- Email: [email protected]
The identification of KRAS mutations at codon 12 in plasma DNA is not a prognostic factor in advanced non-small cell lung cancer patients
Lung Cancer 72 (2011) 365–369
Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
The identification of KRAS mutations at codon 12 in plasma DNA is not a prognostic factor in advanced non-small cell lung cancer patients Carlos Camps a,b,1 , Eloisa Jantus-Lewintre b,1 , Andrea Cabrera b , Ana Blasco a , Elena Sanmartín b , Sandra Gallach b , Cristina Caballero a , Nieves del Pozo a , Rafael Rosell c , Ricardo Guijarro d , Rafael Sirera b,e,∗ a
Servicio de Oncología Médica, Hospital General Universitario, Valencia, Spain Laboratorio de Oncología Molecular, Fundación Investigación, Hospital General Universitario, Valencia, Spain c Institut Català d’Oncología, Hospital Universitari Germans Trias i Pujol, Badalona, Barcelona, Spain d Servicio de Cirugía Torácica, Hospital General Universitario, Valencia, Spain e Departamento de Biotecnología, Universidad Politécnica de Valencia, Valencia, Spain b
a r t i c l e
i n f o
Article history: Received 30 July 2010 Received in revised form 1 September 2010 Accepted 6 September 2010 Keywords: KRAS Molecular markers Prognostic factors Non-small cell lung cancer (NSCLC)
a b s t r a c t Background: Qualitative analysis of circulating DNA in the blood is a promising non-invasive diagnostic and prognostic tool. Our aim was to study the association between the presence of KRAS mutations at codon 12 and several clinical variables in advanced non-small cell lung cancer (NSCLC) patients. Methods: We examined 308 stage IIIB and IV NSCLC patients who were treated with cisplatin and docetaxel. Blood samples were collected before chemotherapy, and circulating DNA was extracted from the plasma using commercial adsorption columns. The KRAS mutational status was determined by an RT-PCR method that is based on allelic discrimination. Results: The median age of the patients was 60 years [31–80], 84% were male, 98% had a performance status of 0–1 and 84% of the patients were in stage IV. The histological subtypes were as follows: 30% squamous cell carcinoma (SCC), 51% adenocarcinoma (ADC) and 19% others. Of the 277 response-evaluated patients, 1% achieved a complete response (CR), 26% achieved a partial response (PR), 34% had stable disease (SD) and 39% had progressive disease (PD). Additionally, 27 (8.8%) patients had KRAS mutations; 26 had a KRAS codon 12 TGT mutation, and 1 had a codon 12 GTT mutation. Plasmatic KRAS mutations were found in patients presenting SCC or ADC. Patients with KRAS mutations in plasma DNA had a median progression free survival (PFS) of 5.77 months [3.39–8.14], whereas for patients with wild-type (wt) KRAS, the PFS was 5.43 months [4.65–6.22] (p = 0.277). The median overall survival (OS) in KRAS-mutated patients was 9.07 months [4.43–13.70] vs 10.03 months [8.80–11.26] in wt patients (p = 0.514). Conclusions: In advanced NSCLC patients, there were no significant differences between patients with or without KRAS mutations in plasma-free DNA with respect to the baseline characteristics, response rates, PFS or OS. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Due to a low cure rate (6–15%) and the lack of adequate screening measures, lung cancer is the leading cause of cancer death [1]. These facts have motivated the search for new ways to detect, predict, and monitor lung cancer [2–6]. Cancer is a multi-step process involving key cancer-related genes [7,8]; mutations in the KRAS
∗ Corresponding author at: Departamento de Biotecnología, Universidad Politécnica de Valencia, Camino de Vera s/n 46022, Valencia, Spain. Tel.: +34 963 879 556; fax: +34 961 972 151. E-mail addresses: [email protected], sirera [email protected] (R. Sirera). 1 These authors contributed equally to this work. 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.09.005
gene have recently gained attention because KRAS is part of several signaling pathways, and genetic alterations in KRAS might thus lead to tumor development [9]. In fact, KRAS mutations are found in up to 30% of non-small cell lung cancer (NSCLC) tumors, are primarily (90%) in codon 12 [6,10–12], occur early in the development of malignancy and in some cancers have been detected in free DNA in the blood before clinical diagnosis [13]. NSCLC KRAS mutations have been associated with larger tumors, lymph node metastases, and poorer progression and survival [14,15]. Bremnes et al., reviewed the utility of analyzing circulating tumor derived DNA in blood for the development of sensitive molecular biology approaches to analyze gene mutations (such as in KRAS) in these kinds of samples [16]. This technique is of interest because there are reports of correlation between the presence
366
C. Camps et al. / Lung Cancer 72 (2011) 365–369
of KRAS mutations in the serum/plasma DNA and the mutational status of KRAS in tumors [17–23]. Recently, several NSCLC trials demonstrated that KRAS mutations in circulating DNA correlated with survival and response to treatment [19,22,24]; on the other hand, a study conducted by our group failed to demonstrate this prognostic role [23], and another NSCLC study did not reveal any circulating mutant KRAS [25]. Therefore, because there are substantial discrepancies in several studies on KRAS mutational status in both tumor and blood, the goal of this study was to investigate the prognostic significance of the codon 12 KRAS mutations in the plasma DNA from 308 wellcharacterized advanced NSCLC patients. 2. Patients and methods 2.1. Patients This study was performed retrospectively with blood samples from 308 patients diagnosed with advanced NSCLC who were enrolled in a multicentric clinical trial of the Spanish Lung Cancer Group. All patients had clinical stage IIIB or IV cancer and had not undergone previous chemotherapy treatment. Patients were excluded if they had two primary tumors at the time of diagnosis. The histological diagnosis of the tumor was based on the World Health Organization criteria. The TNM classification and the clinical stage were determined according to the revised criteria published in 1997 [26]. Patients were treated with cisplatin (75 mg/m2 ) and docetaxel (75 mg/m2 ) on day 1 every 3 weeks. The evaluation of response was performed after the first 3 cycles of chemotherapy. Responses were categorized according to RECIST criteria [27] into four groups: complete response (CR), partial response (PR), stable disease (SD) and progression of disease (PD), and reported as best response achieved per patient. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria. Patients with CR, PR or stabilization continued treatment until disease progression or a maximum of 8 cycles in the absence of unacceptable toxicity. Patients progressing at or before their first evaluation were shifted to a second-line treatment. All patients provided informed consent. The study was conducted in accordance with the Declaration of Helsinki and applicable local regulatory requirements and laws. The institutional ethical review board approved the study protocol. 2.2. Samples Peripheral blood samples were collected from patients prior to chemotherapy. Ten milliliters of blood were collected in tubes containing EDTA as anticoagulant (BD Vacutainer® , UK). Plasma was isolated after centrifugation at 2500 × g for 10 min and stored at −80 ◦ C until further analysis. Plasma DNA was extracted using the QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations for serum or plasma processing. 2.3. Mutation analysis of KRAS codon 12 Plasma KRAS codon12 gene mutations were detected by an allelic discrimination method using fluorogenic RT-PCR, with a GeneAmp 7000 SDS for detection and custom designed primers as probes (Applied Biosystems). To detect two of the most common KRAS mutations in codon 12, Gly12Cys (GGT > TGT) and Gly12Val (GGT > GTT), a region from the KRAS gene was amplified with the following primers: forward, 5-AGGCCTGCTGAAAATGACTGAATAT3, and reverse, 5-GCTGTATCGTCAAGGCACTCTT-3. To detect the Gly12Cys mutation, two MGB probes were used: VIC 5-TCCAACTACCACAAGTT-3 and FAM 5-TCCAACTACAACAAGTT3. To detect the Gly12Val mutation, two different MGB
Table 1 Baseline characteristics of the studied patients.
