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ORIGINAL ARTICLE

EGFR Mutations Detected in Plasma Are Associated with Patient Outcomes in Erlotinib Plus Docetaxel-Treated Non-small Cell Lung Cancer Philip C. Mack, PhD, William S. Holland, MS, Rebekah A. Burich, MS, Randeep Sangha, MD, Leslie J. Solis, MS, Yueju Li, MA, Laurel A. Beckett, PhD, Primo N. Lara, Jr., MD, Angela M. Davies, MD, and David R. Gandara, MD

Purpose: Activating mutations in the epidermal growth factor receptor (EGFR) are associated with enhanced response to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC), whereas KRAS mutations translate into poor patient outcomes. We hypothesized that analysis of plasma for EGFR and KRAS mutations from shed tumor DNA would have clinical utility. Methods: An allele-specific polymerase chain reaction assay using Scorpion-amplification refractory mutation system (DxS, Ltd) was used to detect mutations in plasma DNA from patients with advanced stage NSCLC treated as second- or third-line therapy on a phase I/II trial of docetaxel plus intercalated erlotinib. Results: EGFR mutations were detected in 10 of 49 patients (20%). Six (12%) had single activating mutations in EGFR, associated with improved progression-free survival (median, 18.3 months), compared with all other patients (median, 3.9 months; p ⫽ 0.008), or those with wild-type EGFR (median, 4.0 months; p ⫽ 0.012). Four of 49 patients harbored a de novo T790M resistance mutation (median progressionfree survival, 3.9 months). EGFR mutational status was associated with clinical response (45 assessable, p ⫽ 0.0001); in the six patients with activating mutations, all achieved complete (33%) or partial (67%) response. All CR patients had E19del detectable in both tumor and plasma. KRAS mutations were detected in two of 49 (4%) patients, both of whom had rapid progressive disease. Conclusions: Activating EGFR mutations detected in shed DNA in plasma are significantly associated with favorable outcomes in patients with advanced NSCLC receiving docetaxel plus intercalated erlotinib. The addition of docetaxel in this schedule did not diminish the efficacy of erlotinib against patients with EGFR activating mutations. University of California, Davis Cancer Center, Sacramento, California. Disclosure: Dr. Davies is a current employee of OSI Pharmaceuticals. The other authors declare no conflicts of interest. This data was presented at the 2008 American Society of Clinical Oncology Meeting. Address for correspondence: Philip C. Mack, PhD, Division of Hematology and Oncology, Suite 3016, UC Davis Cancer Center, 4501 X Street, Suite 3016, Sacramento, CA 95817. E-mail: pcmack@ucdavis.edu Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.jto.org). Copyright © 2009 by the International Association for the Study of Lung Cancer ISSN: 1556-0864/09/0412-0001

Key Words: EGFR, NSCLC, Erlotinib, Plasma. (J Thorac Oncol. 2009;4: 000–000)

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ung cancer is the leading cause of cancer-related death in the United States.1 A current target for therapeutics is the epidermal growth factor receptor (EGFR). Commonly expressed in non-small cell lung cancer (NSCLC), EGFR abnormalities may lead to constitutive signaling through downstream tyrosine kinase pathways involved in cell proliferation and survival. Small molecule inhibitors of EGFR such as gefitinib and erlotinib have been introduced into the clinic. Large randomized phase III trials testing these agents in unselected patients have demonstrated mixed results.2– 8 Experience with EGFR tyrosine kinase inhibitor (TKI) therapy has led to the recognition of clinical characteristics associated with higher likelihood of response, notably Asian patients, females, never-smokers, and adenocarcinoma histology.8 –11 However, the presence of certain activating EGFR mutations seems to be a better predictor of response. Tumor molecular analyses including EGFR protein expression, gene copy number, tyrosine kinase domain mutations, and polymorphisms have also been reported as potential predictive biomarkers.2,12–18 Additional studies have indicated that mutated KRAS is associated with poor outcomes after therapy with these agents.19,20 Unfortunately, there is often a lack of sufficient tumor material available for biomarker analysis, including EGFR mutation status, in patients with advanced stage NSCLC. However, shed tumor DNA circulating in patient plasma may provide a viable alternative for identification of tumor mutations and other alterations.21 Patients with advanced cancer generally have higher plasma concentrations of circulating DNA,22,23 which has been shown to contain tumor-specific alterations.24,25 However, conventional assays may lack the requisite sensitivity to fully translate this concept into clinical utility.26 Newer methodologies such as Scorpion probes have been used in combination with allele-specific amplification (amplification refractory mutation system 关ARMS兴) in fluorescence-based real-time PCR offer the potential for increased sensitivity in detecting circulating DNA mutations.27–29 Using this technique, as a follow-up, we report detection of EGFR and

