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Does KRAS mutational status predict chemoresistance in advanced non-small cell lung cancer (NSCLC)?
Lung Cancer 83 (2014) 383–388
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Does KRAS mutational status predict chemoresistance in advanced non-small cell lung cancer (NSCLC)? M. Macerelli a,b,∗ , C. Caramella c , L. Faivre d , B. Besse a , D. Planchard a , V. Polo a,e , M. Ngo Camus a , A. Celebic d , V. Koubi-Pick f , L. Lacroix f , J.P. Pignon d , J.C. Soria a a
Department of Medical Oncology, Gustave Roussy, Villejuif, France Department of Medical Oncology, University Hospital, Udine, Italy c Department of Radiology, Gustave Roussy, Villejuif, France d Department of Biostatistics and Epidemiology, Gustave Roussy, Villejuif, France e Department of Second Medical Oncology, Oncologic Venetian Institute, Padua, Italy f Translational Research Laboratory, Gustave Roussy, Villejuif, France b
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 15 December 2013 Accepted 20 December 2013 Keywords: KRAS Advanced non-small cell lung cancer Platinum-based chemotherapy Chemoresistance Tumor aggressiveness Specific point mutations
a b s t r a c t Background: Clinical implications of KRAS mutational status in advanced non-small cell lung cancer (NSCLC) remain unclear. To clarify this point, we retrospectively explored whether KRAS mutations could impact tumor response, and disease control rate (DCR) to first-line platinum-based chemotherapy (CT) as well as progression-free survival (PFS) or overall survival (OS). Methods: Between June 2009 and June 2012, 340 patients with advanced (stage IIIB/IV) NSCLC were reviewed in a single institution (Institut Gustave Roussy). Two hundred and one patients had a biomolecular profile and received a platinum-based first-line CT. Patients with an unknown mutational status or with actionable alterations were excluded. We retained two groups: patients with KRAS mutated tumor (MUT) and patients with wild-type KRAS/EGFR (WT). Multivariate analyses with Cox model were used. Survival curves were calculated with Kaplan–Meier method. Results: One hundred and eight patients were included in the analysis: 39 in the MUT group and 69 in the WT group. Baseline radiological assessment demonstrated more brain (P = 0.01) and liver (P = 0.04) metastases in MUT patients. DCR was 76% for MUT vs. 91% for WT group (P = 0.03), regardless of the type of platinum-based CT (use of pemetrexed or not). Although no statistically significant differences were found, shorter PFS (4.9 vs. 6.0 months; P = 0.79) and OS (10.3 vs. 13.2 months; P = 0.40) were observed for patients with KRAS mutated tumors in univariate analysis. Conclusions: KRAS mutant tumors had a lower DCR after the first-line platinum-based CT, but this difference did not translate in PFS or OS. The presence of KRAS mutations may confer a more aggressive disease, with greater baseline incidence of hepatic and cerebral metastases. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death worldwide with more than one million deaths every year. However, over the last ten years new therapeutic options have enriched the lung anti-cancer armamentarium, and today in the metastatic setting multiple lines of therapy have become available [1–3]. Large scale studies showed that nearly
∗ Corresponding author at: Department of Medical Oncology, University Hospital Santa Maria della Misericordia, Piazzale Santa Maria della Misericordia, 33100 Udine, Italy. Tel.: +39 0432 552750 51; fax: +33 0432 55 27 51; mobile: +39 388 17 29 003. E-mail addresses: [email protected], [email protected] (M. Macerelli). 0169-5002/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2013.12.013
