EGFR will be assigned to receive single-agent erlotinib, whereas those with wild-type (WT) EGFR will have ERCC1 mRNA expression to determine treatment. Patients with high expression of ERCC1 mRNA will be assigned to no treatment whereas those with low expression of ERCC1 mRNA will be assigned to receive pemetrexed/cisplatin.13 Although some studies have used ERCC1 protein levels as determined by IHC and others have used ERCC1 gene expression as determined by PCR to predict response to treatment, to date there are no studies comparing the use of these two modalities. This is an area which will require further research.

vated by nucleoside kinases to gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP) within the cell. The cytotoxic effect is thought to be due to a combination of inhibition of DNA synthesis and subsequent apoptosis. dFdCDP first inhibits ribonulceotide reductase; then, gemcitabine triphosphate competes with deoxycyktidine triphosphate for incorporation into DNA which ultimately results in termination of DNA elongation.17 Given this mechanism, it is easy to understand that levels of RRM1 (which is the regulatory subunit of ribonucleotide reductase) predict response to treatment with gemcitabine. Rosell et al showed that patients who had low levels of RRM1 mRNA expression had a statistically significant increased survival of 15.5 versus 6.8 months in those who had high RRM1 mRNA expression (P ⫽ .0028) when treated with gemcitabine/ cisplatin.18 Recently, Bepler et al confirmed these findings showing that in situ RRM1 levels were significantly correlated with response to treatment with gemcitabine.7 Based on the findings that ERCC1 and RRM1 expression predicts response to platinum-based regimens and gemcitabine, respectively, the Molecular Analysis Individualized Therapy (MADeIT) trial was conducted to assess the feasibility and efficacy of tailoring therapy based on these two biomarkers.19 In this study, patients with advanced NSCLC who had low levels of RRM1 were enrolled in the arm that would receive gemcitabine with either carboplatin or docetaxel depending on ERCC1 levels. Those with high levels of RRM1 were enrolled in the arm that would not receive gemcitabine and based on ERCC1 expression would be treated with either carboplatin/docetaxel or docetaxel/vinorelbine. The RR was 44% with an OS of 59% and progression-free survival (PFS) of 14% at 12 months. MADeIT showed that therapeutic decision-making based on RRM1 and ERCC1 levels is both feasible and a promising option for the future.19

Ribonucleotide Reductase M1 Protein (RRM1)

Breast Cancer Susceptibility Gene 1 (BRCA1)

RRM1 is the regulatory subunit of the enzyme ribonucleotide reductase. Ribonucleotide reductase catalyzes the reaction which forms ribonucleotides from deoxyribonucleotides. Ribonucleotides are in turn used in the synthesis of DNA. Similar to ERCC1, RRM1 is both a prognostic and predictive biomarker in patients who have NSCLC. Bepler et al observed that in patients with resected NSCLC who did not receive preoperative chemotherapy or radiation, higher levels of RRM1 correlated with longer disease-free survival and OS.14 It is thought that increased RRM1 results in decreased tumor invasion and metastatic potential consequently predicting a more indolent tumor course.15 Interestingly, it has been shown that coexpression of RRM1 and ERCC1 predicts a subgroup of patients that has a better prognosis than expression of only one of these prognostic markers. Zheng et al showed that RRM1 mRNA expression correlated with ERCC1 mRNA expression. Those patients with high RRM1 expression had an OS of 120 months versus 60.2 months in those with low RRM1 expression (HR 0.61; P ⫽ .02). On subgroup analysis, patients with coexpression of both had the greatest survival advantage.16 RRM1 has also been shown to be predictive of response to treatment with gemcitabine-based therapy; however, before exploring this topic, it is important to understand how gemcitabine is thought to exert its cytotoxic effects. Gemcitabine is a prodrug that is acti-