Total Age Median Range Gender Male Female Histology SCC ADC Others Stage IIIB IV ECOG-PS 0 1 2 NA
N
%
308
100
60 31–80 258 50
83.8 16.2
94 156 58
30.5 50.6 18.8
49 259
15.9 84.1
79 223 4 2
25.6 72.4 1.3 0.6
ADC, adenocarcinomas; SCC, squamous cell carcinomas; NA, not available.
probes were used, VIC 5-TCCAACTACCACAAGTT-3 and FAM 5TCCAACTAACACAAGTT-3. The reaction was done in 96-well plates with a total volume of 50 l/well. Each well contained the following reagents: 25 l of TaqMan Universal Master Mix (Applied Biosystems), 3 l of a mix consisting of two probes and two primers, 5 l of purified DNA from each sample and water up to a total volume of 50 l. The PCR conditions were as follows: pre-heated at 50 ◦ C for 2 min (to allow for the AmpErase activity to degrade all of the mRNA), a hotstart at 95 ◦ C for 10 min, followed by 40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 1 min. Reactions were performed on a Gene Amp 7000 Sequence Detection System (Applied Biosystems). All samples were analyzed in duplicate. DNA from the NCI-H23 cell line was used as an endogenous control for KRAS-mutated gene. 2.4. Statistical analysis Categorical variables were analyzed using Pearson’s chi-square test and the comparisons and correlations between continuous and categorical variables were conducted using the non-parametric Mann–Whitney U-test or Kruskal–Wallis test. OS was calculated from the date of diagnosis and PFS was calculated from the date treatment started. OS and PFS curves were plotted according to the Kaplan–Meier method, and differences between groups were assessed using the log-rank test. The significance level was set at p < 0.05 (two-sided). 3. Results The most relevant demographic and baseline clinicopathological characteristics of the 308 studied patients are summarized in Table 1. The median age was 60 years (range [31–80]), and 84% of the patients were men. Sixteen percent of the patients’ cancers were defined as stage IIIB, and the remaining 84% were defined as stage IV. There were 75 patients (24.4%) with clinical responses (CR or PR), and 202 patients (65.6%) with SD or PD (see Table 2). Some of these patients (158/308, 51%) received second-line chemotherapy. 3.1. KRAS mutational status KRAS mutational status was analyzed in plasma samples from the 308 advanced NSCLC, prior to chemotherapy treatment with a combination of cisplatin and docetaxel. The wt KRAS gene was
C. Camps et al. / Lung Cancer 72 (2011) 365–369
367
Table 2 Objective response evaluation data for 308 patients with advanced NSCLC. Responsea
n
%
CR PR SD PD NA Total
3 72 94 108 31 308
1.0 23.4 30.5 35.1 10.1 100.0
CR, complete response; PR, partial response; SD, stable disease; PD; progressive disease; NA, not available. a Tumor response was evaluated according to the Response Evaluation Criteria for Solid Tumors (RECIST) [27].
detected in 224 patients (72.7%), while in 27 patients (8.8%), a codon 12 KRAS mutation was found in the circulating DNA. The allelic discrimination analyses of the KRAS mutations showed that a G > T transversion in the first nucleotide (GGT > TGT), causing a Gly to Cys substitution was observed in 26 of the 27 KRAS-mutated samples. The other observed mutation (3.1%) was caused by a transversion of the second nucleotide (GGT > GTT), resulting in a Gly to Val substitution (Table 3). 3.2. KRAS mutations: correlation with clinico-pathological and prognostic variables KRAS-mutated samples were found in all the histological subtypes; in ADC, 15 samples (55.6%) had mutated KRAS, while 9 (33.3%) KRAS-mutated samples were found among the SCC and 3 (11.1%) among the other histological subtypes. There were no statistically significant differences among KRAS genotype for any of the pre-treatment characteristics, including age (p = 0.118), gender (p = 0.632), ECOG-PS (p = 0.428), stage (p = 0.504), number of metastatic locations (p = 0.541) or smoking status (p = 0.520). The influence of pre-treatment plasma DNA KRAS mutations on prognosis was also investigated. In our cohort, after a median follow-up time of 9.68 months [0.83–39.13], 108 (35.1%) patients progressed (Table 2). Fig. 1 shows that there were no differences in PFS between KRAS wt and KRAS mutated patients (p = 0.277). Patients harboring mutations in plasma DNA had a median PFS of 5.77 months [95% CI, 3.39–8.14], whereas for patients with wt KRAS, the PFS was 5.43 months [95% CI, 4.65–6.22]. The OS was similar for the two KRAS genotype groups (p = 0.514). The median OS was 9.07 months [95% CI, 4.43–13.70] and 10.03 months [95% CI, 8.80–11.26] in patients with mutant and wt KRAS, respectively (Fig. 2). Due to the fact that second line treatments could have effects on OS of the studied patients, we also performed the same analysis, in a subgroup of patients who did not receive a different treatment regimen. Again, no significant differences were found between wt and mutant KRAS patients (6.07 months [95% CI, 4.56–7.57] vs 4.10 months [95% CI, 2.58–5.62], respectively, p = 0.329). From the whole cohort, 158 patients received a second line treatment, and only 8 patients were treated with EGFR-TKIs (6 received gefitinib, and the other 2, erlotinib). Among these 8 EGFR-
Fig. 1. PFS according to KRAS mutational status in advanced NSCLC patients. PFS of 251 advanced NSCLC patients treated with platinum-based combination chemotherapy. The patients were divided into mutant and wt KRAS genotype groups based on the analysis of KRAS genotype on plasma DNA samples.
TKIs treated patients, there were 6 patients with KRAS wt and, in the other 2 cases, the KRAS mutational status in plasma could not be determined. In the 6 patients that harboured KRAS wt, the response to TKI treatment was PR or SD in 4 of them and PD in 2 patients. Considering the small number of TKI-treated patients, no statistical analyses could be done, but we can observe that KRAS wt patients seem to be more likely responder to TKI-based therapies. We were unable to detect KRAS mutations in 57 (18.5%) of the analyzed samples. However, in 53 of these cases (93%), neither of the KRAS alleles could be amplified by RT-PCR, most likely due to degradation of the starting genetic material rather than a failure of the allelic discrimination assay.
Table 3 Summary of the KRAS mutational status in plasma DNA in the studied population. KRAS mutational status
n
wt (GGT) mut (TGT) mut (GTT) Unknown Total
224 26 1 57 308
% 72,7 8.4 0.3 18.5 100.0
Wt, wild type; mut TGT, Gly12Cys mutation (GGT > TGT); mut GTT, Gly12Val mutation (GGT > GTT).
Fig. 2. OS according to KRAS mutational status in advanced NSCLC patients. OS analysis of 251 advanced NSCLC patients treated with platinum-based combination chemotherapy. The patients were divided into mutant and wt KRAS genotype groups based on the analysis of KRAS genotype of plasma DNA samples.