all lung cancer cells exhibit inactivation of growth inhibitor pathways (TP53, RB1, p16, STK11 and CDKN2A tumor suppressors) [4] or mutations of growth regulatory genes (KRAS, EGFR, BRAF, MEK-1, HER2, MET, EML-4-ALK, KIF5B-RET, and NKX2.1). Beyond EGFR mutations and ALK rearrangements [5,6], other “actionable” molecular mutations may be explored and used to select targeted therapies such as amplification of HER2, FGFR1 and c-MET, rearrangements of RET or ROS1, activating mutations of HER2, FGFR2, and PI3K [7–13]. Among the most common molecular alterations observed in NSCLC lie the mutations of KRAS. These mutations occur in 15–30% of NSCLC and are more frequent in adenocarcinoma (20–50%) [14]. KRAS mutations are usually mutually exclusive with EGFR mutation and ALK rearrangements [15,16]. The prognostic value of KRAS mutations has been investigated in both the adjuvant and the metastatic setting, but its value remains controversial
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[17,18]. Alongside, the predictive value of KRAS mutations to define chemosensitivity to specific chemotherapeutic agents is an area of intense debate [19]. We performed a monocentric retrospective study regarding the chemosensitivity of KRAS mutant (MUT) vs. KRAS/EGFR wild-type (WT) advanced NSCLC. We secondarily analyzed the relationship between specific KRAS mutations and patient’s outcomes. The treatment response was assessed evaluating kinetics of tumor growth as well as with RECIST criteria 1.1 [22,23]. 2. Patients and methods 2.1. Study population Consecutive patients with advanced NSCLC treated at Gustave Roussy between June 2009 and June 2012 were retrospectively reviewed. All patients with a determination of KRAS and EGFR status and treated with a platinum-based chemotherapy (CT) in the 1st line setting were included in this analysis. Patients with unknown mutational traits or with actionable alterations, when known, were excluded (i.e. PI3K, HER2, BRAF, FGFR4, ERBB4, PTEN, NRAS, and STK11 mutations; HER2, FGFR1, and MET amplification; ALK translocation). A baseline CT-scan (S0 ) and at least two further CT-scan or one before and one after S0 were deemed necessary for TGR evaluation. Patient data were collected from medical records including sex, age, smoking status, cardiovascular comorbidities, histological type performance status (PS), stage (TNM) at diagnosis and at 1st line, number and site of metastasis. The 7th lung cancer TNM edition has been used for staging at diagnosis and at 1st line. The platinum-based chemotherapy used for patients with a stage II–IIIA at diagnosis who relapsed within the following 12 months has been defined as first line therapy as well for patients with a stage IIIB relapsed within the subsequent 6 months after the first administration of therapy. 2.2. Mutations detection To identify EGFR, KRAS, BRAF, PI3K, HER2, FGFR4, ERBB4, PTEN, NRAS and STK11 mutations we used the Sanger sequencing approach. To detect ALK translocations and HER2, FGFR1 and MET amplifications we utilized the FISH (fluorescent in situ hybridization) method. 2.3. Tumor growth rate Tumor growth (GR) is calculated with GR = Log10 (Vt /V0 )/t where Vt is the tumor volume (V) at time t and V0 is the tumor volume at baseline (V = (D3 )/2 where D is the tumor size). Tumor growth rate (TGR) is defined as an increase in tumor volume during one month, expressed in percentage, obtained by the following formula: TGR = 100 (exp(GR) − 1) [21]. We defined four different periods: pre-treatment period (time between the last tumor evaluation before baseline and at baseline, n = 41), experimental period 1 (time between the first CT-scan evaluation after baseline and at baseline, n = 63), experimental period 2 and 3 for the second and third CT-scan after baseline (n = 56 and n = 39, respectively). TGR is calculated for each period and for each patient with CT-scan available. 2.4. Statistical analysis Objective response rate (ORR) was assessed according to RECIST 1.1 criteria; progression-free survival (PFS) was calculated from the start of the first line of platinum-based chemotherapy until the first evidence of disease progression or death whatever the cause or last follow-up, and overall survival (OS) from the same
Fig. 1. Flow chart.
