- Email: [email protected]
Personalized Treatment of Lung Cancer
Personalized Treatment of Lung Cancer Ravi Salgia, Thomas Hensing, Nicholas Campbell, April K. Salama, Michael Maitland, Philip Hoffman, Victoria Villaflor, and Everett E. Vokes Lung cancer is a heterogenous group of disorders, and a difficult disease to treat. The traditional approach of surgical resection for early-stage disease, potentially followed by chemotherapy, as well chemotherapy (with or without radiation) in later stages of disease is being supplemented with a personalized approach. The personalized approach has classically been used by the oncologist based on clinical/pathological parameters such as the performance status of the patient and histology of lung cancer. As molecular mechanisms have been explored in lung cancer more recently, the personalized approach also has incorporated molecular abnormalities. In particular, EGFR, K-ras, ALK, MET, CBL, and COX2, have come to the forefront as potential biomarkers and therapeutic targets. Thus, we review the various molecular mechanisms in lung cancer and the role of novel therapeutics. Semin Oncol 38:274-283 © 2011 Elsevier Inc. All rights reserved.
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ultiple advances have been made in the treatment of lung cancer over the past several years, including the development of novel targeted and chemotherapeutic agents. Despite this, overall survival rates for lung cancer remain low. Lung cancer is a heterogeneous disease, and there is a great deal of interest in the personalization of therapy. This requires careful coordination between the laboratory and the clinic to identify potential novel therapeutic targets, and to better select patients for optimal treatment. The use of molecularly targeted agents in selected patient subsets offers the potential to both improve efficacy and reduce toxicity. EGFR, K-ras, EML4-ALK, and MET along with other key drivers in lung cancer carcinogenesis appear to represent such targets. Additionally, the selection of patients for therapy may be improved with the development of gene signatures to refine prognosis and guide therapeutic decisions.
Department of Medicine, Section of Hematology/Oncology, The University of Chicago, and University of Chicago Cancer Research Center, Chicago, IL. Supported in part by NIH/NCI (5R01CA100750 – 07, 5R01CA125541– 04, 5R01CA129501– 03, 3R01CA129501– 02S1), Cancer Research Foundation, Respiratory Health Association of Chicago, and V-Foundation (to R.S.), and ASCO Cancer Foundation Translational Professorship (to E.E.V.). Address correspondence to Ravi Salgia, MD, PhD, Department of Medicine, Section of Hematology/Oncology, The University of Chicago, 5841 S Maryland Ave, Chicago, IL 60637. E-mail: rsalgia@medicine. bsd.uchiago.edu 0270-9295/ - see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2011.01.012
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Despite many advances in lung cancer research in recent years, lung cancer remains the leading cause of cancer related mortality in the United States.1 Recent efforts have focused on attempts to more clearly define the complex pathophysiology of this disease. An increased understanding of the molecular biology of lung cancer has led to a number of important findings that have the potential to directly impact patient care. This includes the discovery of novel molecules, the validation of predictive and prognostic biomarkers, and the development of novel technologies to aid in diagnosis and to guide treatment decisions. We discuss below some of the most clinically relevant translational advances that have been made in lung cancer research, with an emphasis on developments that have been made within the past year.2
TUMOR HISTOLOGY The distinction between squamous and nonsquamous histology has become the first step in the personalized treatment of patients with advanced nonsmall cell lung cancer (NSCLC). Accurate diagnosis of tumor histology has become essential in treatment decision-making and can impact considerations of both toxicity and potential efficacy of selected agents used in the management of this disease. Bevacizumab is a monoclonal antibody against the vascular endothelial growth factor (VEGF). In the early development of bevacizumab, squamous tumor histology was identified as a predictor of risk for bleeding complications.3 Consequently, patients with squamous NSCLC were excluded from the randomized trial that led to the apSeminars in Oncology, Vol 38, No 2, April 2011, pp 274-283
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proval of this agent.4 In the Eastern Cooperative Oncology Group (ECOG) 4599 trial, patients with nonsquamous NSCLC were randomized to treatment with carboplatin and paclitaxel with or without bevacizumab. Patients on the bevacizumab arm had improved overall survival (12.3 months v 10.3 months; hazard ratio [HR] 0.79; P ⫽ .003) compared to chemotherapy alone.5 Severe (grade ⱖIII) pulmonary hemorrhage was seen in 2.3% of patients (10 of 427 patients) treated with bevacizumab. This is in contrast to 9.1% of patients (six of 66 patients) on the randomized phase II trial that did not exclude patients with squamous tumors. The association between pulmonary hemorrhage and tumor histology also has been seen with other agents that target VEGF.6,7 Nonsquamous histology has been shown to be a predictor of improved survival after treatment with the multitargeted antifolate pemetrexed. In a prespecified subset analysis of the randomized comparison of cisplatin and pemetrexed with cisplatin and gemcitabine in treatment-naive patients with advanced NSCLC, median survival was superior on the pemetrexed arm for patients with nonsquamous tumors (adenocarcinoma and large cell carcinoma v squamous histology, n ⫽ 1,000, 11.8 v 10.4 months, respectively; HR 0.81; 95% confidence interval [CI], 0.70 – 0.84; P ⫽ .05).8 Patients with squamous tumors had inferior survival on the pemetrexed arm compared to cisplatin and gemcitabine (median 9.4 months v 10.8 months; HR 1.23; 95% CI, 1–1.51; P ⫽ .05). The interaction between histology and survival outcomes was maintained when controlled for known clinical prognostic factors.9 Most recently, the interaction between histology and efficacy also was seen in the prospective evaluation of pemetrexed in the maintenance setting. In this trial, patients with advanced NSCLC were randomized to maintenance pemetrexed versus placebo if they had stable or responding disease after four cycles of platinum-based chemotherapy. A significant improvement in overall survival was reported in patients with nonsquamous tumors (median survival 15.5 v 10.3 months, pemetrexed v placebo, respectively; HR 0.70; 95% CI, 0.56 – 0.88; P ⫽ .002).10 In contrast, no benefit with maintenance pemetrexed was seen in patients with squamous tumors (median survival 9.9 v 10.8 months, pemetrexed v placebo, respectively; HR 1.07; 95% CI, 0.77–1.5; P ⫽ .999). Adequate tissue, therefore, is necessary to define tumor histology accurately and guide treatment decision-making. However, the distinction between squamous and nonsquamous histology by hematoxylin and eosin staining alone may not always be straightforward. In a recent prospective analysis, the interobserver reproducibility (kappa coefficient) of the distinction between squamous and nonsquamous histology among 12 community and 12 expert pathologists was assessed using the 2004 World Health Organization (WHO) clas-
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sification.11 The pathologists viewed the same digital images from 96 patients. Confidence in the diagnosis was high in 52%, moderate in 40%, and low in 8% of the cases. There was only moderate interobserver agreement in the diagnosis of squamous versus nonsquamous histology among all pathologists ( ⫽ 0.55; 95% CI, 0.53– 0.58; P ⫽ .001). Although the level of agreement was higher in expert pathologists, it did not reach the level of good clinical agreement ( ⫽ 0.64 v 0.41, expert v community, respectively). Good clinical agreement was only seen when pathologists were confident of their diagnosis ( ⫽0.78). However, the level of interobserver agreement dropped significantly if they were only moderately confident in the diagnosis ( ⫽ 0.028). Diagnostic accuracy can be improved with the incorporation of molecular biomarkers. The addition of antibody-based immunohistochemical staining for protein expression of thyroid transcription factor-1 (TTF-1) and tumor protein 63 (TP63), as well as other markers, can be helpful in defining histological subtypes of NSCLC.12 As molecularly targeted therapeutics are developed, the identification of biomarkers that define the specific subset of patients who may benefit will be required and will likely supplant tumor histology alone in guiding treatment decision-making. Thymidylate synthase (TS) is one of the targets of pemetrexed and is a potential predictive biomarker for this agent. In preclinical testing, overexpression of TS has been associated with resistance to pemetrexed.13,14 Tumor histologies shown to be resistant to this agent in clinical testing also have been shown to have relatively higher TS expression compared to adenocarcinoma, including squamous and small cell carcinoma.15,16 In a recent series of patients with large cell carcinoma, TS expression was highest if there was neuroendocrine differentiation and lowest in tumors without neuroendocrine differentiation that were TTF-1–positive.17 In the randomized trial comparing cisplatin and pemetrexed to cisplatin and gemcitabine, low TS-mRNA expression was associated with improved time to progression and time to treatment failure on the pemetrexed arm, although the results were not statistically significant due to a limited number of tumor samples.18,19
EPIDERMAL GROWTH FACTOR RECEPTOR The family of epidermal growth factor receptors, EGFR/HER1, HER2/neu, HER3 and HER4, are involved in a wide variety of cellular processes, including proliferation, suppression of apoptosis, cell motility, and angiogenesis. EGFR has been shown to be dysregulated by a variety of mechanisms in NSCLC, including overexpression, amplification, or mutation.20
EGFR as a Predictive Marker in NSCLC Anti-EGFR therapy is playing an increasingly important role in the treatment of NSCLC. Therapy with
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EGFR tyrosine kinase inhibitors (TKIs)— erlotinib or gefitinib— has been established to be beneficial in the second-line setting, but previous trials in unselected patient populations have failed to show a benefit in the first-line setting.21 Certain clinical and pathologic features, including Asian ethnicity, female sex, adenocarcinoma histology, and light/never smokers, have been identified as predictors of response to an EGFR TKI, and it is now known that these epidemiologic and clinical factors select for a population with a molecularly distinct subset of NSCLC. These patients are much more likely to have sensitizing mutations in the tyrosine kinase domain.22–25 Recently, a study using these clinicopathologic features to select patients compared gefitinib with standard chemotherapy as first-line treatment for patients with advanced lung adenocarcinoma.26 At 12 months, the progression-free survival (PFS) was 24.9% in the gefitinib arm, versus 6.7% in the carboplatin-paclitaxel arm (HR 0.74; 95% CI, 0.65– 0.85). Furthermore, in the 261 patients with an EGFR mutation, those who received gefitinib had an improved PFS compared to those who received chemotherapy (HR 0.48; 95% CI, 0.36 – 0.64; P ⬍.0001). These data are further supported by other analyses that report a benefit from treatment with an EGFR TKI in selected patients.2,27,28 Importantly, for patients who did not harbor an EGFR mutation, PFS was significantly shorter if they received gefitinib rather than chemotherapy (HR 2.85; 95% CI, 2.05–3.98; P ⬍.001), suggesting a possible deleterious effect or an absence of therapeutic effect of first-line treatment with an EGFR TKI in this patient subset. Together, these data support the use of EGFR mutational testing and consideration of first-line therapy with an EGFR TKI only in patients with a confirmed EGFR mutation.