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KRAS mutations in plasma from patients with NSCLC receiving erlotinib-based therapy, with significant associations observed between the presence of mutations and clinical outcomes.30

MATERIALS AND METHODS Blood Sample Collection and DNA Extraction Whole blood specimens were collected immediately before treatment, in ethylenediaminetetraacetic acid-tubes and centrifuged at 1000 rpm for 15 minutes. DNA was extracted from 0.5 to 2.0 mL of plasma using QIAamp DNA Blood mini kit (Qiagen, Valencia, CA). Estimated final DNA concentrations generally ranged from 1 to 9 ng/␮l, with an average of 2.3 ng/␮l.

Tissue Specimens All slides were H&E stained and underwent histologic review for tumor content by a certified pathologist. Slides were deparaffinized by xylene and dehydrated by 100% ethanol. DNA was extracted using the QIAamp DNA mini kit (Qiagen).

PCR and Direct Sequencing for EGFR To detect mutations in EGFR exons 19, 20, and 21, a nested PCR assay was used. External primer sequences for each exon have been previously published.16 Additional primers and methodology are provided in Supplementary Material (see Supplementary Digital Content 1, http://links.lww.com/JTO/A11.

Scorpion-ARMS for EGFR and KRAS Mutations EGFR and KRAS Scorpion test kits (DxS Ltd, Manchester, UK), were used to detect mutations in DNA using real-time PCR following the manufacturer’s protocol. Additional methodology and analyses are provided in Supplementary Material (see Supplementary Digital Content 1, http://links.lww.com/JTO/A11).

TABLE 1. Baseline Patient and Disease Characteristics of Advanced Patients with Non-small Cell Lung Cancer Treated with Docetaxel and Erlotinib No. of Patients (N ⴝ 48)

%

7 12 29

14.6 25.0 60.4

59 34–79 31 17

64.6 35.4

27 21

56.3 43.7

16 32

33.3 66.7

24 24

50.0 50.0

2 44 2

4.2 91.6 4.2

32 7 9

66.6 14.6 18.8

Treatment arma Phase I: arm A Phase I: arm B Phase II Characteristic Age, yr Median Range Age ⬍65 yr Age ⱖ65 yr Sex Female Male Smoking status Never-smoker Former/current smoker ECOG performance status 0 ⱖ1 Race African descent White East/South East Asian Histological type Adenocarcinoma Squamous cell carcinoma Other: NSCLC, NOS

a Phase I, Arm A: Docetaxel 70 mg day 1 and erlotinib 600 – 800 mg days 2, 9, and 16 on a 21-d cycle. Phase I, Arm B: Docetaxel 70 –75 mg day 1 and erolotinib 150 –300 mg days 2–16 on a 21-d cycle. Phase II: Docetaxel 70 mg day 1 and erlotinib 200 mg days 2–16 on a 21-d cycle. ECOG, Eastern Cooperative Oncology Group; NSCLC, non-small cell lung cancer; NOS, not otherwise specified.