starting date until death from any cause or last follow-up. Patients’ characteristics were compared between MUT and WT groups using Chi-square tests. Median follow-up was computed using inverted Kaplan–Meier method. The prognostic role of KRAS in patients treated by platinum-based CT was assessed using univariate and multivariate analysis. Survival rates were estimated by Kaplan–Meier method and univariate and multivariate analysis conducted by Cox regression model. Covariates with a P-value inferior or equal to 0.10 in univariate analysis were included in the multivariate model as well as variables with unbalance between KRAS MUT and WT groups. Survival rates and hazard ratio were reported with their 95% confidence interval (CI). To study tumor response, univariate and multivariate analyses were performed using Chi-square tests and logistic models. TGR difference between the two groups during different periods is compared with Kruskal–Wallis test. TGR difference between the pre-treatment period and the experimental periods is compared with Wilcoxon signed rank test. To study the impact of the type of mutations on radiological responses, PFS and OS, the rare types of mutation were excluded. 3. Results 3.1. Patient characteristics Among a total of 340 patients with NSCLC included from June 1, 2009 to June 30, 2012, 108 assessable patients were eligible for this analysis, 39 in MUT group (mutated KRAS and wild-type EGFR) and 69 WT group (wild-type KRAS and wild-type EGFR) (Fig. 1). Patient baseline demographics and disease characteristics are summarized in Table 1. The groups were balanced by sex, age, smoking status, PS and TNM. There was a trend for more adenocarcinomas in the MUT group (P = 0.10). Cardiovascular comorbidities were more frequent in WT group (P = 0.001). At baseline there was an excess of brain (P = 0.01) and liver (P = 0.04) metastases in the MUT group compared to the WT group (Table 1). A higher percentage of MUT patients has been treated with platinum/pemetrexed in the 1st line (72% vs. 49% in WT group; P = 0.02). In MUT group, 36% of patients received only one line of chemotherapy, 28% received only two lines, and 33% more than two lines, compared with 22, 22 and 55% in the WT group, respectively (Table 2). Moreover, in WT group a greater percentage of patients (30% vs. 16%) received a bi-chemotherapy at second line of treatment. 3.2. Responses and disease control The 1st line ORR of the MUT and WT groups, respectively 21 and 39%, were not statistically significant (P = 0.056). However, MUT
M. Macerelli et al. / Lung Cancer 83 (2014) 383–388 Table 1 Patient baseline demographics and disease characteristics. KRAS mutation (N = 39), N (%) Sex 26 (67) Male 13 (33) Female Age 59 (45–78) Median (range) 28 (72) <65 ≥65 11 (28) Smoking history Never smoker 1 (3) Ex smoker 29 (76) Current smoker 8 (21) Cardiovascular comorbidity 7 (18) Yes No 32 (82) Histological type Adenocarcinoma 33 (85) Squamous cell carcinoma 3 (8) Other 3 (8) Performance status at first-line 0–1 32 (82) 2–3 7 (18) TNM at diagnosis II/IIa/IIb/IIIa 4 (10) IIIb/IV 35 (90) TNM at first-line 2 (5) IIIb 37 (95) IV Number of organ with metastasis 0–1 11 (28) 2 16 (41) 12 (31) ≥3 Adrenal gland metastasis 11 (28) Yes No 28 (72) Bone metastasis 18 (46) Yes No 21 (54) Brain metastasis Yes 13 (33) No 26 (67) Liver metastasis Yes 8 (21) No 31 (79) Lung metastasis Yes 12 (31) 27 (69) No Other metastasis 17 (44) Yes 22 (56) No
KRAS wild type (N = 69), N (%)
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Table 2 Treatment received according to KRAS status. Chi2 test 1.00
46 (67) 23 (33) 0.40 61 (34–85) 44 (64) 25 (36) 0.25 8 (12) 50 (72) 11 (16) 0.001 34 (49) 35 (51) 0.10 46 (67) 7 (10) 16 (23) 0.64 59 (86) 10 (14) 0.24 13 (19) 56 (81) 0.85 3 (4) 66 (96) 0.18 31 (45) 25 (36) 13 (19) 0.45 15 (22) 54 (78) 0.57 28 (41) 41 (59) 0.01 9 (13) 60 (87) 0.04 5 (7) 64 (93) 0.49 17 (25) 52 (75) 0.32 37 (54) 32 (46)