New EGFR TKIs Recently, two studies were presented that evaluated a new group of irreversible EGFR TKIs. The LUX-Lung 2 trial was a nonrandomized, phase II trial that treated patients with advanced NSCLC and known EGFR mutations with BIBW 2992—an oral, irreversible, dual EGFR and HER2 TKI. Ninety-four percent of patients had disease control with a median PFS of 12 months, supporting further investigations of the agent.29 The second agent tested was PF 299804, an oral, irreversible EGFR/HER-2/HER-4 TKI, which was compared to erlotinib in a randomized, phase II trial for patients with advanced NSCLC who had progressed with standard, cytotoxic chemotherapy. While patients were stratified based on EGFR mutational status, it was not a requirement for enrollment and only 16% of patients had such a mutation. Perhaps not surprisingly in this unselected patient population, response rates were low but significantly higher in those patients who had
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received PF 299804 compared to erlotinib (17% v 4%, respectively; P ⫽ .008).30
Resistance to EGFR TKIs Acquired resistance to EGFR TKIs is not uncommon and may develop in a number of ways such as through other mutations within the EGFR gene or through activation of other pathways.31,32 MET amplification can occur in EGFR-resistant cells in NSCLC and may explain secondary resistance in up to 20% of patients treated with an EGFR TKI. A secondary mutation involving the substitution of methionine for threonine at position 790 (T790M) accounts for approximately 50% of cases in which acquired resistance to erlotinib or gefitinib occurs.33 The T790M mutation is intriguing, however, in that the development of such a mutation actually may confer a relatively improved survival, as tumors that possess it appear to have a more indolent course than tumors with EGFR TKI resistance due to other causes. In a recent study, 93 patients with known EGFR-activating mutations who were treated with EGFR TKIs underwent repeat biopsies at the time of progression. Sixty-two percent of patients had developed a T790M mutation but had a significantly improved survival compared to those who had not (39 months v 26 months, P ⫽ .007).34
K-ras Oncogenes of the ras family encode for proteins on the cytoplasmic surface of cell membranes and have been implicated as regulators of the signal transduction pathways that control cell growth. Although mutations in each of the three distinct ras genes—H-ras, K-ras, and N-ras— have been linked with malignancy, K-ras is the most closely associated with NSCLC.35 K-ras proteins are activated when bound to guanosine triphosphate (GTP) and are inactivated by GTPase-activating protein (GAP), which causes the hydrolysis of GTP to guanosine diphosphate (GDP). K-ras proteins possess intrinsic GTPase activity, but point mutations at codons 12, 13, or 61 in the K-ras oncogene lead to a constitutively active protein production via changes at the GTP-binding domain, which prevents the conversion of GTP to GDP.36
K-ras Mutations and Patient Characteristics K-ras mutations in NSCLC have been described at a varying rate depending on the patient population studied. Overall, K-ras mutations have been reported in 15% to 20% of all patients with NSCLC and approximately 30% to 50% of those with adenocarcinoma histology.36 K-ras mutations are seen almost exclusively in smokers, with one study reporting that 43% of smokers who developed NSCLC exhibited such a mutation versus 0% of life-long nonsmokers with NSCLC (P ⫽ .001). Interestingly, the chance of having a K-ras mutation
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was not affected by the level or timing of tobacco abuse as heavy, light, current, and former smokers who developed NSCLC had statistically similar rates of mutation. There was also a persistent risk for K-ras mutation long after smoking cessation, which at least implies that K-ras mutations occur quite early in the carcinogenic process.37
The Prognostic and Predictive Nature of K-ras Mutations The impact of K-ras mutations on prognosis is likely negative. Several trials have found that K-ras mutations lead to decreased survival,35,38,39 while others have been unable to confirm this,40,41 Due to these disparate results, Mascaux and colleagues performed a meta-analysis that included 28 trials with 3,620 patients and reported worse outcomes for patients with K-ras mutations (HR 1.35; 95% CI, 1.16 –1.56) and particularly for those patients with an adenocarcinoma histology (HR 1.59; 95% CI, 1.26 –2.02).36 As for predictive impact, the possession of a K-ras mutation in NSCLC is associated with decreased response to EGFR TKIs. However, EGFR mutations and K-ras mutations are almost always mutually exclusive and the lack of response to an EGFR TKI is likely more closely linked and better predicted by the lack of an EGFR mutation than the existence of a K-ras mutation.42 In fact, in the exceedingly rare instances in which both an EGFR mutation and K-ras mutation have been reported in the same patient, the response to an EGFR TKI was excellent when that EGFR mutation was an exon 19 deletion—a known activating mutation— but exceedingly poor when that mutation was a T790M mutation.43 While EGFR TKIs clearly have no activity in almost all patients with a K-ras mutation, the response to EGFR monoclonal antibodies such as cetuximab— unlike the colorectal cancer population44—is less clearly impacted in NSCLC when a K-ras mutation is present.45
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stained positive for EGFR by immunohistochemistry, the molecular subset of patients most likely to benefit from cetuximab has not been defined. In contrast to treatment with EGFR TKIs, efficacy after treatment with cetuximab does not appear to correlate with EGFR or KRAS-related biomarkers.48 In the Southwest Oncology Group (SWOG) 0819 trial, cetuximab is now being tested with carboplatin and paclitaxel alone or combined with bevacizumab in newly diagnosed patients with advanced NSCLC. Biomarker studies are included in this trial in an effort to define further the optimal patient subset to treat with this agent.
EML4-ALK The EML4-ALK fusion protein represents one of the newest molecular targets in NSCLC. Anaplastic lymphoma kinase (ALK) was originally identified as part of a chromosomal translocation that is known to be associated with anaplastic large cell lymphoma, which results in a nucelophosmin–ALK fusion product (NPMALK). A number of other translocations resulting in unique ALK fusion proteins also have been identified, and may have differential effects on downstream signaling.49 ALK subsequently has been shown to be dysregulated in both hematologic and solid malignancies by a number of different mechanisms, including translocations, as well as mutations in the tyrosine kinase domain.49
Role in Oncogenesis The EML4-ALK translocation was first identified in 2007 from a surgically resected lung adenocarcinoma specimen.50,51 The fusion product results from an inversion in chromosome 2p, in which the echinoderm microtubule-associated protein-like 4 (EML4) becomes fused to the intracellular kinase domain of ALK. It has been shown that this fusion has clear oncogenic activity, and transgenic mice that express EML4-ALK specifically in lung alveolar cells develop hundreds of adenocarcinoma nodules within weeks.50
CETUXIMAB Cetuximab is an anti-EGFR immunoglobulin G1 monoclonal antibody. In the FLEX (First-Line Erbitux in Lung Cancer) trial, patients with treatment-naive stage IIIB or IV NSCLC were randomized to six cycles of cisplatin and vinorelbine with or without cetuximab. Patients on the cetuximab arm had improved overall survival (HR 0.87; 95% CI, 0.762– 0.996; P ⫽ .044).46 In BMS099, cetuximab was tested in combination with carboplatin and paclitaxel. A comparable improvement in overall survival was seen with the addition of cetuximab to chemotherapy (HR 0.89; 95% CI, 0.754 – 1.051), but the difference was not statistically significant (P ⫽ .169).47 Although the FLEX trial required patients to have tumors with at least one cell that
Clinicopathologic Features of EML4-ALK NSCLC The incidence of EML4-ALK in NSCLC appears to be low, with a frequency of 1% to 7% in reported studies.52 Despite the relatively low frequency, EML4-ALK NSCLC appears to represent a distinct subset of patients. In the largest series to date, NSCLC patients with at least two of the following characteristics were selected for genetic screening: female sex, Asian ethnicity, never/light smoking history, and adenocarcinoma histology.52 A total of 141 tumors were screened, and 19 (13%) patients were found to be EML4-ALK mutant; 31 (22%) were EGFR mutant. Consistent with the previous literature, EML4-ALK and EGFR mutations were mutually
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exclusive. Interestingly, the patients who harbored EML4-ALK translocation were significantly younger and more likely to be men compared to patients with EGFR mutations or to those who were wild-type (WT) for both genes. While EML4-ALK has been reported in smokers, in this particular study, patients with EML4ALK, like EGFR mutants, were more likely to be never/ light smokers when compared to WT patients (P ⬍.001). More studies are needed to define the clinical characteristics.