Statistical Analysis Survival distributions were summarized with KaplanMeier plots and differences were assessed with the log-rank test. A two-sided Fisher exact test was used to assess differences in patient response versus mutation status.

institutional review boards of participating institutions. All patients gave informed consent and the trial was conducted with adherence to the Helsinki Declaration. Patient characteristics for the entire cohort are summarized in Table 1.

Patients and Clinical Trial Methodology The phase I/II trial of docetaxel plus intercalated erlotinib that forms the basis for this correlative science study stemmed from preclinical data suggesting that pharmacodynamic separation of these two agents resulted in superior antitumor activity, as previously reported.30 –33 The phase I portion consisted of two arms conducted simultaneously with docetaxel given every 21 days (70 –75 mg/m2 intravenously) to a maximum of six cycles. Arm B, where erlotinib was administered on days 2 to 16 (150 –300 mg), was chosen for further study in a phase II expansion, enrolling patients with previously treated stages IIIB and IV NSCLC. Forty-nine patients with cytologically or histologically defined NSCLC from either the phase I arms or the phase II study were analyzed here. This study was approved by the respective

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RESULTS In Vitro Evaluation of Scorpion-ARMS for Detection of EGFR and KRAS Mutations The sensitivity and specificity of Scorpions-ARMS was tested using serial dilutions of EGFR-mutant, KRAS-mutant, and wild-type (WT) NSCLC cell lines (Figure 1A, B). The scorpions approach was able to detect each mutation at percentages as low as 0.1% of total DNA content. In contrast, detection by direct sequencing was negligible at 10% (Figure 1C). No false positive signals were observed for any probe in this setting.

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FIGURE 1. Scorpion-amplification refractory mutation system (ARMS) assay sensitivity. Non-small cell lung cancer (NSCLC) lines harboring epidermal growth factor receptor (EGFR) and KRAS mutations were serially diluted and analyzed by ScorpionARMS. A, Genomic DNA was extracted from H1975 cells (mutations at L858R and T790M), H1650 (del746 –750 mutation in exon 19), and A549 cells (wild type 关WT兴 EGFR). DNA from the EGFR-mutant cell lines was diluted with DNA from A549 at the following ratios: 0, 0.5, 0.33, 0.1, 0.01, and 0.001. B, A dilution series for the KRAS codon 12 GGT3 AGT using DNA from A549 cells and H460 cells (WT at codon 12). C, Comparison between Scorpion-ARMS and DNA sequencing for the L858R mutation.

EGFR Activating Mutations Are Predictive of Response to Docetaxel Plus Erlotinib The Scorpions-ARMS approach was used to detect EGFR and KRAS mutations in plasma and tumor specimens from patients with NSCLC enrolled on a phase I/II clinical trial of docetaxel plus intermittent erlotinib. Archival paraffin blocks or slides with analyzable material were available from 14 patients, whereas fresh frozen plasma specimens were available from all 49 patients with NSCLC in this cohort. The absence or presence of mutations was confirmed in an independent run, and for analysis of plasma—from a separate aliquot. A patient was considered positive if a mutation was detected either in plasma or in tumor. Twelve EGFR mutations were detected in 10 patients; whereas KRAS mutations were detected in two patients (Table 2). Of the 10 patients that tested positive for EGFR mutations, six were histologically classified as adenocarcinoma, one as squamous cell carcinoma, and three as not otherwise specified. The KRAS and EGFR mutations were exclusive. Four

patients were positive for the exon 20 T790M mutation, associated with resistance to erlotinib; two of which also harbored additional EGFR activating mutations. Representative examples of EGFR and KRAS mutations are shown in Figure 2. Tumor mutational status was compared with patient response to treatment (Figure 3). Both complete responders harbored exon 19 deletions, readily detectable in both tumor and plasma (Figure 2A). Of the patients with PR as best response, four of 10 harbored activating mutations (two in exon 19 and two in exon 21). All four patients with T790M mutations had stable disease as best response. No activating mutations were detected in any of the 13 patients with progressive disease; however, in two of these patients, codon 12 KRAS mutations were observed. Patients with activating mutations (absent the T790M resistant mutation) were significantly more likely to respond to treatment (p ⫽ 0.0002). Table 2 shows individual characteristics for patients with either an EGFR or a KRAS mutation.