368
C. Camps et al. / Lung Cancer 72 (2011) 365–369
4. Discussion High levels of DNA derived from the primary tumor are present in the serum or plasma of cancer patients. In fact, patients with larger tumors have more DNA in the plasma. We have focused on KRAS mutations in the plasma DNA because point mutations appear early in cancer development and the detection of these mutations in plasma DNA has been demonstrated to be feasible [23,28,29]. Also, KRAS mutational status analysis, together with other molecular markers may have value in determining which patients will benefit from adjuvant platinum-based chemotherapy [30] or even from using targeted therapy with TK-inhibitors [24]. In this study, we developed a new molecular detection method based on an allelic discrimination assay by means of RT-PCR, which allows us to assess free circulating tumor DNA. KRAS mutations in DNA in the blood were first reported in 1994 [31,32] and since then, many investigations have attempted to elucidate the prevalence of these mutations and their correlation with KRAS mutations in the tumor tissue [19,25]. In our analysis, we determined that there were KRAS mutations in the plasma DNA in 8.8% of the analyzed cases; in the literature, this percentage ranges from 0 to 24% [19,22–25]. One possible limitation of this study is the low amount of DNA extracted form the plasma of the patients. Two alternative or combined strategies could be used to increase the resolution of our assay in the future: (1) to use centrifugal filter devices that simply and efficiently concentrate the sample; (2) the use of peptide nucleic acid (PNA) probes, capable of detecting mutations in the presence of 100-fold background levels of wildtype allele and suitable for serum/plasma samples [33]. Although the above described alternative methodologies that may improve the sensitivity of the assay, if we take into account that in 53 out of the 57 samples with undetermined results, either the wild-type or any of both mutations analyzed could be found, reflecting an important degradation of the starting genetic material rather than a failure of the allelic discrimination assay. However, our study is, to date, the largest study that has investigated the prevalence and the prognostic role of mutant KRAS in the plasma DNA of NSCLC patients. In 26 of the 27 KRAS-mutated samples (96.3%), the change consisted of a G > T transversion in the first nucleotide of codon 12 (GGT > TGT), resulting in a Gly to Cys substitution. The other observed mutation (1/27, 3.7%) was caused by another transversion, in the second nucleotide of the triplet (GGT > GTT), resulting in a Gly to Val substitution, consistent with previous studies [19]. The Gly to Cys substitution is the most frequent KRAS mutation in lung cancer described in the literature [34]. In a previous study on NSCLC patient serum samples [23], we analyzed other codon 12 KRAS gene mutations, and we found that Gly to Cys and Gly to Val were the most commonly found mutations, covering the 100% of the detected KRAS mutations in serum in our studied population, in concordance with the results published by Ramirez et al. [19]. In contrast, Gautschi et al. [35] reported that Gly to Cys and Gly to Val mutations represent the 64% of circulating KRAS mutations detected in lung cancer, and therefore, it is possible that in present study we underestimate the KRAS mutation rate in circulating DNA samples from advanced NSCLC patients, and this point should be considered in the design of upcoming studies. According to the literature, KRAS mutations are primarily present in ADC and LCC [36]. We detected 24 samples harboring KRAS mutations in patients with ADC or LCC (88.9% of cases) but also 3 samples (11.1%) from patients with SCC. Importantly, this difference could be attributed to the large variation in the number of cases that were included in the studies, as well as other demographic or geographical differences [37]. The patient characteristics and clinical baseline variables, such as age, gender, performance status, smoking history, histological subgroups, or stage, did not appear to have an impact on the KRAS
genotype. In addition, our results support the idea that the KRAS mutations were equally distributed among patients with regional and distant metastases. In contrast to previous studies, our data did not indicate a poor prognosis for patients whose circulating DNA contained a KRAS mutation. This discrepancy in the prognostic value of KRAS mutations might be due to the limited number of cases that were analyzed in other studies and to the analytical differences of the research groups and experimental procedures. Several studies have demonstrated a controversial correlation between the prognostic potential of KRAS mutations in serum/plasma DNA in NSCLC; there have been numerous negative and positive reports, and some reports have even demonstrated associations between the presence of KRAS mutations in serum/plasma DNA and chemotherapy response rates [19,22–24,38,39]. However, the number of patients in these studies was small, and it is important to note that our present study has statistical power due to the large number of patients included. Recently, it has been reported that plasma KRAS mutation status is associated with a poor tumor response to EGFRTKIs in NSCLC patients and may be used as a predictive marker in selecting patients for such treatment [24]. In relation to this result, there have been proposals to isolate circulating tumor cells from peripheral blood of NSCLC, and analyze the KRAS mutational status of these cells as a more accurate method of assessing KRAS mutations [40]. In summary, we observed that in previous reports several molecular biology detection methods have been used to assess KRAS mutational status, and the methods vary widely in complexity and sensitivity. These facts might explain the heterogeneity and discrepancies in the results on the prognostic impact of KRAS mutations on NSCLC; on the other hand, their predictive value of selecting patients who could benefit more from the use of EGFR-TKI therapies is interesting, but needs to be investigated further. 5. Conclusion We have developed a molecular biology technology that is affordable for large-scale studies and is not time consuming. In our cohort of NSCLC patients, the presence of mutant KRAS in the plasma DNA did not correlate with the disease stage, performance status, objective response rates, or survival. Based on this work, the prognostic relevance of KRAS mutations in the plasma/serum DNA of NSCLC patients remains controversial. Conflict of interest statement The authors declare no conflicts of interest or any financial disclosure. Funding This work was sponsored in part by a grant from the Spanish Society of Medical Oncology (SEOM) and by a grant (RD06/0020/1024) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation & European Regional Development Fund (ERDF) “Una manera de hacer Europa”. None of the funding agencies were involved in the design, data management, data analysis, manuscript preparation and review, or decision to submit. Acknowledgements The authors gratefully acknowledge the collaboration of the following investigators from the Spanish Lung Cancer Group (GECP):
C. Camps et al. / Lung Cancer 72 (2011) 365–369