patients had lower DCR than WT patients (76% vs. 91%; P = 0.03). This difference was also observed in the 2nd line setting but not significant (46% vs. 61%). The TGR did not confirm the difference in DCR between the two groups (Suppl. Fig. 1) in the first line setting during the experimental period 1 (P = 0.08). Platinum/pemetrexed drug association did not reveal significant differences in objective response and disease control rates within the two groups in the 1st and 2nd line (Suppl. Table 1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan. 2013.12.013. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan. 2013.12.013. 3.3. Survival outcomes With a median follow-up not reached but superior to 24 months, 78 deaths and 107 events for PFS were observed. In univariate
Number of lines 1 2 ≥3 Data missinga First-line treatment Bi-CT Platinum–pemetrexed Platinum–vinorelbine Platimum–gemcitabine Platimum–paclitaxel CT with bevacizumab Platinum–paclitaxel–bevacizumab Platinum–gemcitabine–bevacizumab Platinum–pemetrexed–bevacizumab CT with target therapyb Second-line treatment Mono-CT Paclitaxel Pemetrexed Docetaxel Gemcitabine Bi-CT Platinum–paclitaxel Platinum–pemetrexed Platinum–taxotere Platinum–gemcitabine Others Target therapy EGFR inhibitors MEK inhibitor Others CT with bevacizumab Carboplatin/paclitaxel/beva Paclitaxel/beva Pemetrexed/beva
MUT group (N = 39), number of patients (%)
WT group (N = 69), number of patients (%)
14 (36) 11 (28) 13 (33) 1 (3)
15 (22) 15 (22) 38 (55) 1 (1)
32 (82) 25 (64) 2 (5) 3 (7) 2 (5) 4 (10) 2 (50) – 2 (50) 3 (7) (N = 25) 13 (52) 7 (54) 1 (8) 5 (38) – 4 (16) 2 (50) 1 (25) – – 1 (25)c 3 (12) 2 (66) 1 (33) – 5 (20) 3 (60) 2 (40) –
59 (85) 33 (48) 13 (19) 6 (9) 7 (10) 9 (13) 7 (78) – 2 (22) 1 (1) (N = 53) 15 (28) 5 (33) 7 (47) 2 (13) 1 (7) 16 (30) 8 (50) 4 (25) 1 (6) 1 (6) 2 (13)d 15 (28) 12 (80) – 3 (20)e 7 (13) 3 (43) 3 (43) 1 (14)
CT, chemotherapy. a These patients received at least one line. b CT administered within clinical trials: 2 patients (MUT and WT): platinum–pemetrexed–cetuximab; 1 (MUT): platinum–paclitaxel–PI3K inhibitor; 1 (MUT): platinum–taxotere–antiangiogenic agent (ombrabulin). c Platinum–vinorelbine. d 1 patient: platinum–etoposide; 1 patient: cisplatin–adriamicine–cyclophosphamide. e Drugs administered within clinical trials: 1 patient: Aurora kinases inhibitor; 1 patient: cMET inhibitor (tivantinib)/erlotinib; 1 patient: platinum–paclitaxel–PI3K inhibitor.
analysis, the PFS was not significantly different (P = 0.79, HR = 1.06 [95% CI 0.71–1.57]) between the MUT (median: 4.9 [95% CI 2.6–7.1] months) and the WT group (6.0 [4.7–7.5] months) (Fig. 2A). The OS was not significantly different (P = 0.40, HR = 1.22 [0.77–1.94]) between the MUT (median: 10.3 [5.67–16.6] months) and WT group (13.2 [10.6–18.1] months) (Fig. 2B). In multivariate analyses, performance status >1 and more than 2 metastatic sites at baseline predicted a shorter PFS and OS, but KRAS was not associated to these endpoints (Suppl. Table 2). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan. 2013.12.013. 3.4. KRAS mutations Overall, the most common sites of KRAS mutations were found at codon 12 in 28 patients (72%) and at codon 13 in 7 patients (18%, Suppl. Table 3). Between these two subgroups (codon 12 vs. 13) no significant difference was found in ORR (0% vs. 26%; P = 0.13) and DCR (78% vs. 71%; P = 0.72). The median PFS were
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Fig. 2. Kaplan–Meier curves for progression-free survival (A) and overall survival (B) in patients with non-small cell lung cancer treated with platinum-based chemotherapy in first line. Curves are shown for patients harboring KRAS mutations vs. KRAS wild-type/EGFR wild-type tumors.