The BRCA1 is part of a family of proteins involved in DNA repair and, when mutated, is involved in oncogenesis in breast cancer patients. Mouse models showed an increased sensitivity to cisplatin in BRCA1-deficient mice and development of resistance to cisplatin when BRCA1-deficient cells undergo through reconstitution of its function.20,21 Thus, it was proposed that tumors with the mutated BRCA1 gene and subsequent under-expression of its protein had increased sensitivity to platinum agents. Importantly, it was also noted that while conferring resistance to cisplatin there was an increase in tumor susceptibility to taxanes.21 This relationship has been explained as a function of the ability of BRCA1 to restore spindle assembly checkpoint function which allows taxanes to stabilize microtubules and, therefore, cause mitotic arrest.22 Based on the role of BRCA1 in DNA repair and its effects in resistance to platinum and sensitivity to taxanes, clinical trials were developed to look into its usefulness as predictive biomarker in NSCLC. Thus far, BRCA1 IHC staining in biopsy specimens has failed to predict response to platinum agents23; meanwhile, the results have been different using mRNA level expression of BRCA1. In a clinical trial involving early-stage NSCLC patients who underwent surgical resection, higher levels of BRCA1 mRNA showed a significant decrease in median survival compared to patients with lower levels.24 Another series of 126 patients (stage I-IIIA) treated

Clinical Lung Cancer November 2011

Akshay Sudhindra et al with surgery alone showed a significant improvement in time-toprogression (TTP) and median survival in patients with low BRCA1 mRNA (P ⫽ .01).25 Because these results were found in patients not treated with chemotherapy, it suggested a different biological behavior in tumors related to their BRCA1 gene expression. Also, in the early-stage clinical setting, Taron et al conducted a study of 55 patients who were treated with neoadjuvant chemotherapy (cisplatin/ gemcitabine) stratified by their BRCA1 mRNA levels.26 Patients in the lowest quartile of BRCA1 mRNA expression had a significant difference in median survival (not reached for the lowest quartile group compared to 12 months in higher quartile group), a significant difference in down-staging after neoadjuvant chemotherapy, and a significant reduction in risk of death (RR 0.2; 95% CI, 0.050.83; P ⫽ .02).26 BRCA1 mRNA expression has been associated with in vitro resistance to platinum-based therapy, but sensitivity to taxanes in samples obtained from patients with gastric and NSCLC with malignant pleural effusions.27 These results were corroborated in a clinical trial in which 56 patients with metastatic NSCLC showed a significant difference in RR and OS when treated with taxanes; overall response rate (ORR) was 15% and 52% in patients with low and high BRCA1 mRNA expression, respectively. OS was statistically different in favor of those expressing high BRCA1 mRNA levels (13 versus 8.7 months; P ⫽ .03).28 A larger study which included 102 metastatic chemotherapy-naïve NSCLC patients showed a significant increase in RR for those who had high BRCA1 mRNA levels treated with a non-platinum doublet (gemcitabine/paclitaxel) in first-line treatment; in this group, there was also a decrease in risk of progression.29 In a landmark study from the Spanish group, BRCA1 was tested as a predictive biomarker by selecting initial treatment for metastatic NSCLC patients.30 Patient stratification was based on two biomarkers: EGFR mutation and BRCA1 mRNA levels. Thus, low-level patients (n ⫽ 38) were treated with cisplatin/gemcitabine; intermediate- level patients (n ⫽ 40) were treated with cisplatin/docetaxel, and high-level patients were treated with docetaxel alone (n ⫽ 33). Median survival was 11, 9, and 11 months for the low-, intermediate-, and high-level groups, respectively. Although RRs were lower in patients with low BRCA1 mRNA, median survival and TTP were not different.30 An ongoing phase III randomized clinical trial is currently underway to evaluate whether BRCA1 can be used to determine initial chemotherapy in patients with NSCLC.