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TABLE 2. Demographics and Tumor Histology of Patients Positive for EGFR or KRAS Mutations Patient ID 25 76 11 52 57 66 23 8 63 19 59 28

Mutation

Response

Smoking History

Age

Sex

Histology

Detected in

del746-750 del746-750 L858R del746-750 L858R del746-750 del and T790M L858R& T790M T790M T790M KRAS KRAS

CR CR PR PR PR PR SD SD SD SD PD PD

Ex, 10 pack/yr Ex, 2 packs/yr Never Never Never Current, 30 pack/yr Ex, 40 pack/yr Unknown Ex, 50 pack/yr Ex Never Unknown

47 61 44 45 67 47 66 54 73 70 38 52

F F M M F F F F F M M M

Adeno NOS Adeno Adeno NOS Squam Adeno Adeno Adeno NOS NOS Adeno

P, T P, T P T P P, T T P P, T P P P, T

For patients 11, 57, 8, 19, and 59, only plasma was available for analysis. CR, complete response; PR, partial response; SD, stable disease; PD, progressive disease; P, plasma; T, tumor.

FIGURE 2. Representative examples of patient specimens positive for EGFR or KRAS mutations. A, Two patients with E19del identified in both plasma and tumor DNA. Both patients had complete response to therapy. B, A patient specimen with de novo positivity for both E19del and T790M. C, A patient with a KRAS mutation identified in both plasma and tumor.

EGFR Activating Mutations Are Associated with Improved PFS Following Treatment with Docetaxel and Intermittent Erlotinib The relationship between the EGFR/KRAS mutation status and patient overall survival (OS) and progression-free survival (PFS) times was investigated. For all 49 patients, the median PFS was 5.4 months and the median OS was 18.8 months. Patients were stratified into subgroups based on the

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following mutational genotypes: (1) positive for a single EGFR activating mutation in exon 19 or 21 (E19del or L858R), (2) WT EGFR, (3) presence of an exon 20 T790M resistance mutation, and (4) positive for a KRAS codon 12 or 13 mutation. The PFS for each of these groups is graphed in Figure 4A. Patients with activating mutations, absent a resistance factor, had a favorable median PFS of 18.3 months (n ⫽ 6). Patients harboring a T790M mutation had a median PFS

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FIGURE 3. Relationship between mutation-positivity and patient response. Clinical response data, as determined using RECIST criteria, was available for 45 patients. Those positive for activating mutations (E19del or L858R) were significantly associated with response to therapy (p ⫽ 0.0002) (labeled green). Four specimens were positive for T790M mutations, two of which also contained activating mutations (labeled yellow), occurring in patients with SD. Two KRAS mutations were identified in patients with progressive disease (labeled red).

of 3.9 months (n ⫽ 4), comparable with the 4.0 month PFS of those with WT EGFR (n ⫽ 37). Both patients with KRAS mutations had poor outcomes, with a median PFS of less than 1.5 months. Patients with single EGFR activating mutations (i.e., absent a resistance factor) had a significantly improved PFS when compared with patients with WT EGFR (p ⫽ 0.012), or when compared with all other patients (p ⫽ 0.0079). Patients with EGFR activating mutations had a numerically superior OS (median of 39.6 months); however, it was not significantly superior to that of patients with WT EGFR or T790M (17.8 and 24.2 months, respectively; Figure 4B). The two patients harboring KRAS mutations performed poorly, with a median OS of 2.5 months.