EML4-ALK as a Therapeutic Target Most importantly, EML4-ALK may potentially serve as a novel therapeutic target in NSCLC. Currently, patients with EML4-ALK appear to have both a similar response rate to platinum-based chemotherapy and overall survival compared to patients who are WT for both EML4-ALK and EGFR. Most recently, a phase I/II trial of the oral ALK inhibitor crizotinib (PF-02341066) has shown promising activity in patients with NSCLC whose tumors harbor the EML4-ALK fusion gene.19 In the phase I part of the study, 250 mg twice daily was established as the maximum tolerated dose (MTD) of this agent. In phase II, a molecularly enriched cohort of 82 EML4-ALK–positive patients was treated at the MTD. The majority of patients in this cohort had tumors with adenocarcinoma histology (96%) and were former (23%) or never smokers (76%). Most patients had received at least one prior regimen and the mean age was 51years. The objective response rate was 57% (95% CI, 46%– 68%) with an overall disease control rate of 87% (95% CI, 77%–93%). The response rate did not differ based on the number of prior regimens (52%, 67%, and 56% for 1, 2, and ⱖ3 prior regimens, respectively). The median duration of treatment was 5.7 months and 72% of patients were progression-free at 6 months. A randomized trial is currently ongoing comparing crizotinib to standard second-line chemotherapy (pemetrexed or docetaxel), as well as the role of crizotinib in the first-line setting for the appropriate population.
MET MET is a receptor tyrosine kinase that is expressed in both normal and malignant cells.53 MET, via signaling through hepatocyte growth factor/scatter factor (HGF/ SF) is important for many cellular processes, including cell proliferation, angiogenesis, invasion, and metastasis.53 MET signaling becomes dysregulated in both NSCLC and small cell lung cancer through a number of different mechanisms, including overexpression, mutations, and amplification, and through autocrine/paracrine activation of HGF.54,55
Potential Role as a Prognostic Marker Prognostic markers such as age, sex, performance status, weight loss, and other comorbidities have pro-
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vided some information in predicting the clinical course of patients with lung cancer. It is clear that these parameters are limited in their clinical utility, and there is some evidence to suggest that MET may be a potential prognostic marker in lung cancer. Increased MET gene copy number as detected by fluorescence in situ hybridization (FISH) may be a poor prognostic factor in patients with resected NSCLC.56 In a study of 447 patients with resected NSCLC, patients whose tumors were MET-positive (ⱖ5 copies/cell) had a shorter overall survival compared to those whose tumors were MET-negative (25.8 v 47.5 months, P ⫽ .005.) Further studies are ongoing to confirm the use of MET amplification as a prognostic marker in lung cancer.
MET-Mediated Therapy There also has been considerable interest in the role MET amplification may play in resistance to therapeutic inhibition with EGFR TKIs. Resistance to therapy is a complex process, and can occur via a number of different mechanisms. In addition to the T790M mutation, MET amplification may lead to acquired resistance to EGFR TKIs through ERBB3 activation of PI3K in up to 20% of patients.57 This finding potentially has important therapeutic implications for patients treated with EGFR TKIs whose tumors do not respond to treatment. A number of MET inhibitors are in early phase clinical trials, and studies looking at strategies for dual inhibition of both EGFR and MET are currently ongoing. ARQ 197 is a novel non–adenosine triphosphate (non-ATP) competitive inhibitor of MET. In a recently reported randomized phase II trial, erlotinib plus ARQ 197 was compared to erlotinib plus placebo in patients with advanced NSCLC who had received at least one prior regimen.58 The primary endpoint was PFS. On the combination arm the median PFS was 16.1 weeks compared to 9.7 weeks in patients who received erlotinib alone (adjusted HR 0.68; 95% CI, 0.47– 0.98; P ⬍.05). Although there was no difference in overall survival (median 36.6 weeks v 29.4 weeks, combination v erlotinib alone, adjusted HR 0.88; P ⫽ 0.52), when analyzed by histology, patients on the combination arm with nonsquamous tumors had a significantly prolonged median PFS (18.9 v 9.7 weeks, adjusted HR 0.61; P ⬍.05) and overall survival (43.1 v 29.4 weeks, adjusted HR 0.58; P ⬍.05). PFS also was prolonged significantly in patients with K-ras mutant tumors (unadjusted HR 0.18), with a trend for improved PFS in patients with wild-type EGFR (unadjusted HR 0.70). Crossover was allowed on the placebo arm. Of the 34 patients who did crossover to the combination, 23 were evaluable for response. Two patients achieved a partial response and nine had stable disease. In the two patients who achieved a partial response, one had a tumor with an EGFR mutation and both had
Personalized treatment of lung cancer
tumors that were K-ras wild-type with amplification of MET.58
ETHNICITY AND LUNG CANCER While lung cancer remains a leading cause of mortality for all races, recent research has focused on ethnic variations in this disease. One of the most striking disparities seen is the difference in lung cancer risk and survival for African Americans. When compared to their Caucasian counterparts, African Americans have a significantly higher risk of developing and dying from lung cancer. Epidemiologic research has focused on behavioral, cultural, and socioeconomic factors that may influence risk, although no clear link has been established.59
Molecular Heterogeneity and Race As the heterogeneity of lung cancer becomes more clearly defined on a molecular level, understanding the relationship that race may have with this variation has become more critical than ever. A number of studies have investigated the role that genetic susceptibility may play in lung cancer carcinogenesis. Previous studies have focused on race-specific genetic polymorphisms involved in the modulation of tobacco smoke carcinogens, although results have not been consistent.59 Additionally, polymorphisms in TP53 that have been associated with an increased lung cancer risk are seen in African Americans at a higher frequency when compared to other races.60 A recent analysis of MET and EGFR mutations in NSCLC patients also highlights clear ethnic variation.61 In a study comparing 141 Asian, 76 Caucasian, and 66 African American lung cancer patients, the incidence of MET mutations was highest in the Asian population, at 13%, and no MET mutations were detected in African Americans.61 The most common mutation detected was N375S, which represents a mutation in the semaphorin domain that appears to confer resistance to MET inhibition. It is well established that the frequency of EGFR mutations is higher in East Asian populations, which has important therapeutic implications. The differential frequency of MET mutations highlights the need for larger population-based studies to better characterize this disease.
CBL CBL is an E3 ubiquitin ligase that regulates receptor tyrosine kinase (RTK) activity in a number of different receptors, including MET, EGFR, platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR). It is expressed in both normal and malignant tissues, with the highest levels seen in hematopoietic tissues.62
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Function and Role in Oncogenesis CBL is required for ubiquitination and degradation of RTKs.63 However, in addition to its role as a regulator of RTK signaling, evidence suggests that CBL may have a dual role as a signal transduction molecule. Recent data show that alterations in the CBL gene occur in NSCLC, and this pathway may play a critical role in lung cancer carcinogenesis. Tumor samples from 119 patients with NSCLC were analyzed for CBL mutations.64 Eight novel mutations in CBL were identified with specific mutations being confined to a particular ethnicity (the study included Caucasian, African American, and Taiwanese patients). Additionally, 22% of patients demonstrated a loss of heterozygosity (LOH) at the CBL locus. In vitro data showed that these alterations in CBL resulted in increased cell viability and motility but did not alter the E3 ubiquitin ligase activity of CBL. The Taiwanese cohort (50 patients) was also analyzed for EGFR, MET, P53, and K-ras mutations. Interestingly, EGFR and MET mutations were not mutually exclusive with CBL mutations, although there was no overlap with P53 or K-ras mutations. Taken together, these results suggest that CBL may contribute to oncogenesis independent of its role as a regulator of kinase activity. Future studies are needed to define further the key components of this pathway, and the potential for therapeutic intervention.
COX-2 Overexpression of cyclooxygenase-2 (COX-2) is common in NSCLC.65,66 In preclinical models, inhibition of COX-2 has been shown to inhibit lung cancer growth.67 In Cancer and Leukemia Group B (CALGB) 30203, 140 treatment-naive patients with advanced NSCLC were randomized to treatment with carboplatin and gemcitabine with the COX-2 inhibitor, celecoxib, a 5-lipoxygenase (5-LOX) inhibitor, zileuton, or both.68 Tissue was obtained from 76% of the patients (107 of 140 patients). COX-2 expression by immunohistochemistry was shown to be a negative prognostic marker for survival (HR 2.51; P ⫽ .019). Although there was no difference in overall survival between the treatment arms, patients who had tumors with increased COX-2 expression had improved overall survival (HR 0.342; P ⫽ .005) if they received celecoxib. CALGB 30801 is an ongoing randomized trial investigating the role of COX-2 inhibition in patients with COX-2– expressing advanced NSCLC.
GENE EXPRESSION PROFILING Developments in gene expression profiling now allow for the measurement of the expression of thousands of genes simultaneously through a microarray platform. This technology has afforded researchers the ability to identify unique profiles that may be associated
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with high or low risk of disease progression. Information derived from these signatures may be applicable in a number of different clinical settings, and may be used to guide treatment decisions in the future. Several profiles have been developed that have been shown to risk stratify patients with NSCLC.69,70 In a study of 101 tumor samples from patients with resected NSCLC, a five-gene signature was used to identify patients as either high or low risk of an adverse outcome (relapse or death from lung cancer). With a median follow-up of 20 months, 59 patients who were identified as having high-risk disease had a significantly shorter survival (20 months v 40 months, P ⬍.001) when compared to those who had a low-risk signature. This association with survival was confirmed in the validation cohort of 60 patients. Although this technology has the potential to guide treatment decision-making in the future, validation of gene signatures as either prognostic or predictive biomarkers in prospective clinical trials is required.