Thymidilate Synthase (TS) Critical in the de novo synthesis of DNA is the production of the pyrimidine nucleotide thymidine monophosphate from deoxyuridine monophosphate. This reaction is catalyzed by TS. dTMP is subsequently phosphorylated and used for DNA synthesis and repair. For a long time, TS has been recognized as a target for anticancer drugs and can be inhibited directly by inhibitors such as 5-fluorouracil or folate analogs such as pemetexed. In patients with NSCLC, it has been observed that TS expression varies with the histology of the tumor. Ceppi et al noted that TS mRNA expression was higher in patients with squamous cell carcinoma than in patients with adenocarcinoma.31 Although TS expression is not prognostic in patients with NSCLC, it has been shown pre-clinically to be predictive of response

to treatment with pemetrexed (cell lines that exhibited low levels of TS were shown to be more sensitive to pemetrexed).32 Based on these preclinical findings, a treatment-by-histology approach has been suggested and is the basis of a number of clinical studies nowadays. To date, one retrospective analysis performed over the pemetrexed indication trial33 and two prospective randomized trials (initial treatment for metastatic disease and maintenance therapy) have shown the difference in RR by histologic type and chemotherapy agent used.34-36 One theory for these differences is that TS is expressed by different lung cancer histologies.

EGFR Pathway EGFR is a transmembrane protein that belongs to a family of three other related proteins (the ErbB family of receptors consisting of Her-2, Her-3, and Her-4). EGFR is over-expressed in approximately 40% to 80% of cases of NSCLC.37 Downstream activation of intracellular pathways results from ligand binding to a single-chain EGFR causing a conformational change and dimer formation with resultant autophosphorylation of the intracellular tyrosine kinase (TK) domain.38 Downstream inhibition can be achieved in two ways: (1) by monoclonal antibodies that bind to the extracellular domain preventing ligand binding and subsequent dimerization; and (2) by small-molecule TKIs which compete reversibly with adenosine triphosphate to bind to the intracellular domain of the EGFR TK, thus inhibiting autophosphorylation. EGFR exists as a WT in which ligand binding to receptor triggers dimer formation and subsequent downstream activation or in a mutant type. The latter one is the focus of our discussion as it has proved to be both a prognostic and a predictive biomarker in NSCLC. The most common mutations observed in lung cancer are a leucine-to-arginine substitution at position 858 of exon 21 (L858R) or a deletion in exon 19.39 These mutations result in constitutive activation of the EGFR TK because of destabilization of the autoinhibition conformation. EGFR status and response to targeted therapies can be evaluated by protein expression via IHC, gene copy number via fluorescent in situ hybridization (FISH), or identification of mutant EGFR by direct sequencing. To date, the majority of data favors EGFR mutation analysis as the preferred biomarker.

EGFR By IHC It has been noted that IHC detects EGFR expression in anywhere from 40% to 80% of NSCLC cases.40,41 A number of studies have focused on EGFR protein expression and whether this predicts response to treatment with a TKI. Among the two most significant studies were the BR.21 trial conducted by the National Cancer Institute of Canada42 and the Iressa Survival Evaluation in Lung cancer (ISEL) trial.43 In the BR.21 trial, there was a significantly higher ORR in patients with tumors that were EGFR IHC positive versus those that were EGFR IHC negative (7.5% versus 3.8%, P ⫽ .03) to treatment with erlotinib. The ISEL trial showed similar results with gefitinib; however, there was no reported P value (8.2% versus 1.4%).43 Despite the results of these two large trials, EGFR protein expression is not currently used as a test to determine if a patient will benefit from treatment with an EGFR TKI,7 even though it was the chosen test for the sequential tarceva in unresectable NSCLC trial in which patients whose tumors expressed EGFR by IHC were ran-

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Biomarkers in Lung Cancer domized to erlotinib versus placebo; this study approved erlotinib as maintenance therapy in NSCLC in 2010.