5.8 (95% CI 2.8–8.8) and 1.08 months (1.0–14.3) months, and the median OS were 11.7 (5.7–19.6) and 6.4 (4.4–16.6) months, respectively. Nonetheless, no significant difference in PFS and OS was found between common mutations at codons 12 and 13 (P = 0.61, HR = 0.79 [95% CI 0.31–1.99]; P = 0.37, HR = 1.52 [0.61–3.83]), and among these two subgroups and WT group (Fig. 3A and B). There was not significant variation of the metastasis localization according to mutated codons (12 vs. 13; data not shown). Among specific KRAS mutations observed in at least 2 patients, median OS ranged from 5 to 21 months (Suppl. Table 3). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.lungcan.2013.12.013. 4. Discussion The prognostic and predictive role of KRAS tumor mutations has been debated for years. Some studies showed a poorer outcome for patients with KRAS mutant tumor, but other failed to confirm it [14,24–27]. The resistance to treatment with EGFR tyrosin-kinase inhibitors (TKIs) of tumor harboring KRAS mutations is also partially controversial, considering potential escape pathways downstream [28,29] and different response according to type of KRAS mutations (codon 12 vs. 13) [30]. In our study, patients harboring a KRAS mutation presented a worse DCR in 1st line platinum-based therapy.
Nonetheless, no statistically significant differences were found in ORR, with a trend of worse responses in MUT group, consistent with previous studies in advanced NSCLC and in the adjuvant setting [31–34]. Significant difference in DCR did not translate in worse PFS and OS for MUT group as in these previous analyses [19]. Preclinical and clinical data on chemosensitivity of NSCLC cellular clones showed a different response to various antineoplastic drugs depending on the type of KRAS point mutations [35] (i.e. tumor cells with G12V mutant alleles may respond better to cisplatinum and with less sensitivity to pemetrexed unlike G12C mutant alleles [20]). In our study no significant difference was found among the various platinum-based treatments in ORR (19% vs. 27%; pemetrexed vs. others – paclitaxel, bevacizumab, vinorelbine, gemcitabine) and in DCR (94% vs. 89%). Kandar et al. showed that the efficacy of pemetrexed was superior in patients with specific KRAS subtypes such as those with G12C, G12A and G13A point mutations compared to G12V in 1st line and maintenance therapy for advanced NSCLC [35]. Patients treated with pemetrexed and with KRAS G12A, G12V and G13D mutations had a better survival (21, 12 and 12 months, respectively) compared to those with G12C and G13C mutations (both 6 months). Shepherd et al. demonstrated a deleterious impact of adjuvant CT for codon 13 KRAS mutant NSCLC compared to codon 12 and wild-type KRAS disease (HR = 5.78; 95% CI 2.06–16.2; P < .001) [18]. We found no statistically significant difference in response rate and survival between
Fig. 3. Kaplan–Meier curves for progression-free survival (A) and overall survival (B) for patients harboring codon 12 KRAS mutations (MUT) vs. codon 13 KRAS mutations (MUT) vs. wild-type (WT) patients. The P-values correspond to the comparison of the 3 curves by a log-rank test.