CIRCULATING TUMOR CELLS It is has been well established that epithelial cells, presumed to be tumor cells, can be detected in the blood of patients with metastatic cancer.71 Methods that have been used previously to detect circulating tumor cells (CTCs) isolated low numbers of cells, which limited their use for subsequent molecular analyses. A recent development has been the use of the CTC-chip, a microfluidic device that isolates and analyzes CTCs with a high purity.72 Using microchip technology, the CTC-chip separates viable CTCs in peripheral blood samples by binding to microposts coated with an epithelial-cell adhesion molecule (EpCAM). Genomic DNA from CTCs collected using this method has been used for mutational analyses. In a study of 27 patients with metastatic NSCLC, EGFR mutational analysis was performed on DNA collected from CTCs. In 12 patients who were known to have a primary tumor with an EGFR mutation, detection of the EGFR mutation was more sensitive in the circulating tumor cells (11 of 12 patients) than in the plasma (four of 12 patients).73 Interestingly, the T790M mutation was seen not only in patients who had prior therapy with an EGFR TKI, but it was detected also in pretreatment biopsy specimens. The presence of this mutation pretreatment was significantly associated with a decrease in PFS (7.7 v 16.5 months, P ⬍.001). Additionally, the types of mutations seen in the CTCs changed throughout a patient’s clinical course; for example, while the T790M mutation was detected at low levels in the initial CTC specimen, it appeared to increase in frequency over time, correlating clinically with drug resistance.73 This technology represents a notable advance for a number of reasons. Available tissue is often limited, and
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this technique allows for reliable tumor genotyping with a less invasive procedure. Additionally, it allows for real time tumor genotyping, as it is clear that the genomic profile of a patient’s original tumor may change throughout the course of therapy. CTCs also may be used to identify other predictive biomarkers that can help clinicians further tailor treatment recommendations. In a recent series, CTCs from 75 patients with late-stage NSCLC were analyzed for ERCC1 mRNA expression using real-time reverse transcriptase–polymerase chain reaction (RT-PCR).74 In this series, ERCC1 expression levels in CTCs were significantly correlated with response (P ⫽ .003) to platinum-based chemotherapy.
CONCLUSIONS While the goal of delivering personalized therapy for patients with lung cancer is certainly not new, clinicians now have more factors to consider that can help guide therapeutic decision-making. In addition to considering traditional factors such as a patient’s performance status, gender and age, tumor histology has now been established as an important predictive factor for both toxicity and efficacy to available therapeutics. Accurate diagnosis of tumor histology is, therefore, essential. A case now can be made also to test for specific molecular biomarkers in selected patients with advanced adenocarcinoma of the lung, including EGFR mutations and possibly EML4-ALK translocations in newly diagnosed and previously treated never (⬍100 cigarettes in a lifetime) or former (stopped ⱖ15 years ago) light (smoked ⱕ10 pack years) smokers, respectively. K-ras mutation status also may be used to predict resistance to EGFR TKIs in patients with adenocarcinoma of the lung, although the association between K-ras mutations and survival after treatment with these agents has not been established. In the future, additional molecular biomarkers may be incorporated into the diagnostic algorithm, including MET, CBL, and COX-2. Adequate tissue is often the limiting factor in establishing biomarker status, which may be overcome through genotyping of CTCs. The treatment of lung cancer remains complex and challenging. While significant advances have been made, much remains unknown about the molecular biology of this disease. Novel approaches to diagnosis and therapy are needed, and the findings reviewed here represent clear promise for the future.
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3. Johnson D, Fehrenbacher L, Novotny W, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-smallcell lung cancer. J Clin Oncol. 2004;22:2184 –91. 4. Sandler A, Gray R, Brahmer J, et al. Randomized phase II/III trial of paclitaxel plus carboplatin with or without bevacizumab in patients with advanced non-squamous non-small cell lung cancer: an Eastern Cooperative Oncology Group trial--E4599. J Clin Oncol. 2005;23 Suppl:4. 5. Sandler A, Gray R, Perry MC, et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med. 2006;355:2542–50. 6. Sandler AB, Schiller JH, Gray R, et al. Retrospective evaluation of the clinical and radiographic risk factors associated with severe pulmonary hemorrhage in firstline advanced, unresectable non–small-cell lung cancer treated with carboplatin and paclitaxel plus bevacizumab. J Clin Oncol. 2009;27:1405–12. 7. Socinski MA, Novello S, Brahmer JR, et al. Multicenter, phase II trial of sunitinib in previously treated, advanced non-small-cell lung cancer. J Clin Oncol. 2008;26:650 – 6. 8. Scagliotti GV, Parikh P, von Pawel J, et al. Phase III study comparing cisplatin plus gemcitabine with cisplatin plus pemetrexed in chemotherapy-naive patients with advanced-stage non-small-cell lung cancer. J Clin Oncol. 2008;26:3543–51. 9. Syrigos KN, Vansteenkiste J, Parikh P, et al. Prognostic and predictive factors in a randomized phase III trial comparing cisplatin-pemetrexed v cisplatin-gemcitabine in advanced non-small-cell lung cancer. Ann Oncol. 2010;21:556 – 61. 10. Ciuleanu T, Brodowicz T, Zielinski C, et al. Maintenance pemetrexed plus best supportive care v placebo plus best supportive care for non-small-cell lung cancer: a randomised, double-blind, phase 3 study. Lancet. 2009; 374:1432– 40. 11. Grilley-Olson JE, Hayes D, Qaqish BF, et al. Diagnostic reproducibility of squamous cell carcinoma in the ear of histology-directed non-small cell chemothearpy: a large prospective study. J Clin Oncol. 2009;27:abstract 8008. 12. Ring BZ, Seitz RS, Beck RA, et al. A novel five-antibody immunohistochemical test for subclassification of lung carcinoma. Mod Pathol. 2009;22:1032– 43. 13. Giovannetti E, Mey V, Nannizzi S, et al. Cellular and pharmacogenetics foundation of synergistic interaction of pemetrexed and gemcitabine in human non-small-cell lung cancer cells. Mol Pharmacol. 2005;68:110 – 8. 14. Schultz RM, Chen VJ, Bewley JR, Roberts EF, Shih C, Dempsey JA. Biological activity of the multitargeted antifolate, MTA (LY231514), in human cell lines with different resistance mechanisms to antifolate drugs. Semin Oncol. 1999;26:68 –73. 15. Socinski MA, Smit EF, Lorigan P, et al. Phase III study of pemetrexed plus carboplatin compared with etoposide plus carboplatin in chemotherapy-naive patients with extensive-stage small-cell lung cancer. J Clin Oncol. 2009;27:4787–92. 16. Ceppi P, Volante M, Saviozzi S, et al. Squamous cell carcinoma of the lung compared with other histotypes shows higher messenger RNA and protein levels for thymidylate synthase. Cancer. 2006;107:1589 –96.
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17. Monica V, Scagliotti GV, Ceppi P, et al. Differential thymidylate synthase expression in different variants of large-cell carcinoma of the lung. Clin Cancer Res. 2009; 15:7547–52. 18. Scagliotti G, Kaiser C, Biesma B, et al. Correlations of biomarker expression and clinical outxome in a large phase III trial of pemetrexed plus cisplatin or gemcitabine plus cisplatin in chemonaive patients with locally advanced or metastatic non-small cell lung cancer. J Thorac Oncol. 2007;2:S375. 19. Bang Y, Kwak EL, Shaw AT, et al. Clinical activity of the oral ALK inhibitor PF-02341066 in ALK-positive patients with non-small cell lung cancer (NSCLC). J Clin Oncol (Meeting Abstracts). 2010;28:3. 20. Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al. Epidermal growth factor receptor in non-small-cell lung carcinomas: correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol. 2003;21:3798 – 807. 21. Ray M, Salgia R, Vokes EE. The role of EGFR inhibition in the treatment of non-small cell lung cancer. Oncologist. 2009;14:1116 –30. 22. Ahmed SM, Salgia R. Epidermal growth factor receptor mutations and susceptibility to targeted therapy in lung cancer. Respirology. 2006;11:687–92. 23. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350:2129 –39. 24. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004; 101:13306 –11. 25. Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung cancer—molecular and clinical predictors of outcome. N Engl J Med. 2005;353:133– 44. 26. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med. 2009;361:947–57. 27. Jackman DM, Miller VA, Cioffredi L-A, et al. Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: results of an online tumor registry of clinical trials. Clin Cancer Res. 2009;15:5267– 73. 28. Janne PA, Wang XF, Socinski MA, et al. Randomized phase II trial of erlotinib (E) alone or in combination with carboplatin/paclitaxel (CP) in never or light former smokers with advanced lung adenocarcinoma: CALGB 30406. J Clin Oncol (Meeting Abstracts). 2010;28:7503. 29. Yang C, Shih J, Su W, et al. A phase II study of BIBW 2992 in patients with adenocarcinoma of the lung and activating EGFR mutations (LUX-Lung 2). J Clin Oncol (Meeting Abstracts). 2010;28:7521. 30. Boyer MJ, Blackhall FH, Park K, et al. Efficacy and safety of PF299804 v erlotinib (E): a global, randomized phase II trial in patients (pts) with advanced non-small cell lung cancer (NSCLC) after failure of chemotherapy (CT). J Clin Oncol (Meeting Abstracts). 2010;28:LBA7523. 31. Engelman JA, Jänne PA. Mechanisms of acquired resistance to epidermal growth factor receptor tyrosine ki-
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nase inhibitors in non–small cell lung cancer. Clin Cancer Res. 2008;14:2895–9. Sierra JR, Cepero V, Giordano S. Molecular mechanisms of acquired resistance to tyrosine kinase targeted therapy. Mol Cancer. 2010;9:75. Kosaka T, Yatabe Y, Endoh H, et al. Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib. Clin Cancer Res. 2006;12:5764 –9. Oxnard GR, Riely GJ, Arcila MH, et al. Clinical course of patients (pts) with acquired resistance (AR) to EGFR tyrosine kinase inhibitors (TKI). J Clin Oncol (Meeting Abstracts). 2010;28:7520. Slebos RJ, Kibbelaar RE, Dalesio O, et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N Engl J Med. 1990;323:561–5. Mascaux C, Iannino N, Martin B, et al. The role of RAS oncogene in survival of patients with lung cancer: a systematic review of the literature with meta-analysis. Br J Cancer. 2005;92:131–9. Ahrendt SA, Decker PA, Alawi EA, et al. Cigarette smoking is strongly associated with mutation of the K-ras gene in patients with primary adenocarcinoma of the lung. Cancer. 2001;92:1525–30. Mitsudomi T, Steinberg SM, Oie HK, et al. ras gene mutations in non-small cell lung cancers are associated with shortened survival irrespective of treatment intent. Cancer Res. 1991;51:4999 –5002. Nelson HH, Christiani DC, Mark EJ, Wiencke JK, Wain JC, Kelsey KT. Implications and prognostic value of K-ras mutation for early-stage lung cancer in women. J Natl Cancer Inst. 1999;91:2032– 8. Tsao MS, Aviel-Ronen S, Ding K, et al. Prognostic and predictive importance of p53 and RAS for adjuvant chemotherapy in non small-cell lung cancer. J Clin Oncol. 2007;25:5240 –7. Lu C, Soria JC, Tang X, et al. Prognostic factors in resected stage I non-small-cell lung cancer: a multivariate analysis of six molecular markers. J Clin Oncol. 2004;22: 4575– 83. Suda K, Tomizawa K, Mitsudomi T. Biological and clinical significance of KRAS mutations in lung cancer: an oncogenic driver that contrasts with EGFR mutation. Cancer Metast Rev. 2010;29:49 – 60. Jackman DM, Miller VA, Cioffredi LA, et al. Impact of epidermal growth factor receptor and KRAS mutations on clinical outcomes in previously untreated non-small cell lung cancer patients: results of an online tumor registry of clinical trials. Clin Cancer Res. 2009;15:5267–73. Van Cutsem E, Lang I, D’haens G, et al. KRAS status and efficacy in the first-line treatment of patients with metastatic colorectal cancer (mCRC) treated with FOLFIRI with or without cetuximab: the CRYSTAL experience. J Clin Oncol (Meeting Abstracts). 2008;26:2. O’Byrne KJ, Bondarenko I, Barrios C, et al. Molecular and clinical predictors of outcome for cetuximab in nonsmall cell lung cancer (NSCLC): data from the FLEX study. J Clin Oncol (Meeting Abstracts). 2009;27:8007. Pirker R, Pereira JR, Szczesna A, et al. Cetuximab plus chemotherapy in patients with advanced non-small-cell lung cancer (FLEX): an open-label randomised phase III trial. Lancet. 2009;373:1525–31.