˜ R. de las Penas, G. Alonso, G. López Vivanco, M. Provencio, R. Rosell, ˜ J.L. González Larriba, A. Artal, R. García Gómez, N. Vinolas, J. Terrasa, F. Barón, B. Massuti, E. Pujol Obis, A. Carrato, R. Colomer, J.M. PuertoPica, P. Martínez, P. Diz, P. Bueso, P. Lianes, B. Medina, I. Barreto, D. ˜ Gutierrez Abad, C. Mesía, I. Moreno, C. Madronal, T. de Portugal, ˜ M. Viricuela, M. López Brea, L. Jolis, R. Pérez Carrión, M. Saldana, I. Manchegs, A. Arizcun, F. Arranz, Glez-Ageitos, J. García García, J. Gracia Marco, I. Porras, I. Maestu, C. Santander, and Álvarez de Mon, Burillo. References [1] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin 2009;59:225–49. [2] Schatzkin A, Freedman LS, Schiffman MH, Dawsey SM. Validation of intermediate end points in cancer research. J Natl Cancer Inst 1990;82:1746–52. [3] Birrer MJ, Brown PH. Application of molecular genetics to the early diagnosis and screening of lung cancer. Cancer Res 1992;52:2658s–64s. [4] Mao L, Hruban RH, Boyle JO, Tockman M, Sidransky D. Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res 1994;54:1634–7. [5] Mills NE, Fishman CL, Scholes J, Anderson SE, Rom WN, Jacobson DR. Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis. J Natl Cancer Inst 1995;87:1056–60. [6] Keohavong P, DeMichele MA, Melacrinos AC, Landreneau RJ, Weyant RJ, Siegfried JM. Detection of K-ras mutations in lung carcinomas: relationship to prognosis. Clin Cancer Res 1996;2:411–8. [7] Harris CC. Chemical and physical carcinogenesis: advances and perspectives for the 1990s. Cancer Res 1991;51:5023s–44s. [8] Stanley LA. Molecular aspects of chemical carcinogenesis: the roles of oncogenes and tumour suppressor genes. Toxicology 1995;96:173–94. [9] Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ. Increasing complexity of Ras signaling. Oncogene 1998;17:1395–413. [10] Rodenhuis S, Slebos RJ. Clinical significance of ras oncogene activation in human lung cancer. Cancer Res 1992;52:2665s–9s. [11] Huncharek M, Muscat J, Geschwind JF. K-ras oncogene mutation as a prognostic marker in non-small cell lung cancer: a combined analysis of 881 cases. Carcinogenesis 1999;20:1507–10. [12] Graziano SL, Gamble GP, Newman NB, Abbott LZ, Rooney M, Mookherjee S, et al. Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer. J Clin Oncol 1999;17:668–75. [13] Mulcahy HE, Lyautey J, Lederrey C, qi CX, Anker P, Alstead EM, et al. A prospective study of K-ras mutations in the plasma of pancreatic cancer patients. Clin Cancer Res 1998;4:271–5. [14] Cho JY, Kim JH, Lee YH, Chung KY, Kim SK, Gong SJ, et al. Correlation between Kras gene mutation and prognosis of patients with nonsmall cell lung carcinoma. Cancer 1997;79:462–7. [15] Fukuyama Y, Mitsudomi T, Sugio K, Ishida T, Akazawa K, Sugimachi K. K-ras and p53 mutations are an independent unfavourable prognostic indicator in patients with non-small-cell lung cancer. Br J Cancer 1997;75:1125–30. [16] Bremnes RM, Sirera R, Camps C. Circulating tumour-derived DNA and RNA markers in blood: a tool for early detection, diagnostics, and follow-up? Lung Cancer 2005;49:1–12. [17] Kopreski MS, Benko FA, Borys DJ, Khan A, McGarrity TJ, Gocke CD. Somatic mutation screening: identification of individuals harboring K-ras mutations with the use of plasma DNA. J Natl Cancer Inst 2000;92:918–23. [18] Ryan BM, McManus RO, Daly JS, Keeling PW, Weir DG, Lefort F, et al. Serum mutant K-ras in the colorectal adenoma-to-carcinoma sequence, Implications for diagnosis, postoperative follow-up, and early detection of recurrent disease. Ann N Y Acad Sci 2000;906:29–30. [19] Ramirez JL, Sarries C, de Castro PL, Roig B, Queralt C, Escuin D, et al. Methylation patterns and K-ras mutations in tumor and paired serum of resected non-smallcell lung cancer patients. Cancer Lett 2003;193:207–16.
369
[20] Sorenson GD. Detection of mutated KRAS2 sequences as tumor markers in plasma/serum of patients with gastrointestinal cancer. Clin Cancer Res 2000;6:2129–37. [21] Anker P, Lefort F, Vasioukhin V, Lyautey J, Lederrey C, Chen XQ, et al. K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology 1997;112:1114–20. [22] Kimura T, Holland WS, Kawaguchi T, Williamson SK, Chansky K, Crowley JJ, et al. Mutant DNA in plasma of lung cancer patients: potential for monitoring response to therapy. Ann N Y Acad Sci 2004;1022:55–60. [23] Camps C, Sirera R, Bremnes R, Blasco A, Sancho E, Bayo P, et al. Is there a prognostic role of K-ras point mutations in the serum of patients with advanced non-small cell lung cancer? Lung Cancer 2005;50:339–46. [24] Wang S, An T, Wang J, Zhao J, Wang Z, Zhuo M, et al. Potential clinical significance of a plasma-based KRAS mutation analysis in patients with advanced non-small cell lung cancer. Clin Cancer Res 2010;16:1324–30. [25] Bearzatto A, Conte D, Frattini M, Zaffaroni N, Andriani F, Balestra D, et al. p16(INK4A) Hypermethylation detected by fluorescent methylationspecific PCR in plasmas from non-small cell lung cancer. Clin Cancer Res 2002;8:3782–7. [26] Mountain CF. Revisions in the international system for staging lung cancer. Chest 1997;111:1710–7. [27] Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000;92:205–16. [28] Yakubovskaya MS, Spiegelman V, Luo FC, Malaev S, Salnev A, Zborovskaya I, et al. High frequency of K-ras mutations in normal appearing lung tissues and sputum of patients with lung cancer. Int J Cancer 1995;63:810–4. [29] Dziadziuszko R, Jassem E, Jassem J. Clinical implications of molecular abnormalities in lung cancer. Cancer Treat Rev 1998;24:317–30. [30] Custodio AB, Gonzalez-Larriba JL, Bobokova J, Calles A, Alvarez R, Cuadrado E, et al. Prognostic and predictive markers of benefit from adjuvant chemotherapy in early-stage non-small cell lung cancer. J Thorac Oncol 2009;4:891–910. [31] Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL. Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomarkers Prev 1994;3:67–71. [32] Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun M. Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia. Br J Haematol 1994;86:774–9. [33] Taron M, Ichinose Y, Rosell R, Mok T, Massuti B, Zamora L, et al. Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas. Clin Cancer Res 2005;11:5878–85. [34] Hunt JD, Strimas A, Martin JE, Eyer M, Haddican M, Luckett BG, et al. Differences in KRAS mutation spectrum in lung cancer cases between African Americans and Caucasians after occupational or environmental exposure to known carcinogens. Cancer Epidemiol Biomarkers Prev 2002;11:1405–12. [35] Gautschi O, Huegli B, Ziegler A, Gugger M, Heighway J, Ratschiller D, et al. Origin and prognostic value of circulating KRAS mutations in lung cancer patients. Cancer Lett 2007;254:265–73. [36] Mitsudomi T, Viallet J, Mulshine JL, Linnoila RI, Minna JD, Gazdar AF. Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines. Oncogene 1991;6:1353–62. [37] Gao HG, Chen JK, Stewart J, Song B, Rayappa C, Whong WZ, et al. Distribution of p53 and K-ras mutations in human lung cancer tissues. Carcinogenesis 1997;18:473–8. [38] Slebos RJ, Kibbelaar RE, Dalesio O, Kooistra A, Stam J, Meijer CJ, et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N Engl J Med 1990;323:561–5. [39] Iyengar P, Tsao MS. Clinical relevance of molecular markers in lung cancer. Surg Oncol 2002;11:167–79. [40] Tsao DA, Yang MJ, Chang HJ, Yen LC, Chiu HH, Hsueh EJ, et al. A fast and convenient new technique to detect the therapeutic target, K-ras mutant, from peripheral blood in non-small cell lung cancer patients. Lung Cancer 2010;68:51–7.