M. Macerelli et al. / Lung Cancer 83 (2014) 383–388
KRAS mutations at codons 12 and 13, although there is a trend toward decreased OS for codon 13 mutations (Fig. 3B). Further, we found a more important tumor burden with a greater incidence of liver and brain metastases at baseline for MUT patients. Previous data did not show any difference between MUT and WT patients with respect to the presence of specific metastatic sites [36,37]. Likely, tumor biology guides not only the chemosensitivity but also metastatic localizations of mutant disease. A different behavior of KRAS mutant disease could also explain the larger number of MUT patients (23% vs. 16%; data not shown) who cannot receive even a second line therapy. Our study has some limitations. First, we used a retrospective design. Second, the power of this study is limited and our population has been selected from a large local database where all patients with stage IIIB and IV and treated with platinum-based CT has been included in order to obtain a comprehensive molecular profile. Thus, we removed for our analysis 32% of reviewed patients that had unknown molecular traits and this exclusion may have affected the final analysis of our data. We also excluded 16% of the patients because of known actionable alterations beyond KRAS mutation (Fig. 1) to avoid confounding effects related to the presence of other mutations and to compare as homogeneous and pure as possible groups. The small size of the MUT cohort does not allow to analyze the impact of the numerous different KRAS mutations. Treatment strategy was different between the two groups (type of regimen, number of lines) and might be a confounding factor for the outcome. In conclusion, this study did not show a different outcome for patient with KRAS mutated tumors respect to WT tumors. It suggests that KRAS phenotype might be a more aggressive pattern and that the different KRAS mutations might be linked to a different chemosensitivity and behavior. Conflict of interest statement None declared. Acknowledgements During this project, Marianna Macerelli was supported by a grant from the Italian Association of Thoracic Oncology (AIOT). This work was supported by the European Community’s Seventh Framework Programme (FP7/2007–2013) under Grant agreement number HEALTH-F2-2010-258677 – CURELUNG project. References [1] Jacoulet NP, Gainet M, Elleuch R. Third-line chemotherapy in advanced non-small cell lung cancer: identifying the candidates for routine. J Thorac Oncol 2009;4:1544–9. [2] Sekine HI, Horinouchi H, Nokihara H, Yamamoto N, Kubota K, Tamura T. Retrospective analysis of third-line and fourth-line chemotherapy for advanced non-small-cell lung cancer. Clin Lung Cancer 2012;13:39–43. [3] Langer CJ, Mok T, Postmus PE. Targeted agents in the third-/fourth-line treatment of patients with advanced (stage III/IV) non-small cell lung cancer (NSCLC). Cancer Treat Rev 2013;39:252–60. [4] Liu P, Morrison C, Wang L, Xiong D, Vedell P, Cui P, et al. Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing. Carcinogenesis 2012;33:1270–6. [5] Li T, Kung HJ, Mack PC, Gandara DR. Genotyping and genomic profiling of non-small-cell lung cancer: implications for current and future therapies. J Clin Oncol 2013;31:1039–49. [6] Hirsch FR, Jänne PA, Eberhardt WE, Cappuzzo F, Thatcher N, Pirker R, et al. Epidermal growth factor receptor inhibition in lung cancer: status 2012. J Thorac Oncol 2013;8:373–84. [7] Reungwetwattana T, Weroha SJ, Molina JR. Oncogenic pathways, molecularly targeted therapies, and highlighted clinical trials in non-small-cell lung cancer (NSCLC). Clin Lung Cancer 2012;13:252–66.