47. Lynch TJ, Patel T, Dreisbach L, et al. Cetuximab and first-line taxane/carboplatin chemotherapy in advanced non-small-cell lung cancer: results of the randomized multicenter phase III trial BMS099. J Clin Oncol. 2010; 28:911–7. 48. Khambata-Ford S, Harbison CT, Hart LL, et al. Analysis of potential predictive markers of cetuximab benefit in BMS099, a phase III study of cetuximab and first-line taxane/carboplatin in advanced non-small-cell lung cancer. J Clin Oncol. 2010;28:918 –27. 49. Mosse YP, Wood A, Maris JM. Inhibition of ALK signaling for cancer therapy. Clin Cancer Res. 2009;15:5609 –14. 50. Mano H. Non-solid oncogenes in solid tumors: EML4ALK fusion genes in lung cancer. Cancer Sci. 2008;99: 2349 –55. 51. Soda M, Choi YL, Enomoto M, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature. 2007;448:561– 6. 52. Shaw AT, Yeap BY, Mino-Kenudson M, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol. 2009; 27:4247–53. 53. Christensen JG, Burrows J, Salgia R. c-Met as a target for human cancer and characterization of inhibitors for therapeutic intervention. Cancer Lett. 2005;225:1–26. 54. Ma PC, Jagadeeswaran R, Jagadeesh S, et al. Functional expression and mutations of c-Met and its therapeutic inhibition with SU11274 and small interfering RNA in non-small cell lung cancer. Cancer Res. 2005;65: 1479 – 88. 55. Kim ES, Salgia R. MET pathway as a therapeutic target. J Thorac Oncol. 2009;4:444 –7. 56. Cappuzzo F, Marchetti A, Skokan M, et al. Increased MET gene copy number negatively affects survival of surgically resected non-small-cell lung cancer patients. J Clin Oncol. 2009;27:1667–74. 57. Engelman JA, Zejnullahu K, Mitsudomi T, et al. MET amplification leads to gefitinib resistance in lung cancer by activating ERBB3 signaling. Science. 2007;316: 1039 – 43. 58. Schiller JH, Akerley WL, Brugger W, et al. Results from ARQ 197–209: A global randomized placebo-controlled phase II clinical trial of erlotinib plus ARQ 197 v erlotinib plus placebo in previously treated EGFR inhibitor-naive patients with locally advanced or metastatic non-small cell lung cancer (NSCLC). J Clin Oncol (Meeting Abstracts). 2010;28:LBA7502. 59. Abidoye O, Ferguson MK, Salgia R. Lung carcinoma in African Americans. Nat Clin Pract Oncol. 2007;4: 118 –29. 60. Wu X, Zhao H, Amos CI, et al. p53 genotypes and haplotypes associated with lung cancer susceptibility and ethnicity. J Natl Cancer Inst. 2002;94:681–90. 61. Krishnaswamy S, Kanteti R, Duke-Cohan JS, et al. Ethnic differences and functional analysis of MET mutations in lung cancer. Clin Cancer Res. 2009;15:5714 –23. 62. Peschard P, Fournier TM, Lamorte L, et al. Mutation of the c-Cbl TKB domain binding site on the Met receptor tyrosine kinase converts it into a transforming protein. Mol Cell. 2001;8:995–1004. 63. Peschard P, Park M. From Tpr-Met to Met, tumorigenesis and tubes. Oncogene. 2007;26:1276 – 85.
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64. Tan C, Krishnaswamy S, Nandi S, et al. CBL is frequently altered in lung cancers: its relationship to mutations in MET and EGFR tyrosine kinases. PLoS One. 2010;5:e8972. 65. Wolff H, Saukkone NK, Anttila S, Karjalainen A, Vainio H, Ristimaki A. Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res. 1998;58:4997–5001. 66. Khuri F, Wu H, Lee J, et al. Cyclooxygenase-2 overexpression is a marker of poor prognosis in stage I nonsmall cell lung cancer. Clin Cancer Res. 2001;7:861–7. 67. Masferrer JL, Leahy KM, Koki AT, et al. Antiangiogenic and antitumor activities of cyclooxygenase-2 inhibitors. Cancer Res. 2000;60:1306 –11. 68. Edelman MJ, Watson D, Wang X, et al. Eicosanoid modulation in advanced lung cancer: cyclooxygenase-2 expression is a positive predictive factor for celecoxib ⫹ chemotherapy—Cancer and Leukemia Group B trial 30203. J Clin Oncol. 2008;26:848 –55. 69. Chen H-Y, Yu S-L, Chen C-H, et al. A five-gene signature and clinical outcome in non–small-cell lung cancer. N Engl J Med. 2007;356:11–20.
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70. Skrzypski M, Jassem E, Taron M, et al. Three-gene expression signature predicts survival in early-stage squamous cell carcinoma of the lung. Clin Cancer Res. 2008; 14:4794 –9. 71. Allard WJ, Matera J, Miller MC, et al. Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004;10:6897–904. 72. Nagrath S, Sequist LV, Maheswaran S, et al. Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007;450:1235–9. 73. Maheswaran S, Sequist LV, Nagrath S, et al. Detection of mutations in EGFR in circulating lung-cancer cells. N Engl J Med. 2008;359:366 –77. 74. Hoffmann AC, Gauler T, Parr A, Eberhardt WE, Schuler MH. Use of ERCC1 expression levels in circulating tumor cells to predict response to platinum-based chemotherapy in patients with non-small cell lung cancer. J Clin Oncol (Meeting Abstracts). 2010;28:7512.
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ultiple advances have been made in the treatment of lung cancer over the past several years, including the development of novel targeted and chemotherapeutic agents. Despite this, overall survival rates for lung cancer remain low. Lung cancer is a heterogeneous disease, and there is a great deal of interest in the personalization of therapy. This requires careful coordination between the laboratory and the clinic to identify potential novel therapeutic targets, and to better select patients for optimal treatment. The use of molecularly targeted agents in selected patient subsets offers the potential to both improve efficacy and reduce toxicity. EGFR, K-ras, EML4-ALK, and MET along with other key drivers in lung cancer carcinogenesis appear to represent such targets. Additionally, the selection of patients for therapy may be improved with the development of gene signatures to refine prognosis and guide therapeutic decisions.
