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Imaging, Diagnosis, Prognosis |
Authors' Affiliations: Departments of 1 Thoracic/Head and Neck Medical Oncology, 2 Biostatistics and Applied Mathematics, and 3 Pathology, The University of Texas M. D. Anderson Cancer Center, Houston, Texas and 4 University of Colorado Health Sciences Center, Cancer Center, Aurora, Colorado
Requests for reprints: Ignacio I. Wistuba, Department of Pathology, M. D. Anderson Cancer Center, Unit 85, 1515 Holcombe Boulevard, Houston, TX 77030-4009. Phone: 713-563-9184; Fax: 713-563-1848; E-mail: iiwistuba{at}mdanderson.org.
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Abstract |
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Experimental Design: Patients with advanced NSCLC treated with EGFR-TKI with available archival tissue specimens were included. EGFR and KRAS mutations were analyzed using PCR-based sequencing. EGFR copy number was analyzed using fluorescence in situ hybridization.
Results: The study included 73 patients, 59 of whom had all three potential markers successfully analyzed. EGFR mutation was detected in 7 of 71 patients (9.8%), increased EGFR copy number in 32 of 59 (54.2%), and KRAS mutation in 16 of 70 (22.8%). EGFR mutation (P < 0.0001) but not increased EGFR copy number (P = 0.48) correlated with favorable response. No survival benefit was detected in patients with either of these features. KRAS mutation correlated with progressive disease (P = 0.04) and shorter median time to progression (P = 0.0025) but not with survival. Patients with both EGFR mutation and increased EGFR copy number had a >99.7% chance of objective response, whereas patients with KRAS mutation with or without increased EGFR copy number had a >96.5% chance of disease progression.
Conclusion: KRAS mutation should be included as indicator of resistance in the panel of markers used to predict response to EGFR-TKIs in NSCLC.
10% of patients with NSCLC that has progressed with prior chemotherapy (2–6). In patients with NSCLC who benefit from gefitinib or erlotinib, the responses can be dramatic and may last for longer than a year (2–6). Several markers have been identified that predict response to the EGFR-specific tyrosine kinase inhibitors (EGFR-TKI) in patients with NSCLC. Activating mutations in the EGFR tyrosine kinase domain (exons 18-21), increased EGFR copy number, and increased EGFR protein expression have been associated with favorable response to EGFR-TKIs (7–17). In contrast, KRAS gene mutation, which occurs in 20% to 30% of NSCLCs, mainly in adenocarcinomas (30%) and smokers (18), has been reported to be associated with poor response to EGFR-TKIs (19–23).
Studies have also investigated potential markers of survival in patients with NSCLC treated with EGFR-TKIs. Whether activating mutations in the EGFR tyrosine kinase domain are associated with a survival advantage from gefitinib or erlotinib, especially in Western populations with NSCLC, is controversial. Several retrospective studies showed prolonged survival in gefitinib-treated patients with tyrosine kinase activating mutations, mostly in Asian populations (8–14), whereas in the BR.21 study, the hazard ratio for death was almost identical in patients with mutated and wild-type EGFR (0.73 and 0.77, respectively; ref. 16). EGFR increased copy number has been shown to predict favorable survival outcomes after EGFR-TKI therapy (7, 13, 15, 17).
Several studies have shown that EGFR mutation and KRAS mutation are mutually exclusive (19, 24, 25), and EGFR mutation and genomic gain are associated (7), but the relationship between increased EGFR copy number and KRAS mutation and the effect of this combination on response to EGFR-TKI therapy have not yet been investigated. The purpose of this retrospective study was to investigate the concomitant presence of increased EGFR copy number and KRAS mutation in tumor specimens from patients with NSCLC and to clarify the predictive value of combinations of EGFR mutation status, EGFR copy number status, and KRAS mutation status in predicting response and survival in patients with NSCLC treated with EGFR-TKIs.
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Materials and Methods |
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This study was approved by the M. D. Anderson Cancer Center. All specimens were histologically classified according to the WHO classification for lung cancer by an experienced thoracic pathologist (I.I.W.; ref. 26). Imaging studies were assessed by a medical oncologist (E.M.), who graded responses according to the Response Evaluation Criteria in Solid Tumors (27). In case of stable disease, measurements had to meet the stable disease criteria at least once after the first evaluation at a minimum interval of 6 to 8 weeks. All the investigators were blinded to patient outcomes.
EGFR and KRAS mutation analysis. Exons 18 through 21 of EGFR and exon 1 of KRAS were PCR amplified using intron-based primers as previously described (25, 28, 29). From microdissected formalin-fixed, paraffin-embedded cells, 200 cells were used for each PCR amplification, as previously described (29). All PCR products were directly sequenced using the Applied Biosystems PRISM dye terminator cycle sequencing method. All sequence variants were confirmed by independent PCR amplifications from at least two independent microdissections and sequenced in both directions, as previously reported (29).