Contents lists available at ScienceDirect
Lung Cancer journal homepage: www.elsevier.com/locate/lungcan
The identification of KRAS mutations at codon 12 in plasma DNA is not a prognostic factor in advanced non-small cell lung cancer patients Carlos Camps a,b,1 , Eloisa Jantus-Lewintre b,1 , Andrea Cabrera b , Ana Blasco a , Elena Sanmartín b , Sandra Gallach b , Cristina Caballero a , Nieves del Pozo a , Rafael Rosell c , Ricardo Guijarro d , Rafael Sirera b,e,∗ a
Servicio de Oncología Médica, Hospital General Universitario, Valencia, Spain Laboratorio de Oncología Molecular, Fundación Investigación, Hospital General Universitario, Valencia, Spain c Institut Català d’Oncología, Hospital Universitari Germans Trias i Pujol, Badalona, Barcelona, Spain d Servicio de Cirugía Torácica, Hospital General Universitario, Valencia, Spain e Departamento de Biotecnología, Universidad Politécnica de Valencia, Valencia, Spain b
a r t i c l e
i n f o
Article history: Received 30 July 2010 Received in revised form 1 September 2010 Accepted 6 September 2010 Keywords: KRAS Molecular markers Prognostic factors Non-small cell lung cancer (NSCLC)
a b s t r a c t Background: Qualitative analysis of circulating DNA in the blood is a promising non-invasive diagnostic and prognostic tool. Our aim was to study the association between the presence of KRAS mutations at codon 12 and several clinical variables in advanced non-small cell lung cancer (NSCLC) patients. Methods: We examined 308 stage IIIB and IV NSCLC patients who were treated with cisplatin and docetaxel. Blood samples were collected before chemotherapy, and circulating DNA was extracted from the plasma using commercial adsorption columns. The KRAS mutational status was determined by an RT-PCR method that is based on allelic discrimination. Results: The median age of the patients was 60 years [31–80], 84% were male, 98% had a performance status of 0–1 and 84% of the patients were in stage IV. The histological subtypes were as follows: 30% squamous cell carcinoma (SCC), 51% adenocarcinoma (ADC) and 19% others. Of the 277 response-evaluated patients, 1% achieved a complete response (CR), 26% achieved a partial response (PR), 34% had stable disease (SD) and 39% had progressive disease (PD). Additionally, 27 (8.8%) patients had KRAS mutations; 26 had a KRAS codon 12 TGT mutation, and 1 had a codon 12 GTT mutation. Plasmatic KRAS mutations were found in patients presenting SCC or ADC. Patients with KRAS mutations in plasma DNA had a median progression free survival (PFS) of 5.77 months [3.39–8.14], whereas for patients with wild-type (wt) KRAS, the PFS was 5.43 months [4.65–6.22] (p = 0.277). The median overall survival (OS) in KRAS-mutated patients was 9.07 months [4.43–13.70] vs 10.03 months [8.80–11.26] in wt patients (p = 0.514). Conclusions: In advanced NSCLC patients, there were no significant differences between patients with or without KRAS mutations in plasma-free DNA with respect to the baseline characteristics, response rates, PFS or OS. © 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Due to a low cure rate (6–15%) and the lack of adequate screening measures, lung cancer is the leading cause of cancer death [1]. These facts have motivated the search for new ways to detect, predict, and monitor lung cancer [2–6]. Cancer is a multi-step process involving key cancer-related genes [7,8]; mutations in the KRAS
∗ Corresponding author at: Departamento de Biotecnología, Universidad Politécnica de Valencia, Camino de Vera s/n 46022, Valencia, Spain. Tel.: +34 963 879 556; fax: +34 961 972 151. E-mail addresses: [email protected], sirera [email protected] (R. Sirera). 1 These authors contributed equally to this work. 0169-5002/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.lungcan.2010.09.005
gene have recently gained attention because KRAS is part of several signaling pathways, and genetic alterations in KRAS might thus lead to tumor development [9]. In fact, KRAS mutations are found in up to 30% of non-small cell lung cancer (NSCLC) tumors, are primarily (90%) in codon 12 [6,10–12], occur early in the development of malignancy and in some cancers have been detected in free DNA in the blood before clinical diagnosis [13]. NSCLC KRAS mutations have been associated with larger tumors, lymph node metastases, and poorer progression and survival [14,15]. Bremnes et al., reviewed the utility of analyzing circulating tumor derived DNA in blood for the development of sensitive molecular biology approaches to analyze gene mutations (such as in KRAS) in these kinds of samples [16]. This technique is of interest because there are reports of correlation between the presence
366
C. Camps et al. / Lung Cancer 72 (2011) 365–369
of KRAS mutations in the serum/plasma DNA and the mutational status of KRAS in tumors [17–23]. Recently, several NSCLC trials demonstrated that KRAS mutations in circulating DNA correlated with survival and response to treatment [19,22,24]; on the other hand, a study conducted by our group failed to demonstrate this prognostic role [23], and another NSCLC study did not reveal any circulating mutant KRAS [25]. Therefore, because there are substantial discrepancies in several studies on KRAS mutational status in both tumor and blood, the goal of this study was to investigate the prognostic significance of the codon 12 KRAS mutations in the plasma DNA from 308 wellcharacterized advanced NSCLC patients. 2. Patients and methods 2.1. Patients This study was performed retrospectively with blood samples from 308 patients diagnosed with advanced NSCLC who were enrolled in a multicentric clinical trial of the Spanish Lung Cancer Group. All patients had clinical stage IIIB or IV cancer and had not undergone previous chemotherapy treatment. Patients were excluded if they had two primary tumors at the time of diagnosis. The histological diagnosis of the tumor was based on the World Health Organization criteria. The TNM classification and the clinical stage were determined according to the revised criteria published in 1997 [26]. Patients were treated with cisplatin (75 mg/m2 ) and docetaxel (75 mg/m2 ) on day 1 every 3 weeks. The evaluation of response was performed after the first 3 cycles of chemotherapy. Responses were categorized according to RECIST criteria [27] into four groups: complete response (CR), partial response (PR), stable disease (SD) and progression of disease (PD), and reported as best response achieved per patient. Toxicity was graded according to the National Cancer Institute Common Toxicity Criteria. Patients with CR, PR or stabilization continued treatment until disease progression or a maximum of 8 cycles in the absence of unacceptable toxicity. Patients progressing at or before their first evaluation were shifted to a second-line treatment. All patients provided informed consent. The study was conducted in accordance with the Declaration of Helsinki and applicable local regulatory requirements and laws. The institutional ethical review board approved the study protocol. 2.2. Samples Peripheral blood samples were collected from patients prior to chemotherapy. Ten milliliters of blood were collected in tubes containing EDTA as anticoagulant (BD Vacutainer® , UK). Plasma was isolated after centrifugation at 2500 × g for 10 min and stored at −80 ◦ C until further analysis. Plasma DNA was extracted using the QIAamp Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations for serum or plasma processing. 2.3. Mutation analysis of KRAS codon 12 Plasma KRAS codon12 gene mutations were detected by an allelic discrimination method using fluorogenic RT-PCR, with a GeneAmp 7000 SDS for detection and custom designed primers as probes (Applied Biosystems). To detect two of the most common KRAS mutations in codon 12, Gly12Cys (GGT > TGT) and Gly12Val (GGT > GTT), a region from the KRAS gene was amplified with the following primers: forward, 5-AGGCCTGCTGAAAATGACTGAATAT3, and reverse, 5-GCTGTATCGTCAAGGCACTCTT-3. To detect the Gly12Cys mutation, two MGB probes were used: VIC 5-TCCAACTACCACAAGTT-3 and FAM 5-TCCAACTACAACAAGTT3. To detect the Gly12Val mutation, two different MGB
Table 1 Baseline characteristics of the studied patients.