Department of Medicine, Section of Hematology/Oncology, The University of Chicago, and University of Chicago Cancer Research Center, Chicago, IL. Supported in part by NIH/NCI (5R01CA100750 – 07, 5R01CA125541– 04, 5R01CA129501– 03, 3R01CA129501– 02S1), Cancer Research Foundation, Respiratory Health Association of Chicago, and V-Foundation (to R.S.), and ASCO Cancer Foundation Translational Professorship (to E.E.V.). Address correspondence to Ravi Salgia, MD, PhD, Department of Medicine, Section of Hematology/Oncology, The University of Chicago, 5841 S Maryland Ave, Chicago, IL 60637. E-mail: rsalgia@medicine. bsd.uchiago.edu 0270-9295/ - see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1053/j.seminoncol.2011.01.012
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Despite many advances in lung cancer research in recent years, lung cancer remains the leading cause of cancer related mortality in the United States.1 Recent efforts have focused on attempts to more clearly define the complex pathophysiology of this disease. An increased understanding of the molecular biology of lung cancer has led to a number of important findings that have the potential to directly impact patient care. This includes the discovery of novel molecules, the validation of predictive and prognostic biomarkers, and the development of novel technologies to aid in diagnosis and to guide treatment decisions. We discuss below some of the most clinically relevant translational advances that have been made in lung cancer research, with an emphasis on developments that have been made within the past year.2
TUMOR HISTOLOGY The distinction between squamous and nonsquamous histology has become the first step in the personalized treatment of patients with advanced nonsmall cell lung cancer (NSCLC). Accurate diagnosis of tumor histology has become essential in treatment decision-making and can impact considerations of both toxicity and potential efficacy of selected agents used in the management of this disease. Bevacizumab is a monoclonal antibody against the vascular endothelial growth factor (VEGF). In the early development of bevacizumab, squamous tumor histology was identified as a predictor of risk for bleeding complications.3 Consequently, patients with squamous NSCLC were excluded from the randomized trial that led to the apSeminars in Oncology, Vol 38, No 2, April 2011, pp 274-283
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proval of this agent.4 In the Eastern Cooperative Oncology Group (ECOG) 4599 trial, patients with nonsquamous NSCLC were randomized to treatment with carboplatin and paclitaxel with or without bevacizumab. Patients on the bevacizumab arm had improved overall survival (12.3 months v 10.3 months; hazard ratio [HR] 0.79; P ⫽ .003) compared to chemotherapy alone.5 Severe (grade ⱖIII) pulmonary hemorrhage was seen in 2.3% of patients (10 of 427 patients) treated with bevacizumab. This is in contrast to 9.1% of patients (six of 66 patients) on the randomized phase II trial that did not exclude patients with squamous tumors. The association between pulmonary hemorrhage and tumor histology also has been seen with other agents that target VEGF.6,7 Nonsquamous histology has been shown to be a predictor of improved survival after treatment with the multitargeted antifolate pemetrexed. In a prespecified subset analysis of the randomized comparison of cisplatin and pemetrexed with cisplatin and gemcitabine in treatment-naive patients with advanced NSCLC, median survival was superior on the pemetrexed arm for patients with nonsquamous tumors (adenocarcinoma and large cell carcinoma v squamous histology, n ⫽ 1,000, 11.8 v 10.4 months, respectively; HR 0.81; 95% confidence interval [CI], 0.70 – 0.84; P ⫽ .05).8 Patients with squamous tumors had inferior survival on the pemetrexed arm compared to cisplatin and gemcitabine (median 9.4 months v 10.8 months; HR 1.23; 95% CI, 1–1.51; P ⫽ .05). The interaction between histology and survival outcomes was maintained when controlled for known clinical prognostic factors.9 Most recently, the interaction between histology and efficacy also was seen in the prospective evaluation of pemetrexed in the maintenance setting. In this trial, patients with advanced NSCLC were randomized to maintenance pemetrexed versus placebo if they had stable or responding disease after four cycles of platinum-based chemotherapy. A significant improvement in overall survival was reported in patients with nonsquamous tumors (median survival 15.5 v 10.3 months, pemetrexed v placebo, respectively; HR 0.70; 95% CI, 0.56 – 0.88; P ⫽ .002).10 In contrast, no benefit with maintenance pemetrexed was seen in patients with squamous tumors (median survival 9.9 v 10.8 months, pemetrexed v placebo, respectively; HR 1.07; 95% CI, 0.77–1.5; P ⫽ .999). Adequate tissue, therefore, is necessary to define tumor histology accurately and guide treatment decision-making. However, the distinction between squamous and nonsquamous histology by hematoxylin and eosin staining alone may not always be straightforward. In a recent prospective analysis, the interobserver reproducibility (kappa coefficient) of the distinction between squamous and nonsquamous histology among 12 community and 12 expert pathologists was assessed using the 2004 World Health Organization (WHO) clas-
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sification.11 The pathologists viewed the same digital images from 96 patients. Confidence in the diagnosis was high in 52%, moderate in 40%, and low in 8% of the cases. There was only moderate interobserver agreement in the diagnosis of squamous versus nonsquamous histology among all pathologists ( ⫽ 0.55; 95% CI, 0.53– 0.58; P ⫽ .001). Although the level of agreement was higher in expert pathologists, it did not reach the level of good clinical agreement ( ⫽ 0.64 v 0.41, expert v community, respectively). Good clinical agreement was only seen when pathologists were confident of their diagnosis ( ⫽0.78). However, the level of interobserver agreement dropped significantly if they were only moderately confident in the diagnosis ( ⫽ 0.028). Diagnostic accuracy can be improved with the incorporation of molecular biomarkers. The addition of antibody-based immunohistochemical staining for protein expression of thyroid transcription factor-1 (TTF-1) and tumor protein 63 (TP63), as well as other markers, can be helpful in defining histological subtypes of NSCLC.12 As molecularly targeted therapeutics are developed, the identification of biomarkers that define the specific subset of patients who may benefit will be required and will likely supplant tumor histology alone in guiding treatment decision-making. Thymidylate synthase (TS) is one of the targets of pemetrexed and is a potential predictive biomarker for this agent. In preclinical testing, overexpression of TS has been associated with resistance to pemetrexed.13,14 Tumor histologies shown to be resistant to this agent in clinical testing also have been shown to have relatively higher TS expression compared to adenocarcinoma, including squamous and small cell carcinoma.15,16 In a recent series of patients with large cell carcinoma, TS expression was highest if there was neuroendocrine differentiation and lowest in tumors without neuroendocrine differentiation that were TTF-1–positive.17 In the randomized trial comparing cisplatin and pemetrexed to cisplatin and gemcitabine, low TS-mRNA expression was associated with improved time to progression and time to treatment failure on the pemetrexed arm, although the results were not statistically significant due to a limited number of tumor samples.18,19
EPIDERMAL GROWTH FACTOR RECEPTOR The family of epidermal growth factor receptors, EGFR/HER1, HER2/neu, HER3 and HER4, are involved in a wide variety of cellular processes, including proliferation, suppression of apoptosis, cell motility, and angiogenesis. EGFR has been shown to be dysregulated by a variety of mechanisms in NSCLC, including overexpression, amplification, or mutation.20
EGFR as a Predictive Marker in NSCLC Anti-EGFR therapy is playing an increasingly important role in the treatment of NSCLC. Therapy with
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EGFR tyrosine kinase inhibitors (TKIs)— erlotinib or gefitinib— has been established to be beneficial in the second-line setting, but previous trials in unselected patient populations have failed to show a benefit in the first-line setting.21 Certain clinical and pathologic features, including Asian ethnicity, female sex, adenocarcinoma histology, and light/never smokers, have been identified as predictors of response to an EGFR TKI, and it is now known that these epidemiologic and clinical factors select for a population with a molecularly distinct subset of NSCLC. These patients are much more likely to have sensitizing mutations in the tyrosine kinase domain.22–25 Recently, a study using these clinicopathologic features to select patients compared gefitinib with standard chemotherapy as first-line treatment for patients with advanced lung adenocarcinoma.26 At 12 months, the progression-free survival (PFS) was 24.9% in the gefitinib arm, versus 6.7% in the carboplatin-paclitaxel arm (HR 0.74; 95% CI, 0.65– 0.85). Furthermore, in the 261 patients with an EGFR mutation, those who received gefitinib had an improved PFS compared to those who received chemotherapy (HR 0.48; 95% CI, 0.36 – 0.64; P ⬍.0001). These data are further supported by other analyses that report a benefit from treatment with an EGFR TKI in selected patients.2,27,28 Importantly, for patients who did not harbor an EGFR mutation, PFS was significantly shorter if they received gefitinib rather than chemotherapy (HR 2.85; 95% CI, 2.05–3.98; P ⬍.001), suggesting a possible deleterious effect or an absence of therapeutic effect of first-line treatment with an EGFR TKI in this patient subset. Together, these data support the use of EGFR mutational testing and consideration of first-line therapy with an EGFR TKI only in patients with a confirmed EGFR mutation.