EGFR By FISH Several studies have shown the association between a high EGFR copy number by FISH and the response rate to EGFR TKIs.7 Both BR.21 and ISEL trials showed a significant association between high EGFR copy numbers and increased survival in those patients treated with EGFR TKIs.42,43 Goss et al reported similar findings in their phase II study when they compared gefitinib to placebo in patients with advanced NSCLC who were chemotherapy-naïve and had poor performance status (PS).44 Overall, there was no significant difference in PFS, OS, or ORR between the two groups. However, on subgroup analysis, patients who were EGFR-positive by FISH had a statistically significant increase in PFS when treated with gefitinib versus placebo (HR 0.29; CI 95%; 0.11 to 0.73). Despite this, great discordance still exists. For example, the Iressa in Non-small-cell lung cancer Trial Evaluating Response and Survival versus Taxotere® study (INTEREST) was unable to demonstrate that FISH positivity is predictive of response or survival in patients treated with gefitinib.45 In this trial, 1,466 patients who had already been treated with a platinum-based regimen were randomized to receive either gefitinib or docetaxel. In terms of OS, non-inferiority between gefitinib and docetaxel was shown. Moreover, a planned subgroup analysis in patients with high EGFR copy number by FISH did not show superiority of gefitinib over docetaxel (72 versus 71 events; HR 1.09, CI 95% 0.78-1.51; P ⫽ .62; median survival, 8.4 versus 7.5 months). It is also significant that, in a clinical trial conducted by Crino et al in which gefitinib was compared against vinorelbine in chemonaïve elderly patients with advanced NSCLC, patients with FISH- positive tumors actually benefited from treatment with vinorelbine over gefitinib.46 Given these results, the value of EGFR copy number by FISH as a predictor of response to EGFR TKIs remains in question.

EGFR Mutation Mutations in the EGFR TK domain were first reported in 2004.47,48 The landmark study known as the IPASS (Iressa Pan-Asia Study) established gefitinib (and TKIs) as an alternative first-line treatment for those patients whose tumors had EGFR mutations.5 Herein, chemotherapy-naïve stage IIIB or IV NSCLC patients were randomized to front-line therapy with gefitinib or carboplatin/paclitaxel combination. Overall, there was a significantly higher RR in the gefitinib group versus carboplatin/paclitaxel arm (43% versus. 32.2%; P ⫽ .0001). There was also a significantly increased PFS in favor of the geftinib group (HR: 0.74, 95% CI 0.65-0.84; P ⬍ .0001). On subgroup analysis in patients with mutated EGFR, gefitinib improved PFS over chemotherapy (HR: 0.48, 0.36-0.64; P ⬍ 0.0002). On the other hand, in patients without a mutated EGFR, the gefitinib arm was significantly inferior to the chemotherapy group. These results showed that mutation status predicts response to gefitinib, and may suggest harm from gefitinib in those without a mutation. Consequently, given its superiority in predicting response to EGFR TKIs over IHC or FISH, EGFR mutation analysis must be performed in those patients who are considered for TKI as first-line therapy.