Total Age Median Range Gender Male Female Histology SCC ADC Others Stage IIIB IV ECOG-PS 0 1 2 NA
N
%
308
100
60 31–80 258 50
83.8 16.2
94 156 58
30.5 50.6 18.8
49 259
15.9 84.1
79 223 4 2
25.6 72.4 1.3 0.6
ADC, adenocarcinomas; SCC, squamous cell carcinomas; NA, not available.
probes were used, VIC 5-TCCAACTACCACAAGTT-3 and FAM 5TCCAACTAACACAAGTT-3. The reaction was done in 96-well plates with a total volume of 50 l/well. Each well contained the following reagents: 25 l of TaqMan Universal Master Mix (Applied Biosystems), 3 l of a mix consisting of two probes and two primers, 5 l of purified DNA from each sample and water up to a total volume of 50 l. The PCR conditions were as follows: pre-heated at 50 ◦ C for 2 min (to allow for the AmpErase activity to degrade all of the mRNA), a hotstart at 95 ◦ C for 10 min, followed by 40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 1 min. Reactions were performed on a Gene Amp 7000 Sequence Detection System (Applied Biosystems). All samples were analyzed in duplicate. DNA from the NCI-H23 cell line was used as an endogenous control for KRAS-mutated gene. 2.4. Statistical analysis Categorical variables were analyzed using Pearson’s chi-square test and the comparisons and correlations between continuous and categorical variables were conducted using the non-parametric Mann–Whitney U-test or Kruskal–Wallis test. OS was calculated from the date of diagnosis and PFS was calculated from the date treatment started. OS and PFS curves were plotted according to the Kaplan–Meier method, and differences between groups were assessed using the log-rank test. The significance level was set at p < 0.05 (two-sided). 3. Results The most relevant demographic and baseline clinicopathological characteristics of the 308 studied patients are summarized in Table 1. The median age was 60 years (range [31–80]), and 84% of the patients were men. Sixteen percent of the patients’ cancers were defined as stage IIIB, and the remaining 84% were defined as stage IV. There were 75 patients (24.4%) with clinical responses (CR or PR), and 202 patients (65.6%) with SD or PD (see Table 2). Some of these patients (158/308, 51%) received second-line chemotherapy. 3.1. KRAS mutational status KRAS mutational status was analyzed in plasma samples from the 308 advanced NSCLC, prior to chemotherapy treatment with a combination of cisplatin and docetaxel. The wt KRAS gene was
C. Camps et al. / Lung Cancer 72 (2011) 365–369
367
Table 2 Objective response evaluation data for 308 patients with advanced NSCLC. Responsea
n
%
CR PR SD PD NA Total
3 72 94 108 31 308
1.0 23.4 30.5 35.1 10.1 100.0
CR, complete response; PR, partial response; SD, stable disease; PD; progressive disease; NA, not available. a Tumor response was evaluated according to the Response Evaluation Criteria for Solid Tumors (RECIST) [27].
detected in 224 patients (72.7%), while in 27 patients (8.8%), a codon 12 KRAS mutation was found in the circulating DNA. The allelic discrimination analyses of the KRAS mutations showed that a G > T transversion in the first nucleotide (GGT > TGT), causing a Gly to Cys substitution was observed in 26 of the 27 KRAS-mutated samples. The other observed mutation (3.1%) was caused by a transversion of the second nucleotide (GGT > GTT), resulting in a Gly to Val substitution (Table 3). 3.2. KRAS mutations: correlation with clinico-pathological and prognostic variables KRAS-mutated samples were found in all the histological subtypes; in ADC, 15 samples (55.6%) had mutated KRAS, while 9 (33.3%) KRAS-mutated samples were found among the SCC and 3 (11.1%) among the other histological subtypes. There were no statistically significant differences among KRAS genotype for any of the pre-treatment characteristics, including age (p = 0.118), gender (p = 0.632), ECOG-PS (p = 0.428), stage (p = 0.504), number of metastatic locations (p = 0.541) or smoking status (p = 0.520). The influence of pre-treatment plasma DNA KRAS mutations on prognosis was also investigated. In our cohort, after a median follow-up time of 9.68 months [0.83–39.13], 108 (35.1%) patients progressed (Table 2). Fig. 1 shows that there were no differences in PFS between KRAS wt and KRAS mutated patients (p = 0.277). Patients harboring mutations in plasma DNA had a median PFS of 5.77 months [95% CI, 3.39–8.14], whereas for patients with wt KRAS, the PFS was 5.43 months [95% CI, 4.65–6.22]. The OS was similar for the two KRAS genotype groups (p = 0.514). The median OS was 9.07 months [95% CI, 4.43–13.70] and 10.03 months [95% CI, 8.80–11.26] in patients with mutant and wt KRAS, respectively (Fig. 2). Due to the fact that second line treatments could have effects on OS of the studied patients, we also performed the same analysis, in a subgroup of patients who did not receive a different treatment regimen. Again, no significant differences were found between wt and mutant KRAS patients (6.07 months [95% CI, 4.56–7.57] vs 4.10 months [95% CI, 2.58–5.62], respectively, p = 0.329). From the whole cohort, 158 patients received a second line treatment, and only 8 patients were treated with EGFR-TKIs (6 received gefitinib, and the other 2, erlotinib). Among these 8 EGFR-
Fig. 1. PFS according to KRAS mutational status in advanced NSCLC patients. PFS of 251 advanced NSCLC patients treated with platinum-based combination chemotherapy. The patients were divided into mutant and wt KRAS genotype groups based on the analysis of KRAS genotype on plasma DNA samples.
TKIs treated patients, there were 6 patients with KRAS wt and, in the other 2 cases, the KRAS mutational status in plasma could not be determined. In the 6 patients that harboured KRAS wt, the response to TKI treatment was PR or SD in 4 of them and PD in 2 patients. Considering the small number of TKI-treated patients, no statistical analyses could be done, but we can observe that KRAS wt patients seem to be more likely responder to TKI-based therapies. We were unable to detect KRAS mutations in 57 (18.5%) of the analyzed samples. However, in 53 of these cases (93%), neither of the KRAS alleles could be amplified by RT-PCR, most likely due to degradation of the starting genetic material rather than a failure of the allelic discrimination assay.
Table 3 Summary of the KRAS mutational status in plasma DNA in the studied population. KRAS mutational status
n
wt (GGT) mut (TGT) mut (GTT) Unknown Total
224 26 1 57 308
% 72,7 8.4 0.3 18.5 100.0
Wt, wild type; mut TGT, Gly12Cys mutation (GGT > TGT); mut GTT, Gly12Val mutation (GGT > GTT).
Fig. 2. OS according to KRAS mutational status in advanced NSCLC patients. OS analysis of 251 advanced NSCLC patients treated with platinum-based combination chemotherapy. The patients were divided into mutant and wt KRAS genotype groups based on the analysis of KRAS genotype of plasma DNA samples.