Predictive Utility of Plasma Analysis Seven of the 12 patients who were positive for an EGFR or KRAS mutation had tissue available for analysis (Table 2). All cases that were positive for mutations in the plasma were also positive in the tumor specimen. However, two patients had mutations detectable in tumor tissue that were not observed in plasma. When considered without the tumor data, plasma-only analysis remained a significant predictor of patient outcome. Those with plasma-detected mutations in exons 19 and 21 (absent a T790M resistance mutation) had significantly improved PFS compared with patients with WT EGFR (p ⫽ 0.0122) or compared with WT EGFR and T790M-postive patients combined (p ⫽ 0.0088). The association between response rate and activating mutations detected in plasma also remained significant using the dichotomization presented in Figure 3.

DISCUSSION Here, we report the association of EGFR and KRAS mutations in plasma using Scorpion-ARMS with patient out-

FIGURE 4. A, EGFR activating mutations correlate with enhanced progression-free survival (PFS). Kaplan-Meier plot showing PFS of 49 patients stratified by mutation type. Patients with EGFR activating mutations (E19del or L858R) had significantly improved PFS compared with patients with WT EGFR or those harboring a resistance mutation (T790M or KRAS). B, Overall survival by mutation type. Kaplan-Meier plot showing overall survival of 49 patients stratified by mutation type.

comes after erlotinib-based treatment. The presence of EGFR activating mutations correlated significantly with improved tumor response and PFS, but not OS, in patients treated with docetaxel and intercalated erlotinib.30 –33

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In the studies described here, we directed our attention toward detection of mutations of KRAS and the EGFR tyrosine kinase domain. Because tumor tissue was not available for molecular analysis on most patients, we hypothesized that utilization of the recently described Scorpions technology for analysis of plasma DNA would aid in interpretation of the clinical results. Six of 49 patients with NSCLC enrolled on either the phase I or the phase II portion of this trial had activating EGFR mutations in exons 19 or 21, all of whom responded to treatment and as a group had significantly extended PFS. Two of two complete responders harbored exon 19 deletions that were readily detectable both in tissue and in plasma. Unexpectedly, four patients were positive de novo for T790M mutations in exon 20, previously associated with resistance to EGFR TKIs. These patients all had stable disease as best response and did not differ significantly in survival from patients with WT EGFR. KRAS mutations were detected in only two of the 49 patients; however, both of these patients performed poorly, with a mean OS of only 2.5 months. Seven of 12 patients who were positive for an EGFR or KRAS mutation had tissue available for analysis (Table 2). No cases were plasma positive and tumor negative, consistent with absence of false positivity for plasma detection in our study. However, two patients had mutations detectable in tumor tissue that were not observed in plasma, perhaps related to low overall tumor volume. Overall, these data suggest that analysis of shed tumor DNA in plasma can supplement or even replace tumor tissue analysis, adding significant predictive utility when archival specimens are not available. Our data, conducted in a U.S. population treated with an erlotinib-based therapy and inclusive of KRAS and T790M mutation analysis, expand on previous observations regarding use of Scorpion-ARMS to detect EGFR mutations in plasma DNA. Kimura et al.28 detected EGFR mutations in circulating DNA from seven of 42 Japanese patients treated with gefitinib. They noted significant correlations between mutation positivity with patient response and with PFS. A study by Kuang et al.34 evaluating 54 select patients from a U.S. cohort previously treated with EGFR TKIs, found that 70% of patients with EGFR mutations in their tumors also had mutations in their shed DNA in plasma. Using a microfluidics-based digital PCR design to detect EGFR mutations in 35 patients with NSCLC with paired plasma and tumor specimens, Yung et al.35 found mutations in the plasma of 11 of 12 (92%) patients who were tumor-positive for EGFR mutations. Maheswaran et al.36 recently reported on the use of Scorpion-ARMS on circulating lung cancer cells for mutation detection, identifying mutations in 11 of 12 patients known previously to be mutation positive. Similar to our results, these investigators identified a higher than expected number of patients with pretreatment T790M mutations concurrent with an activating mutation. However, none of our T790Mpositive patients had a dramatic response; whereas two of six of theirs did, although the responses were not durable. Unlike archival tissue specimens, collection of patient plasma is easily standardized, limiting variability from han-