EGFR copy number analysis. EGFR copy number per cell was investigated using fluorescence in situ hybridization (FISH) done with the LSI EGFR SpectrumOrange/CEP 7 SpectrumGreen probe (Vysis) according to a published protocol (7, 30). Serial 5-µm-thick tissue sections were incubated at 56°C overnight, deparaffinized, and dehydrated in 100% ethanol. After incubation in 2x saline sodium citrate buffer (2x SSC, pH 7.0) at 75°C for 15 to 25 min, sections were digested with proteinase K (0.25 mg/mL in 2x SSC, pH 7.0) at 37°C for 15 to 25 min, rinsed in 2x SSC (pH 7.0) at room temperature for 5 min, and dehydrated using ethanol in increasing concentrations (70%, 85%, and 100%). The EGFR/CEP 7 probe set was applied per the manufacturer's instructions to an area of the slide containing tumor foci, and the hybridization area was covered with a glass coverslip and sealed with rubber cement. The slides were incubated at 80 °C for 8 to 10 min to permit co-denaturation of chromosomal and probe DNA and were then placed in a humidified chamber at 37°C and left for 20 to 24 h to allow hybridization. Post-hybridization washes were done in 1.5 mol/L urea and 0.1x SSC (pH 7.0-7.5) at 45°C for 30 min and in 2x SSC for 2 min at room temperature. After the samples were dehydrated in ethanol, 4',6-diamidino-2-phenylindole (DAPI; 0.3 mg/mL in Vectashield mounting medium, Vector Laboratories) was applied for chromatin counterstaining.
FISH assessment was done independently by two authors (M.V-G. and A.C.X.) who were blinded to the patients' clinical characteristics and all other molecular variables. Patients were classified into six FISH strata, as previously described (7, 30). High polysomy and gene amplification categories were considered to have increased EGFR copy number, and the categories disomy to low polysomy were considered not to have increased gene copy number.
Statistical analysis. Data were summarized using standard descriptive statistics and frequency tabulation. Associations between categorical variables were assessed using cross-tabulation, the 
2 test, and Fisher's exact test. The Kruskal-Wallis test and Wilcoxon rank-sum test were done to assess differences in continuous variables between clinical-pathologic groups. Logistic regression analysis was applied to estimate the effect of covariates on response (complete response + partial response versus other). Time to disease progression (TTP) and overall survival (OS) were measured for each patient from the first day of treatment with gefitinib or erlotinib. Survival curves were estimated using the Kaplan-Meier method. Univariate and multivariate Cox proportional hazards models were applied to assess the effect of covariates on TTP and OS from the first day of TKI therapy.
One of our interests in this research was in addressing the following question: what is the probability that the response rate in the ith group is greater than the response rates in all other groups? Bayesian methods provide a natural framework to address the above question. Bayesian methods, unlike classic methods, treat the probability of response as a quantity about which the investigator has some degree of uncertainty. This uncertainty is quantified directly via probability. We assume that the response data for each group follow a binomial distribution. The probability of response in the ith group is denoted by Pi. We also assume that the non-informative prior distribution for Pi follows a non-informative ß(0.5,0.5) distribution (for all i). Given these assumptions, we now calculate the posterior probability
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All computations were carried out in SAS or S-plus 2000.
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Results |
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Correlation of EGFR and KRAS abnormalities with patients' clinical and pathologic features. Seventy-one patients were successfully tested for EGFR mutation, 59 for EGFR gene copy number, and 70 for KRAS mutation. Clinical and pathologic characteristics and their correlation with genetic abnormalities are shown in Table 1 .
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KRAS mutation was identified in 16 of the 70 tested patients (22.8%). Fourteen patients had a single-amino-acid substitution in codon 12, and two patients had a codon 13 mutation (Table 2).
None of the tumor samples analyzed harbored concomitant KRAS and EGFR mutation. In the cohort of cases (n = 59) analyzed for all three markers, increased EGFR copy number was detected in 5 of the 6 EGFR mutant cases (83%) and 8 of the 14 KRAS mutant cases (57%).
Correlation of EGFR and KRAS abnormalities with response to EGFR-TKIs. Seven patients (9.6%) had an objective response to TKI therapy (complete response in 1 and partial response in 6); 11 patients (15.1%) had stable disease; and 55 patients (75.3%) had progressive disease. No significant differences in demographic and clinical characteristics were observed between the different response groups (Table 3 ). The presence of EGFR mutation was significantly associated with objective response to TKI treatment (P <.0001; Table 4 ). Four (80%) of the five responders with available FISH data had increased EGFR copy number, but this finding was not statistically significant (Table 4). The presence of KRAS mutation was significantly associated with lack of response to TKI treatment (P = 0.04; Table 4).