368
C. Camps et al. / Lung Cancer 72 (2011) 365–369
4. Discussion High levels of DNA derived from the primary tumor are present in the serum or plasma of cancer patients. In fact, patients with larger tumors have more DNA in the plasma. We have focused on KRAS mutations in the plasma DNA because point mutations appear early in cancer development and the detection of these mutations in plasma DNA has been demonstrated to be feasible [23,28,29]. Also, KRAS mutational status analysis, together with other molecular markers may have value in determining which patients will benefit from adjuvant platinum-based chemotherapy [30] or even from using targeted therapy with TK-inhibitors [24]. In this study, we developed a new molecular detection method based on an allelic discrimination assay by means of RT-PCR, which allows us to assess free circulating tumor DNA. KRAS mutations in DNA in the blood were first reported in 1994 [31,32] and since then, many investigations have attempted to elucidate the prevalence of these mutations and their correlation with KRAS mutations in the tumor tissue [19,25]. In our analysis, we determined that there were KRAS mutations in the plasma DNA in 8.8% of the analyzed cases; in the literature, this percentage ranges from 0 to 24% [19,22–25]. One possible limitation of this study is the low amount of DNA extracted form the plasma of the patients. Two alternative or combined strategies could be used to increase the resolution of our assay in the future: (1) to use centrifugal filter devices that simply and efficiently concentrate the sample; (2) the use of peptide nucleic acid (PNA) probes, capable of detecting mutations in the presence of 100-fold background levels of wildtype allele and suitable for serum/plasma samples [33]. Although the above described alternative methodologies that may improve the sensitivity of the assay, if we take into account that in 53 out of the 57 samples with undetermined results, either the wild-type or any of both mutations analyzed could be found, reflecting an important degradation of the starting genetic material rather than a failure of the allelic discrimination assay. However, our study is, to date, the largest study that has investigated the prevalence and the prognostic role of mutant KRAS in the plasma DNA of NSCLC patients. In 26 of the 27 KRAS-mutated samples (96.3%), the change consisted of a G > T transversion in the first nucleotide of codon 12 (GGT > TGT), resulting in a Gly to Cys substitution. The other observed mutation (1/27, 3.7%) was caused by another transversion, in the second nucleotide of the triplet (GGT > GTT), resulting in a Gly to Val substitution, consistent with previous studies [19]. The Gly to Cys substitution is the most frequent KRAS mutation in lung cancer described in the literature [34]. In a previous study on NSCLC patient serum samples [23], we analyzed other codon 12 KRAS gene mutations, and we found that Gly to Cys and Gly to Val were the most commonly found mutations, covering the 100% of the detected KRAS mutations in serum in our studied population, in concordance with the results published by Ramirez et al. [19]. In contrast, Gautschi et al. [35] reported that Gly to Cys and Gly to Val mutations represent the 64% of circulating KRAS mutations detected in lung cancer, and therefore, it is possible that in present study we underestimate the KRAS mutation rate in circulating DNA samples from advanced NSCLC patients, and this point should be considered in the design of upcoming studies. According to the literature, KRAS mutations are primarily present in ADC and LCC [36]. We detected 24 samples harboring KRAS mutations in patients with ADC or LCC (88.9% of cases) but also 3 samples (11.1%) from patients with SCC. Importantly, this difference could be attributed to the large variation in the number of cases that were included in the studies, as well as other demographic or geographical differences [37]. The patient characteristics and clinical baseline variables, such as age, gender, performance status, smoking history, histological subgroups, or stage, did not appear to have an impact on the KRAS
genotype. In addition, our results support the idea that the KRAS mutations were equally distributed among patients with regional and distant metastases. In contrast to previous studies, our data did not indicate a poor prognosis for patients whose circulating DNA contained a KRAS mutation. This discrepancy in the prognostic value of KRAS mutations might be due to the limited number of cases that were analyzed in other studies and to the analytical differences of the research groups and experimental procedures. Several studies have demonstrated a controversial correlation between the prognostic potential of KRAS mutations in serum/plasma DNA in NSCLC; there have been numerous negative and positive reports, and some reports have even demonstrated associations between the presence of KRAS mutations in serum/plasma DNA and chemotherapy response rates [19,22–24,38,39]. However, the number of patients in these studies was small, and it is important to note that our present study has statistical power due to the large number of patients included. Recently, it has been reported that plasma KRAS mutation status is associated with a poor tumor response to EGFRTKIs in NSCLC patients and may be used as a predictive marker in selecting patients for such treatment [24]. In relation to this result, there have been proposals to isolate circulating tumor cells from peripheral blood of NSCLC, and analyze the KRAS mutational status of these cells as a more accurate method of assessing KRAS mutations [40]. In summary, we observed that in previous reports several molecular biology detection methods have been used to assess KRAS mutational status, and the methods vary widely in complexity and sensitivity. These facts might explain the heterogeneity and discrepancies in the results on the prognostic impact of KRAS mutations on NSCLC; on the other hand, their predictive value of selecting patients who could benefit more from the use of EGFR-TKI therapies is interesting, but needs to be investigated further. 5. Conclusion We have developed a molecular biology technology that is affordable for large-scale studies and is not time consuming. In our cohort of NSCLC patients, the presence of mutant KRAS in the plasma DNA did not correlate with the disease stage, performance status, objective response rates, or survival. Based on this work, the prognostic relevance of KRAS mutations in the plasma/serum DNA of NSCLC patients remains controversial. Conflict of interest statement The authors declare no conflicts of interest or any financial disclosure. Funding This work was sponsored in part by a grant from the Spanish Society of Medical Oncology (SEOM) and by a grant (RD06/0020/1024) from Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (ISCIII), Spanish Ministry of Science and Innovation & European Regional Development Fund (ERDF) “Una manera de hacer Europa”. None of the funding agencies were involved in the design, data management, data analysis, manuscript preparation and review, or decision to submit. Acknowledgements The authors gratefully acknowledge the collaboration of the following investigators from the Spanish Lung Cancer Group (GECP):
C. Camps et al. / Lung Cancer 72 (2011) 365–369