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In their prospective trial of EGFR mutation screening, Rosell et al found that of the 2105 patients with advanced NSCLC, 350 (16.6%) had either of the EGFR mutations (L585R or del exon 19).39 Of these, 217 received treatment with single-agent erlotinib with a PFS and OS of 14 and 27 months, respectively. This trial showed that EGFR mutant lung cancer is actually a distinct class of NSCLC and that large-scale screening for EGFR mutation is feasible and worthy because of improved clinical outcomes.39 Later, Maemondo et al randomly assigned 230 patients with metastatic NSCLC with mutated EGFR who had never received chemotherapy to receive either single-agent gefitinib or carboplatin/paclitaxel combination.49 Planned interim analysis of the first 200 patients showed that PFS was significantly longer in the gefitinib group than the standard chemotherapy group (HR: 0.36; P ⬍ .001). There was also a significantly longer median PFS in the gefitinib group versus the chemotherapy arm (10.8 months versus 5.4 months; HR: 0.30; CI 95%, 0.22-0.41; P ⬍ .001). In addition, the RR was 73.7% versus 30.7% (P ⬍ .001) in favor of gefitinib; altogether, these results confirmed the IPASS observations, and thus, TKIs are now approved as singleagent first-line therapy in those patients with mutated EGFR. Recently, data presented at the annual conference of the European Society for Medical Oncology (ESMO) showed similar data for patients treated with erlotinib. Results from the prospective, randomized, phase III OPTIMAL trial showed increased median PFS (13.1 months versus 4.6 months; HR 0.16; P ⬍ .0001) and objective RR (83% versus 36%; P ⬍ .0001) with erlotinib over chemotherapy consisting of gemcitabine/cisplatin in patients with advanced NSCLC having activating EGFR mutations.50 OS data is not available yet; however, this provides further evidence that EGFR TKIs are superior to chemotherapy as first-line therapy in those patients with mutated EGFR. Still, despite the promising results with EGFR TKIs in patients who have activating EGFR mutations, their clinical efficacy is limited in duration mainly because of the development of resistance. In approximately 50% of tumors, resistance is conferred by a second mutation in which threonine 790 is substituted for methionine (T790M) at the catalytic cleft of the EGFR enzyme.51,52 Preclinically, those tumors that have the T790M mutation continue to be sensitive to irreversible EGFR TKIs.53,54 Currently, a number of phase 2 studies aimed at assessing the clinical efficacy of irreversible EGFR TKIs in those patients who have EGFR activating mutations and who have progressed on erlotinib or gefitinib are underway. In addition to the T790M mutation, amplification of the c-MET protoncogene has also been implicated in the development of acquired resistance to EGFR TKIs. MET encodes for a transmembrane receptor tyrosine kinase through which dimerization leads to downstream signaling. In their sampling of tissue from patients with adenocarcinoma harboring EGFR mutations, Bean et al found MET amplification in 9 of 43 (21%) patients with acquired resistance versus 2 of 62 (3%) patients who were never exposed to an EGFR TKI.55 Likewise, Engleman et al found MET to be amplified in 4 of 18 (22%) tumors after initial partial response to either EGFR TKI.56 They showed that resistance through MET activation may result from downstream signaling of the PI3K/AKT pathway though ErbB3. When they treated gefitinib-resistant clones with mono-

Akshay Sudhindra et al Table 2 Frequency of EML4-ALK Mutation and Demographic Characteristics of Selected Studies Author 6

Soda et al

N

Alkⴙa

% of ADC

Mean Age

Smokers

Female

n/a

n/a

n/a

33

9.1%

9.1%

68

Takeuchi et al

253

4.4%

4.4%

n/a

n/a

n/a

Koivunen et al67

305

3.0%

n/a

n/a

n/a

n/a

Perner et al69

603

2.6%

n/a

n/a

n/a

n/a

Inamura et al71

149

3.4%

3.4%

52

40%

60%

Shaw et al66

141

13.0%

14.0%

52

0%

42%

Takahashi et al70

313

1.6%

2.4%

70

0%

80%

1500

5.4%

n/a

51

24%

48%

74

Kwak et al

Abbreviations: ADC ⫽ adenocarcinoma; Alk ⫽ anaplastic lymphoma kinase; n/a ⫽ not available. a Alk ⫹ tumors in all adenocarcinoma NSCLC cases.

therapy consisting of gefitinib or an MET inhibitor (PHA665752), there was no down-regulation of the ErbB3/PI3K/AKT pathway and no suppression of cell growth. However, when they treated with combined therapy of gefitinib and PHA665752, there was decreased phosphorylation of ErbB3, AKT, and MET with growth suppression.56 As a result of this preclinical data, clinical trials involving dual inhibitors of both MET and EGFR are currently underway.

K-ras Pathway K-ras is a guanosine triphosphate (GTP) binding protein involved in downstream G protein dependent signaling. Inactive Ras guanosine diphosphate is phosphorylated to Ras-GTP with subsequent activation of the mitogen-activated protein kinase (MAPK) cascade ultimately leading to transcription factor activation and cell proliferation. Similar to EGFR, K-Ras can exist in a mutated form in which constitutive activation leads to uncontrolled cell proliferation and immortality. In NSCLC, activating mutations in K-Ras occur predominantly in smokers who have adenocarcinoma.57,58 Most of these mutations are a guanine-to-thymine transversion on exon 12 (90% of cases) or exon 13.59,60 These mutations have been found in 20% to 30% of the patients who have NSCLC and is related with decreased sensitivity to both erlotinib and gefitinib, and poor TTP.61,62 However, the cumulative data from large phase III randomized clinical trials analyses support the concept that K-ras mutation has a prognostic value for EGFR TKIs therapy rather than a predictive value.