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dling and is not subject to formalin-induced artifacts. In addition, serial analysis over time can be performed to look for treatment-related changes, such as resolution of a previously observed plasma EGFR mutation under the influence of erlotinib. However, a limitation of plasma analysis is the potential interpatient variability in the ratio of tumor to WT circulating DNA. In summary, we found that the presence of EGFR activating mutations identified in circulating DNA in plasma, correlated significantly with improved tumor response, and PFS in patients treated with docetaxel plus intermittent erlotinib. The use of docetaxel in this intercalated schedule did not seem to diminish activity of erlotinib in patients harboring EGFR mutations, nor alter the ability to detect favorable patient outcomes associated with such activating mutations. Indeed, a substantial number of patients with no evidence of EGFR activating mutation showed clinical benefit from this treatment paradigm. We conclude that, in the absence of tumor tissue, detection of EGFR and KRAS mutations in cell-free plasma DNA using Scorpion-ARMS is a viable strategy with predictive utility.

ACKNOWLEDGMENTS Supported by The Hope Foundation, P30 CA93373, SanofiAventis, and the Bonnie J. Addario Lung Cancer Foundation. The authors are grateful for the assistance of Nichole Farneth and Natasha Perkins for data and specimen collection. REFERENCES 1. Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, 2007. CA Cancer J Clin 2007;57:43– 66. 2. Tsao MS, Sakurada A, Cutz JC, et al. Erlotinib in lung cancermolecular and clinical predictors of outcome. N Engl J Med 2005; 353:133–144. 3. Thatcher N, Chang A, Parikh P, et al. Gefitinib plus best supportive care in previously treated patients with refractory advanced non-small-cell lung cancer: results from a randomised, placebo-controlled, multicentre study (Iressa Survival Evaluation in Lung Cancer). Lancet 2005;366: 1527–1537. 4. Giaccone G, Herbst RS, Manegold C, et al. Gefitinib in combination with gemcitabine and cisplatin in advanced non-small-cell lung cancer: a phase III trial—INTACT 1. J Clin Oncol 2004;22:777–784. 5. Herbst RS, Giaccone G, Schiller JH, et al. Gefitinib in combination with paclitaxel and carboplatin in advanced non-small-cell lung cancer: a phase III trial–INTACT 2. J Clin Oncol 2004;22:785–794. 6. Herbst RS, Prager D, Hermann R, et al. TRIBUTE: a phase III trial of erlotinib hydrochloride (OSI-774) combined with carboplatin and paclitaxel chemotherapy in advanced non-small-cell lung cancer. J Clin Oncol 2005;23:5892–5899. 7. Gatzemeier U, Pluzanska A, Szczesna A, et al. Phase III study of erlotinib in combination with cisplatin and gemcitabine in advanced non-small-cell lung cancer: the Tarceva Lung Cancer Investigation Trial. J Clin Oncol 2007;25:1545–1552. 8. Shepherd FA, Rodrigues Pereira J, Ciuleanu T, et al.; National Cancer Institute of Canada Clinical Trials Group. Erlotinib in previously treated non-small-cell lung cancer. N Engl J Med 2005;353:123–132. 9. Fukuoka M, Yano S, Giaccone G, et al. Multi-institutional randomized phase II trial of gefitinib for previously treated patients with advanced non-small-cell lung cancer (The IDEAL 1 Trial) 关corrected兴. J Clin Oncol 2003;21:2237– 46. 10. Miller VA, Kris MG, Shah N, et al. Bronchioloalveolar pathologic subtype and smoking history predict sensitivity to gefitinib in advanced non-small-cell lung cancer. J Clin Oncol 2004;22:1103–1109. 11. Kris MG, Natale RB, Herbst RS, et al. Efficacy of gefitinib, an inhibitor of the epidermal growth factor receptor tyrosine kinase, in symptomatic

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