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Correlation of EGFR and KRAS abnormalities with survival. With a median follow-up of 3.2 years, 57 patients had died, and 72 had experienced disease progression. A trend toward better OS was observed for the patients with mutant EGFR, but this did not reach statistical significance (log-rank test, P = 0.08; Table 4). In the multivariate analysis for OS, age [hazard ratio (HR), 1.02; P = 0.07], male gender (HR, 1.96; P = 0.01), and performance status 
2 (HR, 1.68; P = 0.07) were important predictors of OS. Median TTP was significantly shorter in the patients with mutated KRAS (log-rank test, P = 0.0025, Table 4). Longer median TTP was also observed in patients with mutated EGFR, but this finding was not statistically significant (Table 4). The multivariable Cox model indicated that, adjusted for age (HR, 1.02; P = 0.04), KRAS mutation (HR, 2.14; P = 0.01) remained a statistically significant predictor of TTP.
The group of patients with both mutated EGFR and increased EGFR copy number had the longest TTP, whereas the group of patients with mutated KRAS had the shortest TTP (log-rank test, P = 0.008). Similar results were obtained when only patients whose tumor specimens were collected before the start of TKI treatment (n = 62) were included in the analysis, and at multivariable Cox model, KRAS mutation remained a strong predictor of poor TTP (HR, 2.45; P = 0.004).
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The frequencies of EGFR (9.8%) and KRAS (22.8%) mutation, the finding that these mutations were mutually exclusive, and the clinical-pathologic characteristics of the patients in our series are similar to previously published data (21). Our findings that EGFR mutations were detected only in adenocarcinomas and were common in Asian patients and never smokers whereas KRAS mutations were identified mostly in adenocarcinomas and were more common in smokers support the notion that there are at least two molecular pathways involved in the pathogenesis of lung adenocarcinoma: a nonsmoking EGFR signaling-associated pathway and a smoking KRAS signaling-associated pathway (31).
Whereas several recent studies have shown that increased EGFR copy number predicts favorable response to and outcome after treatment with EGFR-TKIs in patients with NSCLC (7, 13, 15, 17), KRAS mutation is known to predict poor outcome after treatment with EGFR-TKIs in patients with NSCLC (19–23). However, to our knowledge, our study is the first to show that activating KRAS mutation overcomes the potential favorable role of increased EGFR copy number in predicting response and survival of NSCLC patients after treatment with EGFR-TKIs. In fact, in our study, all 16 patients with KRAS mutations experienced progressive disease as the best response to treatment with EGFR-TKIs, including eight patients whose tumors had increased EGFR copy number. The strong association between the presence of KRAS mutation and poor response to treatment was also observed when the 11 patients whose specimens tested for the three markers were obtained after the start of TKI therapy (median time of 5 months; range, 2-19 months) were excluded.
Our findings that EGFR mutation was a significant predictor of favorable response to EGFR-TKIs (P < 0.0001) and that EGFR mutation was associated with a trend toward longer OS (P = 0.08; Table 4) are in line with the existing literature (7–17, 32). However, in contrast with previous studies, we did not find significant correlation between EGFR copy number and response or outcome in this cohort of patients (15, 17). Interestingly, eight tumors with increased EGFR copy number also carried KRAS mutation and reported poor response rate (Table 5) and TTP (P = 0.008). Conversely, the group of patients with concomitant EGFR mutation and increased EGFR copy number had the longest TTP interval (P = 0.008). This might be due to the association between EGFR mutation and increased EGFR gene copy number previously described by Cappuzzo et al. (7). In fact, among the five EGFR mutant patients who responded to TKI therapy, four also had increased EGFR copy number (two had high polysomy and two had gene amplification). Of the two EGFR mutant patients who did not respond to TKI therapy, one had high polysomy, and the other had low polysomy. Another interesting finding is the absence of EGFR mutations in tumors from patients who had stable disease (Table 4), three of whom had stable disease for more than 1 year. A similar observation was previously reported by other authors (7) and underscores the importance of defining selection criteria to identify which patients are most likely to benefit from EGFR-TKIs.
In summary, our findings indicate that in patients with NSCLC, KRAS mutation is an important predictor of poor response and outcome to EGFR-TKIs despite the concomitant presence of increased EGFR gene copy number and should be included in the panel of markers to be used to predict response to such therapy.
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Footnotes |
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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 12/21/06; revised 2/14/07; accepted 2/21/07.
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