EML4-ALK Fusion Gene The EML4 is a protein involved in microtubule formation and stabilization, and its gene has been located in the short arm of chromosome 2 (2p21). Anaplastic lymphoma kinase (ALK) gene encodes a TK with several genetic alterations involved in cancer pathogenesis in anaplastic large cell lymphoma, neuroblastoma, and inflammatory myofibroblastic tumors63-65 In 2007, Soda et al described a fusion protein called EML4-ALK in both mouse models of NSCLC and in Japanese NSCLC patients’ tumors.6 It was proposed that an inversion in chromosome 2 leads to the fusion gene and subsequently, a fusion protein that induces a constitutive activation of the intracellular domain of ALK; therefore, it is a downstream cascade of events that lead to carcinogenesis..

Later on, clinical studies confirmed the presence of EML4-ALK mutation in 1.6% to 13% of patients with adenocarcinoma of the lung, leading to the hypothesis that this may represent a different subtype of lung cancer (Table 2).6,66-72 Moreover, there were significant demographic differences between patients with ALK-mutated NSCLC compared to patients with both mutated and WT EGFR. In a case series which included 19 patients who had EML4/ALK mutation and another 122 patients who lack this fusion protein, the ALKpositive patients were significantly younger with a median age at diagnosis of 52 years compared to 66 years in EGFR-mutant patients and 64 years in WT patients.66 A follow-up report that included 477 NSCLC patients of which 43 (9%) were ALK-mutant also found that ALK-mutant patients were significantly younger (mean age, 54 years versus 64 years; P ⬍ .001) and more likely to be never/light smokers (90% versus 37%, P ⬍ .001).64 This difference was not found in a recent report of 211 Japanese patients (2.3% who had EML4-ALK mutation), with a median age of 70 years in five cases of ALK-positive tumors.70 The female predominance reported in EGFR- mutated NSCLC was not found in this novel mutation. Other important distinction is that ALK-positive patients tend to be never or light smokers compared to patients with WT EGFR. No cases have been reported of tumors that have both EML4-ALK and EGFR mutations and it is considered that they are mutually exclusive. Taken together, all these features (young age, never or light smoker, and EGFR WT) drive the possibility that this entity may represent a separate adenocarcinoma of the lung not related to cigarette exposure. Shaw et al proposed the use of EML4-ALK mutation as a predictive marker of resistance to EGFR TKIs.66 Of the 19 patients with ALK mutation, no clinical response (as defined by CR ⫹ PR) to the oral EGFR TKI erlotinib was observed in comparison with 70% RR in patients with EGFR mutations (P ⬍ .001). Although 50% of patients with EGFR mutation had clinical response to platinumbased chemotherapy compared to 25% of patients with ALK mutations, this difference was not significant (P ⫽ .35). It is yet unclear if ALK mutation involves decreased sensitivity to platinum agents. PF-01241066, also known as crizotinib, is an inhibitor of ALK and c-MET receptor TK with activity in ALK mutant tumors reported in a phase I trial73 and recently shown to have significant clinical activity in a phase II trial.72 Bang et al reported a 64% ORR and disease control rate

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Biomarkers in Lung Cancer (CR ⫹ PR ⫹ SD) of 90% in 50 patients with metastatic ALK-mutant lung adenocarcinoma patients treated with crizotininib.74 Although most patients had failed at least one regimen of chemotherapy with different ORR (ORR of 52% with one prior regimen, 67% with two prior regimens, and 56 % with ⱖ3 prior regimens), 5 patients were treated as first-line therapy with an ORR of 80%. Because of these results, two trials are underway to further evaluate the use of crizotinib in metastatic ALK-mutant NSCLC. PROFILE 1005 is a phase II open-label study of crizotinib in patients with metastatic ALK-mutant NSCLC in patients who had failed more than one line of chemotherapy. PROFILE 1007 is a phase III, randomized, open-label study that is currently underway comparing the use of crizotinib versus either docetaxel or pemetrexed in patient who had failed one prior treatment with a platinum-based chemotherapy and it is expected to enroll 318 patients.75 Base on this data, we think that EML4/ALK mutation is an important biomarker that predicts lack of response to EGFR TKIs and has the potential to have a specific targeted drug. Patients should be encouraged to participate in the ongoing trials to clarify whether this new class of drugs should be used as first- or second-line therapy.