˜ R. de las Penas, G. Alonso, G. López Vivanco, M. Provencio, R. Rosell, ˜ J.L. González Larriba, A. Artal, R. García Gómez, N. Vinolas, J. Terrasa, F. Barón, B. Massuti, E. Pujol Obis, A. Carrato, R. Colomer, J.M. PuertoPica, P. Martínez, P. Diz, P. Bueso, P. Lianes, B. Medina, I. Barreto, D. ˜ Gutierrez Abad, C. Mesía, I. Moreno, C. Madronal, T. de Portugal, ˜ M. Viricuela, M. López Brea, L. Jolis, R. Pérez Carrión, M. Saldana, I. Manchegs, A. Arizcun, F. Arranz, Glez-Ageitos, J. García García, J. Gracia Marco, I. Porras, I. Maestu, C. Santander, and Álvarez de Mon, Burillo. References [1] Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics, 2009. CA Cancer J Clin 2009;59:225–49. [2] Schatzkin A, Freedman LS, Schiffman MH, Dawsey SM. Validation of intermediate end points in cancer research. J Natl Cancer Inst 1990;82:1746–52. [3] Birrer MJ, Brown PH. Application of molecular genetics to the early diagnosis and screening of lung cancer. Cancer Res 1992;52:2658s–64s. [4] Mao L, Hruban RH, Boyle JO, Tockman M, Sidransky D. Detection of oncogene mutations in sputum precedes diagnosis of lung cancer. Cancer Res 1994;54:1634–7. [5] Mills NE, Fishman CL, Scholes J, Anderson SE, Rom WN, Jacobson DR. Detection of K-ras oncogene mutations in bronchoalveolar lavage fluid for lung cancer diagnosis. J Natl Cancer Inst 1995;87:1056–60. [6] Keohavong P, DeMichele MA, Melacrinos AC, Landreneau RJ, Weyant RJ, Siegfried JM. Detection of K-ras mutations in lung carcinomas: relationship to prognosis. Clin Cancer Res 1996;2:411–8. [7] Harris CC. Chemical and physical carcinogenesis: advances and perspectives for the 1990s. Cancer Res 1991;51:5023s–44s. [8] Stanley LA. Molecular aspects of chemical carcinogenesis: the roles of oncogenes and tumour suppressor genes. Toxicology 1995;96:173–94. [9] Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ. Increasing complexity of Ras signaling. Oncogene 1998;17:1395–413. [10] Rodenhuis S, Slebos RJ. Clinical significance of ras oncogene activation in human lung cancer. Cancer Res 1992;52:2665s–9s. [11] Huncharek M, Muscat J, Geschwind JF. K-ras oncogene mutation as a prognostic marker in non-small cell lung cancer: a combined analysis of 881 cases. Carcinogenesis 1999;20:1507–10. [12] Graziano SL, Gamble GP, Newman NB, Abbott LZ, Rooney M, Mookherjee S, et al. Prognostic significance of K-ras codon 12 mutations in patients with resected stage I and II non-small-cell lung cancer. J Clin Oncol 1999;17:668–75. [13] Mulcahy HE, Lyautey J, Lederrey C, qi CX, Anker P, Alstead EM, et al. A prospective study of K-ras mutations in the plasma of pancreatic cancer patients. Clin Cancer Res 1998;4:271–5. [14] Cho JY, Kim JH, Lee YH, Chung KY, Kim SK, Gong SJ, et al. Correlation between Kras gene mutation and prognosis of patients with nonsmall cell lung carcinoma. Cancer 1997;79:462–7. [15] Fukuyama Y, Mitsudomi T, Sugio K, Ishida T, Akazawa K, Sugimachi K. K-ras and p53 mutations are an independent unfavourable prognostic indicator in patients with non-small-cell lung cancer. Br J Cancer 1997;75:1125–30. [16] Bremnes RM, Sirera R, Camps C. Circulating tumour-derived DNA and RNA markers in blood: a tool for early detection, diagnostics, and follow-up? Lung Cancer 2005;49:1–12. [17] Kopreski MS, Benko FA, Borys DJ, Khan A, McGarrity TJ, Gocke CD. Somatic mutation screening: identification of individuals harboring K-ras mutations with the use of plasma DNA. J Natl Cancer Inst 2000;92:918–23. [18] Ryan BM, McManus RO, Daly JS, Keeling PW, Weir DG, Lefort F, et al. Serum mutant K-ras in the colorectal adenoma-to-carcinoma sequence, Implications for diagnosis, postoperative follow-up, and early detection of recurrent disease. Ann N Y Acad Sci 2000;906:29–30. [19] Ramirez JL, Sarries C, de Castro PL, Roig B, Queralt C, Escuin D, et al. Methylation patterns and K-ras mutations in tumor and paired serum of resected non-smallcell lung cancer patients. Cancer Lett 2003;193:207–16.
369
[20] Sorenson GD. Detection of mutated KRAS2 sequences as tumor markers in plasma/serum of patients with gastrointestinal cancer. Clin Cancer Res 2000;6:2129–37. [21] Anker P, Lefort F, Vasioukhin V, Lyautey J, Lederrey C, Chen XQ, et al. K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer. Gastroenterology 1997;112:1114–20. [22] Kimura T, Holland WS, Kawaguchi T, Williamson SK, Chansky K, Crowley JJ, et al. Mutant DNA in plasma of lung cancer patients: potential for monitoring response to therapy. Ann N Y Acad Sci 2004;1022:55–60. [23] Camps C, Sirera R, Bremnes R, Blasco A, Sancho E, Bayo P, et al. Is there a prognostic role of K-ras point mutations in the serum of patients with advanced non-small cell lung cancer? Lung Cancer 2005;50:339–46. [24] Wang S, An T, Wang J, Zhao J, Wang Z, Zhuo M, et al. Potential clinical significance of a plasma-based KRAS mutation analysis in patients with advanced non-small cell lung cancer. Clin Cancer Res 2010;16:1324–30. [25] Bearzatto A, Conte D, Frattini M, Zaffaroni N, Andriani F, Balestra D, et al. p16(INK4A) Hypermethylation detected by fluorescent methylationspecific PCR in plasmas from non-small cell lung cancer. Clin Cancer Res 2002;8:3782–7. [26] Mountain CF. Revisions in the international system for staging lung cancer. Chest 1997;111:1710–7. [27] Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, et al. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000;92:205–16. [28] Yakubovskaya MS, Spiegelman V, Luo FC, Malaev S, Salnev A, Zborovskaya I, et al. High frequency of K-ras mutations in normal appearing lung tissues and sputum of patients with lung cancer. Int J Cancer 1995;63:810–4. [29] Dziadziuszko R, Jassem E, Jassem J. Clinical implications of molecular abnormalities in lung cancer. Cancer Treat Rev 1998;24:317–30. [30] Custodio AB, Gonzalez-Larriba JL, Bobokova J, Calles A, Alvarez R, Cuadrado E, et al. Prognostic and predictive markers of benefit from adjuvant chemotherapy in early-stage non-small cell lung cancer. J Thorac Oncol 2009;4:891–910. [31] Sorenson GD, Pribish DM, Valone FH, Memoli VA, Bzik DJ, Yao SL. Soluble normal and mutated DNA sequences from single-copy genes in human blood. Cancer Epidemiol Biomarkers Prev 1994;3:67–71. [32] Vasioukhin V, Anker P, Maurice P, Lyautey J, Lederrey C, Stroun M. Point mutations of the N-ras gene in the blood plasma DNA of patients with myelodysplastic syndrome or acute myelogenous leukaemia. Br J Haematol 1994;86:774–9. [33] Taron M, Ichinose Y, Rosell R, Mok T, Massuti B, Zamora L, et al. Activating mutations in the tyrosine kinase domain of the epidermal growth factor receptor are associated with improved survival in gefitinib-treated chemorefractory lung adenocarcinomas. Clin Cancer Res 2005;11:5878–85. [34] Hunt JD, Strimas A, Martin JE, Eyer M, Haddican M, Luckett BG, et al. Differences in KRAS mutation spectrum in lung cancer cases between African Americans and Caucasians after occupational or environmental exposure to known carcinogens. Cancer Epidemiol Biomarkers Prev 2002;11:1405–12. [35] Gautschi O, Huegli B, Ziegler A, Gugger M, Heighway J, Ratschiller D, et al. Origin and prognostic value of circulating KRAS mutations in lung cancer patients. Cancer Lett 2007;254:265–73. [36] Mitsudomi T, Viallet J, Mulshine JL, Linnoila RI, Minna JD, Gazdar AF. Mutations of ras genes distinguish a subset of non-small-cell lung cancer cell lines from small-cell lung cancer cell lines. Oncogene 1991;6:1353–62. [37] Gao HG, Chen JK, Stewart J, Song B, Rayappa C, Whong WZ, et al. Distribution of p53 and K-ras mutations in human lung cancer tissues. Carcinogenesis 1997;18:473–8. [38] Slebos RJ, Kibbelaar RE, Dalesio O, Kooistra A, Stam J, Meijer CJ, et al. K-ras oncogene activation as a prognostic marker in adenocarcinoma of the lung. N Engl J Med 1990;323:561–5. [39] Iyengar P, Tsao MS. Clinical relevance of molecular markers in lung cancer. Surg Oncol 2002;11:167–79. [40] Tsao DA, Yang MJ, Chang HJ, Yen LC, Chiu HH, Hsueh EJ, et al. A fast and convenient new technique to detect the therapeutic target, K-ras mutant, from peripheral blood in non-small cell lung cancer patients. Lung Cancer 2010;68:51–7.