New EGFR TKIs Recently, two studies were presented that evaluated a new group of irreversible EGFR TKIs. The LUX-Lung 2 trial was a nonrandomized, phase II trial that treated patients with advanced NSCLC and known EGFR mutations with BIBW 2992—an oral, irreversible, dual EGFR and HER2 TKI. Ninety-four percent of patients had disease control with a median PFS of 12 months, supporting further investigations of the agent.29 The second agent tested was PF 299804, an oral, irreversible EGFR/HER-2/HER-4 TKI, which was compared to erlotinib in a randomized, phase II trial for patients with advanced NSCLC who had progressed with standard, cytotoxic chemotherapy. While patients were stratified based on EGFR mutational status, it was not a requirement for enrollment and only 16% of patients had such a mutation. Perhaps not surprisingly in this unselected patient population, response rates were low but significantly higher in those patients who had
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received PF 299804 compared to erlotinib (17% v 4%, respectively; P ⫽ .008).30
Resistance to EGFR TKIs Acquired resistance to EGFR TKIs is not uncommon and may develop in a number of ways such as through other mutations within the EGFR gene or through activation of other pathways.31,32 MET amplification can occur in EGFR-resistant cells in NSCLC and may explain secondary resistance in up to 20% of patients treated with an EGFR TKI. A secondary mutation involving the substitution of methionine for threonine at position 790 (T790M) accounts for approximately 50% of cases in which acquired resistance to erlotinib or gefitinib occurs.33 The T790M mutation is intriguing, however, in that the development of such a mutation actually may confer a relatively improved survival, as tumors that possess it appear to have a more indolent course than tumors with EGFR TKI resistance due to other causes. In a recent study, 93 patients with known EGFR-activating mutations who were treated with EGFR TKIs underwent repeat biopsies at the time of progression. Sixty-two percent of patients had developed a T790M mutation but had a significantly improved survival compared to those who had not (39 months v 26 months, P ⫽ .007).34
K-ras Oncogenes of the ras family encode for proteins on the cytoplasmic surface of cell membranes and have been implicated as regulators of the signal transduction pathways that control cell growth. Although mutations in each of the three distinct ras genes—H-ras, K-ras, and N-ras— have been linked with malignancy, K-ras is the most closely associated with NSCLC.35 K-ras proteins are activated when bound to guanosine triphosphate (GTP) and are inactivated by GTPase-activating protein (GAP), which causes the hydrolysis of GTP to guanosine diphosphate (GDP). K-ras proteins possess intrinsic GTPase activity, but point mutations at codons 12, 13, or 61 in the K-ras oncogene lead to a constitutively active protein production via changes at the GTP-binding domain, which prevents the conversion of GTP to GDP.36
K-ras Mutations and Patient Characteristics K-ras mutations in NSCLC have been described at a varying rate depending on the patient population studied. Overall, K-ras mutations have been reported in 15% to 20% of all patients with NSCLC and approximately 30% to 50% of those with adenocarcinoma histology.36 K-ras mutations are seen almost exclusively in smokers, with one study reporting that 43% of smokers who developed NSCLC exhibited such a mutation versus 0% of life-long nonsmokers with NSCLC (P ⫽ .001). Interestingly, the chance of having a K-ras mutation
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was not affected by the level or timing of tobacco abuse as heavy, light, current, and former smokers who developed NSCLC had statistically similar rates of mutation. There was also a persistent risk for K-ras mutation long after smoking cessation, which at least implies that K-ras mutations occur quite early in the carcinogenic process.37
The Prognostic and Predictive Nature of K-ras Mutations The impact of K-ras mutations on prognosis is likely negative. Several trials have found that K-ras mutations lead to decreased survival,35,38,39 while others have been unable to confirm this,40,41 Due to these disparate results, Mascaux and colleagues performed a meta-analysis that included 28 trials with 3,620 patients and reported worse outcomes for patients with K-ras mutations (HR 1.35; 95% CI, 1.16 –1.56) and particularly for those patients with an adenocarcinoma histology (HR 1.59; 95% CI, 1.26 –2.02).36 As for predictive impact, the possession of a K-ras mutation in NSCLC is associated with decreased response to EGFR TKIs. However, EGFR mutations and K-ras mutations are almost always mutually exclusive and the lack of response to an EGFR TKI is likely more closely linked and better predicted by the lack of an EGFR mutation than the existence of a K-ras mutation.42 In fact, in the exceedingly rare instances in which both an EGFR mutation and K-ras mutation have been reported in the same patient, the response to an EGFR TKI was excellent when that EGFR mutation was an exon 19 deletion—a known activating mutation— but exceedingly poor when that mutation was a T790M mutation.43 While EGFR TKIs clearly have no activity in almost all patients with a K-ras mutation, the response to EGFR monoclonal antibodies such as cetuximab— unlike the colorectal cancer population44—is less clearly impacted in NSCLC when a K-ras mutation is present.45
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stained positive for EGFR by immunohistochemistry, the molecular subset of patients most likely to benefit from cetuximab has not been defined. In contrast to treatment with EGFR TKIs, efficacy after treatment with cetuximab does not appear to correlate with EGFR or KRAS-related biomarkers.48 In the Southwest Oncology Group (SWOG) 0819 trial, cetuximab is now being tested with carboplatin and paclitaxel alone or combined with bevacizumab in newly diagnosed patients with advanced NSCLC. Biomarker studies are included in this trial in an effort to define further the optimal patient subset to treat with this agent.
EML4-ALK The EML4-ALK fusion protein represents one of the newest molecular targets in NSCLC. Anaplastic lymphoma kinase (ALK) was originally identified as part of a chromosomal translocation that is known to be associated with anaplastic large cell lymphoma, which results in a nucelophosmin–ALK fusion product (NPMALK). A number of other translocations resulting in unique ALK fusion proteins also have been identified, and may have differential effects on downstream signaling.49 ALK subsequently has been shown to be dysregulated in both hematologic and solid malignancies by a number of different mechanisms, including translocations, as well as mutations in the tyrosine kinase domain.49
Role in Oncogenesis The EML4-ALK translocation was first identified in 2007 from a surgically resected lung adenocarcinoma specimen.50,51 The fusion product results from an inversion in chromosome 2p, in which the echinoderm microtubule-associated protein-like 4 (EML4) becomes fused to the intracellular kinase domain of ALK. It has been shown that this fusion has clear oncogenic activity, and transgenic mice that express EML4-ALK specifically in lung alveolar cells develop hundreds of adenocarcinoma nodules within weeks.50
CETUXIMAB Cetuximab is an anti-EGFR immunoglobulin G1 monoclonal antibody. In the FLEX (First-Line Erbitux in Lung Cancer) trial, patients with treatment-naive stage IIIB or IV NSCLC were randomized to six cycles of cisplatin and vinorelbine with or without cetuximab. Patients on the cetuximab arm had improved overall survival (HR 0.87; 95% CI, 0.762– 0.996; P ⫽ .044).46 In BMS099, cetuximab was tested in combination with carboplatin and paclitaxel. A comparable improvement in overall survival was seen with the addition of cetuximab to chemotherapy (HR 0.89; 95% CI, 0.754 – 1.051), but the difference was not statistically significant (P ⫽ .169).47 Although the FLEX trial required patients to have tumors with at least one cell that
Clinicopathologic Features of EML4-ALK NSCLC The incidence of EML4-ALK in NSCLC appears to be low, with a frequency of 1% to 7% in reported studies.52 Despite the relatively low frequency, EML4-ALK NSCLC appears to represent a distinct subset of patients. In the largest series to date, NSCLC patients with at least two of the following characteristics were selected for genetic screening: female sex, Asian ethnicity, never/light smoking history, and adenocarcinoma histology.52 A total of 141 tumors were screened, and 19 (13%) patients were found to be EML4-ALK mutant; 31 (22%) were EGFR mutant. Consistent with the previous literature, EML4-ALK and EGFR mutations were mutually
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exclusive. Interestingly, the patients who harbored EML4-ALK translocation were significantly younger and more likely to be men compared to patients with EGFR mutations or to those who were wild-type (WT) for both genes. While EML4-ALK has been reported in smokers, in this particular study, patients with EML4ALK, like EGFR mutants, were more likely to be never/ light smokers when compared to WT patients (P ⬍.001). More studies are needed to define the clinical characteristics.
EML4-ALK as a Therapeutic Target Most importantly, EML4-ALK may potentially serve as a novel therapeutic target in NSCLC. Currently, patients with EML4-ALK appear to have both a similar response rate to platinum-based chemotherapy and overall survival compared to patients who are WT for both EML4-ALK and EGFR. Most recently, a phase I/II trial of the oral ALK inhibitor crizotinib (PF-02341066) has shown promising activity in patients with NSCLC whose tumors harbor the EML4-ALK fusion gene.19 In the phase I part of the study, 250 mg twice daily was established as the maximum tolerated dose (MTD) of this agent. In phase II, a molecularly enriched cohort of 82 EML4-ALK–positive patients was treated at the MTD. The majority of patients in this cohort had tumors with adenocarcinoma histology (96%) and were former (23%) or never smokers (76%). Most patients had received at least one prior regimen and the mean age was 51years. The objective response rate was 57% (95% CI, 46%– 68%) with an overall disease control rate of 87% (95% CI, 77%–93%). The response rate did not differ based on the number of prior regimens (52%, 67%, and 56% for 1, 2, and ⱖ3 prior regimens, respectively). The median duration of treatment was 5.7 months and 72% of patients were progression-free at 6 months. A randomized trial is currently ongoing comparing crizotinib to standard second-line chemotherapy (pemetrexed or docetaxel), as well as the role of crizotinib in the first-line setting for the appropriate population.
MET MET is a receptor tyrosine kinase that is expressed in both normal and malignant cells.53 MET, via signaling through hepatocyte growth factor/scatter factor (HGF/ SF) is important for many cellular processes, including cell proliferation, angiogenesis, invasion, and metastasis.53 MET signaling becomes dysregulated in both NSCLC and small cell lung cancer through a number of different mechanisms, including overexpression, mutations, and amplification, and through autocrine/paracrine activation of HGF.54,55
Potential Role as a Prognostic Marker Prognostic markers such as age, sex, performance status, weight loss, and other comorbidities have pro-
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vided some information in predicting the clinical course of patients with lung cancer. It is clear that these parameters are limited in their clinical utility, and there is some evidence to suggest that MET may be a potential prognostic marker in lung cancer. Increased MET gene copy number as detected by fluorescence in situ hybridization (FISH) may be a poor prognostic factor in patients with resected NSCLC.56 In a study of 447 patients with resected NSCLC, patients whose tumors were MET-positive (ⱖ5 copies/cell) had a shorter overall survival compared to those whose tumors were MET-negative (25.8 v 47.5 months, P ⫽ .005.) Further studies are ongoing to confirm the use of MET amplification as a prognostic marker in lung cancer.