␤-tubulin

Overexpression of class III ␤-tubulin, the target of taxanes, has shown to confer resistance to docetaxel and paclitaxel in lung and ovarian cancer cell lines.76-78 Taxanes bind to ␤-tubulin resulting in stabilization of microtubules and inhibition of their depolymerization. By doing this, it leads to chronic activation of the spindleassembly checkpoint, and ultimately causes mitotic arrest.79 In a retrospective analysis, low levels of class III ␤-tubulin were associated with better RR, PFS, and OS in advanced NSCLC patients treated with a paclitaxel-based regimen.80 Other studies have also seen similar results including outcomes in the neoadjuvant setting.81-83 Furthermore, analysis of samples from patients in the JBR-10 trial, showed that chemotherapy (cisplatin/navelbine) seemed to overcome the negative prognostic effect of high levels of expression of class III ␤-tubulin; the greatest benefit from chemotherapy was seen in those who had high levels of expression of this protein.84 Similar to others, this prognostic and predictive biomarker must be validated in large randomized prospective clinical trials.

Conclusion The future looks very promising and at the same time also intriguing for the treatment of NSCLC. If all these large, prospective, randomized clinical trials validate these needed biomarkers, we will make a major step forward in the treatment of NSCLC with major changes in the management of lung cancer in all stages possibly being near the horizon. The molecular medicine era has arrived and will stay for years to come. Of course, several questions remain unanswered. While we will continue to discover and validate prognostic and predictive biomarkers, we must also define the best assay platforms and make these tests uniform, reliable, and accessible to the population in terms of cost. The concept that all patients with the same diagnosis, gender, stage, and PS are the same must be left behind. This rudimentary patient selection must be replaced by a more accurate and precise method delivered by molecular medicine. It will be the patient’s

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tumor profile driving future clinical decision-making for treatmen of NSCLC.

Clinical Practice Points ● We understand that each patient’s tumor has a different biological and clinical behavior, and thus the importance to differentiate them so we can individualize anticancer therapy. ● To date, only mutation in the epidermal growth factor receptor (EGFR) gene is accepted as a prognostic and predictive biomarker in non-small cell lung cancer. Ideally, patients who have never smoked or have had non-squamous cell histology should be offered an EGFR mutation testing by PCR. If positive, medical oncologists have the option of EGFR tyrosine kinase inhibitor (e.g., erlotinib or gefitinib) as frontline therapy rather than conventional chemotherapy. ● Another promising agent, crizotinib, has shown an impressive overall response rate even in patients previously treated with chemotherapy based on the presence of a new genetic abnormality: EML4/ ALK translocation. If approved by the regulatory authorities, this translocation could be the second predictive biomarker widely accepted for clinical use and another agent will be added to the armamentarium in non-small cell lung cancer. ● Although other biomarkers have shown their utility in retrospective analysis or small phase III trials, they are still under investigation in well-conducted, prospective, randomized phase III clinical trials. If they validate previous observations, we will be able to tailor agents such as platinum, gemcitabine, pemetrexed, and taxanes in patients diagnosed with non-small cell lung cancer. Clinical trials in this regards are being conducted in early and metastatic clinical settings. Hence, we hope that not too far away clinicians will be able to answer questions such as: who should be treated? what therapy? or and in which clinical setting?

Disclosure Conflict of Interest. Dr Santos serves in the Speakers’ Bureau of Genentech and Lilly US Oncology. All other authors state that they have no conflicts of interest.

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