MET-Mediated Therapy There also has been considerable interest in the role MET amplification may play in resistance to therapeutic inhibition with EGFR TKIs. Resistance to therapy is a complex process, and can occur via a number of different mechanisms. In addition to the T790M mutation, MET amplification may lead to acquired resistance to EGFR TKIs through ERBB3 activation of PI3K in up to 20% of patients.57 This finding potentially has important therapeutic implications for patients treated with EGFR TKIs whose tumors do not respond to treatment. A number of MET inhibitors are in early phase clinical trials, and studies looking at strategies for dual inhibition of both EGFR and MET are currently ongoing. ARQ 197 is a novel non–adenosine triphosphate (non-ATP) competitive inhibitor of MET. In a recently reported randomized phase II trial, erlotinib plus ARQ 197 was compared to erlotinib plus placebo in patients with advanced NSCLC who had received at least one prior regimen.58 The primary endpoint was PFS. On the combination arm the median PFS was 16.1 weeks compared to 9.7 weeks in patients who received erlotinib alone (adjusted HR 0.68; 95% CI, 0.47– 0.98; P ⬍.05). Although there was no difference in overall survival (median 36.6 weeks v 29.4 weeks, combination v erlotinib alone, adjusted HR 0.88; P ⫽ 0.52), when analyzed by histology, patients on the combination arm with nonsquamous tumors had a significantly prolonged median PFS (18.9 v 9.7 weeks, adjusted HR 0.61; P ⬍.05) and overall survival (43.1 v 29.4 weeks, adjusted HR 0.58; P ⬍.05). PFS also was prolonged significantly in patients with K-ras mutant tumors (unadjusted HR 0.18), with a trend for improved PFS in patients with wild-type EGFR (unadjusted HR 0.70). Crossover was allowed on the placebo arm. Of the 34 patients who did crossover to the combination, 23 were evaluable for response. Two patients achieved a partial response and nine had stable disease. In the two patients who achieved a partial response, one had a tumor with an EGFR mutation and both had
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tumors that were K-ras wild-type with amplification of MET.58
ETHNICITY AND LUNG CANCER While lung cancer remains a leading cause of mortality for all races, recent research has focused on ethnic variations in this disease. One of the most striking disparities seen is the difference in lung cancer risk and survival for African Americans. When compared to their Caucasian counterparts, African Americans have a significantly higher risk of developing and dying from lung cancer. Epidemiologic research has focused on behavioral, cultural, and socioeconomic factors that may influence risk, although no clear link has been established.59
Molecular Heterogeneity and Race As the heterogeneity of lung cancer becomes more clearly defined on a molecular level, understanding the relationship that race may have with this variation has become more critical than ever. A number of studies have investigated the role that genetic susceptibility may play in lung cancer carcinogenesis. Previous studies have focused on race-specific genetic polymorphisms involved in the modulation of tobacco smoke carcinogens, although results have not been consistent.59 Additionally, polymorphisms in TP53 that have been associated with an increased lung cancer risk are seen in African Americans at a higher frequency when compared to other races.60 A recent analysis of MET and EGFR mutations in NSCLC patients also highlights clear ethnic variation.61 In a study comparing 141 Asian, 76 Caucasian, and 66 African American lung cancer patients, the incidence of MET mutations was highest in the Asian population, at 13%, and no MET mutations were detected in African Americans.61 The most common mutation detected was N375S, which represents a mutation in the semaphorin domain that appears to confer resistance to MET inhibition. It is well established that the frequency of EGFR mutations is higher in East Asian populations, which has important therapeutic implications. The differential frequency of MET mutations highlights the need for larger population-based studies to better characterize this disease.
CBL CBL is an E3 ubiquitin ligase that regulates receptor tyrosine kinase (RTK) activity in a number of different receptors, including MET, EGFR, platelet-derived growth factor receptor (PDGFR), and vascular endothelial growth factor receptor (VEGFR). It is expressed in both normal and malignant tissues, with the highest levels seen in hematopoietic tissues.62
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Function and Role in Oncogenesis CBL is required for ubiquitination and degradation of RTKs.63 However, in addition to its role as a regulator of RTK signaling, evidence suggests that CBL may have a dual role as a signal transduction molecule. Recent data show that alterations in the CBL gene occur in NSCLC, and this pathway may play a critical role in lung cancer carcinogenesis. Tumor samples from 119 patients with NSCLC were analyzed for CBL mutations.64 Eight novel mutations in CBL were identified with specific mutations being confined to a particular ethnicity (the study included Caucasian, African American, and Taiwanese patients). Additionally, 22% of patients demonstrated a loss of heterozygosity (LOH) at the CBL locus. In vitro data showed that these alterations in CBL resulted in increased cell viability and motility but did not alter the E3 ubiquitin ligase activity of CBL. The Taiwanese cohort (50 patients) was also analyzed for EGFR, MET, P53, and K-ras mutations. Interestingly, EGFR and MET mutations were not mutually exclusive with CBL mutations, although there was no overlap with P53 or K-ras mutations. Taken together, these results suggest that CBL may contribute to oncogenesis independent of its role as a regulator of kinase activity. Future studies are needed to define further the key components of this pathway, and the potential for therapeutic intervention.
COX-2 Overexpression of cyclooxygenase-2 (COX-2) is common in NSCLC.65,66 In preclinical models, inhibition of COX-2 has been shown to inhibit lung cancer growth.67 In Cancer and Leukemia Group B (CALGB) 30203, 140 treatment-naive patients with advanced NSCLC were randomized to treatment with carboplatin and gemcitabine with the COX-2 inhibitor, celecoxib, a 5-lipoxygenase (5-LOX) inhibitor, zileuton, or both.68 Tissue was obtained from 76% of the patients (107 of 140 patients). COX-2 expression by immunohistochemistry was shown to be a negative prognostic marker for survival (HR 2.51; P ⫽ .019). Although there was no difference in overall survival between the treatment arms, patients who had tumors with increased COX-2 expression had improved overall survival (HR 0.342; P ⫽ .005) if they received celecoxib. CALGB 30801 is an ongoing randomized trial investigating the role of COX-2 inhibition in patients with COX-2– expressing advanced NSCLC.
GENE EXPRESSION PROFILING Developments in gene expression profiling now allow for the measurement of the expression of thousands of genes simultaneously through a microarray platform. This technology has afforded researchers the ability to identify unique profiles that may be associated
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with high or low risk of disease progression. Information derived from these signatures may be applicable in a number of different clinical settings, and may be used to guide treatment decisions in the future. Several profiles have been developed that have been shown to risk stratify patients with NSCLC.69,70 In a study of 101 tumor samples from patients with resected NSCLC, a five-gene signature was used to identify patients as either high or low risk of an adverse outcome (relapse or death from lung cancer). With a median follow-up of 20 months, 59 patients who were identified as having high-risk disease had a significantly shorter survival (20 months v 40 months, P ⬍.001) when compared to those who had a low-risk signature. This association with survival was confirmed in the validation cohort of 60 patients. Although this technology has the potential to guide treatment decision-making in the future, validation of gene signatures as either prognostic or predictive biomarkers in prospective clinical trials is required.
CIRCULATING TUMOR CELLS It is has been well established that epithelial cells, presumed to be tumor cells, can be detected in the blood of patients with metastatic cancer.71 Methods that have been used previously to detect circulating tumor cells (CTCs) isolated low numbers of cells, which limited their use for subsequent molecular analyses. A recent development has been the use of the CTC-chip, a microfluidic device that isolates and analyzes CTCs with a high purity.72 Using microchip technology, the CTC-chip separates viable CTCs in peripheral blood samples by binding to microposts coated with an epithelial-cell adhesion molecule (EpCAM). Genomic DNA from CTCs collected using this method has been used for mutational analyses. In a study of 27 patients with metastatic NSCLC, EGFR mutational analysis was performed on DNA collected from CTCs. In 12 patients who were known to have a primary tumor with an EGFR mutation, detection of the EGFR mutation was more sensitive in the circulating tumor cells (11 of 12 patients) than in the plasma (four of 12 patients).73 Interestingly, the T790M mutation was seen not only in patients who had prior therapy with an EGFR TKI, but it was detected also in pretreatment biopsy specimens. The presence of this mutation pretreatment was significantly associated with a decrease in PFS (7.7 v 16.5 months, P ⬍.001). Additionally, the types of mutations seen in the CTCs changed throughout a patient’s clinical course; for example, while the T790M mutation was detected at low levels in the initial CTC specimen, it appeared to increase in frequency over time, correlating clinically with drug resistance.73 This technology represents a notable advance for a number of reasons. Available tissue is often limited, and
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this technique allows for reliable tumor genotyping with a less invasive procedure. Additionally, it allows for real time tumor genotyping, as it is clear that the genomic profile of a patient’s original tumor may change throughout the course of therapy. CTCs also may be used to identify other predictive biomarkers that can help clinicians further tailor treatment recommendations. In a recent series, CTCs from 75 patients with late-stage NSCLC were analyzed for ERCC1 mRNA expression using real-time reverse transcriptase–polymerase chain reaction (RT-PCR).74 In this series, ERCC1 expression levels in CTCs were significantly correlated with response (P ⫽ .003) to platinum-based chemotherapy.
CONCLUSIONS While the goal of delivering personalized therapy for patients with lung cancer is certainly not new, clinicians now have more factors to consider that can help guide therapeutic decision-making. In addition to considering traditional factors such as a patient’s performance status, gender and age, tumor histology has now been established as an important predictive factor for both toxicity and efficacy to available therapeutics. Accurate diagnosis of tumor histology is, therefore, essential. A case now can be made also to test for specific molecular biomarkers in selected patients with advanced adenocarcinoma of the lung, including EGFR mutations and possibly EML4-ALK translocations in newly diagnosed and previously treated never (⬍100 cigarettes in a lifetime) or former (stopped ⱖ15 years ago) light (smoked ⱕ10 pack years) smokers, respectively. K-ras mutation status also may be used to predict resistance to EGFR TKIs in patients with adenocarcinoma of the lung, although the association between K-ras mutations and survival after treatment with these agents has not been established. In the future, additional molecular biomarkers may be incorporated into the diagnostic algorithm, including MET, CBL, and COX-2. Adequate tissue is often the limiting factor in establishing biomarker status, which may be overcome through genotyping of CTCs. The treatment of lung cancer remains complex and challenging. While significant advances have been made, much remains unknown about the molecular biology of this disease. Novel approaches to diagnosis and therapy are needed, and the findings reviewed here represent clear promise for the future.
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