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Does KRAS mutational status predict chemoresistance in advanced non-small cell lung cancer (NSCLC)? M. Macerelli a,b,∗ , C. Caramella c , L. Faivre d , B. Besse a , D. Planchard a , V. Polo a,e , M. Ngo Camus a , A. Celebic d , V. Koubi-Pick f , L. Lacroix f , J.P. Pignon d , J.C. Soria a a
Department of Medical Oncology, Gustave Roussy, Villejuif, France Department of Medical Oncology, University Hospital, Udine, Italy c Department of Radiology, Gustave Roussy, Villejuif, France d Department of Biostatistics and Epidemiology, Gustave Roussy, Villejuif, France e Department of Second Medical Oncology, Oncologic Venetian Institute, Padua, Italy f Translational Research Laboratory, Gustave Roussy, Villejuif, France b
a r t i c l e
i n f o
Article history: Received 16 September 2013 Received in revised form 15 December 2013 Accepted 20 December 2013 Keywords: KRAS Advanced non-small cell lung cancer Platinum-based chemotherapy Chemoresistance Tumor aggressiveness Specific point mutations
a b s t r a c t Background: Clinical implications of KRAS mutational status in advanced non-small cell lung cancer (NSCLC) remain unclear. To clarify this point, we retrospectively explored whether KRAS mutations could impact tumor response, and disease control rate (DCR) to first-line platinum-based chemotherapy (CT) as well as progression-free survival (PFS) or overall survival (OS). Methods: Between June 2009 and June 2012, 340 patients with advanced (stage IIIB/IV) NSCLC were reviewed in a single institution (Institut Gustave Roussy). Two hundred and one patients had a biomolecular profile and received a platinum-based first-line CT. Patients with an unknown mutational status or with actionable alterations were excluded. We retained two groups: patients with KRAS mutated tumor (MUT) and patients with wild-type KRAS/EGFR (WT). Multivariate analyses with Cox model were used. Survival curves were calculated with Kaplan–Meier method. Results: One hundred and eight patients were included in the analysis: 39 in the MUT group and 69 in the WT group. Baseline radiological assessment demonstrated more brain (P = 0.01) and liver (P = 0.04) metastases in MUT patients. DCR was 76% for MUT vs. 91% for WT group (P = 0.03), regardless of the type of platinum-based CT (use of pemetrexed or not). Although no statistically significant differences were found, shorter PFS (4.9 vs. 6.0 months; P = 0.79) and OS (10.3 vs. 13.2 months; P = 0.40) were observed for patients with KRAS mutated tumors in univariate analysis. Conclusions: KRAS mutant tumors had a lower DCR after the first-line platinum-based CT, but this difference did not translate in PFS or OS. The presence of KRAS mutations may confer a more aggressive disease, with greater baseline incidence of hepatic and cerebral metastases. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Non-small cell lung cancer (NSCLC) is the most common cause of cancer-related death worldwide with more than one million deaths every year. However, over the last ten years new therapeutic options have enriched the lung anti-cancer armamentarium, and today in the metastatic setting multiple lines of therapy have become available [1–3]. Large scale studies showed that nearly
∗ Corresponding author at: Department of Medical Oncology, University Hospital Santa Maria della Misericordia, Piazzale Santa Maria della Misericordia, 33100 Udine, Italy. Tel.: +39 0432 552750 51; fax: +33 0432 55 27 51; mobile: +39 388 17 29 003. E-mail addresses: [email protected], [email protected] (M. Macerelli). 0169-5002/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.lungcan.2013.12.013
all lung cancer cells exhibit inactivation of growth inhibitor pathways (TP53, RB1, p16, STK11 and CDKN2A tumor suppressors) [4] or mutations of growth regulatory genes (KRAS, EGFR, BRAF, MEK-1, HER2, MET, EML-4-ALK, KIF5B-RET, and NKX2.1). Beyond EGFR mutations and ALK rearrangements [5,6], other “actionable” molecular mutations may be explored and used to select targeted therapies such as amplification of HER2, FGFR1 and c-MET, rearrangements of RET or ROS1, activating mutations of HER2, FGFR2, and PI3K [7–13]. Among the most common molecular alterations observed in NSCLC lie the mutations of KRAS. These mutations occur in 15–30% of NSCLC and are more frequent in adenocarcinoma (20–50%) [14]. KRAS mutations are usually mutually exclusive with EGFR mutation and ALK rearrangements [15,16]. The prognostic value of KRAS mutations has been investigated in both the adjuvant and the metastatic setting, but its value remains controversial
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[17,18]. Alongside, the predictive value of KRAS mutations to define chemosensitivity to specific chemotherapeutic agents is an area of intense debate [19]. We performed a monocentric retrospective study regarding the chemosensitivity of KRAS mutant (MUT) vs. KRAS/EGFR wild-type (WT) advanced NSCLC. We secondarily analyzed the relationship between specific KRAS mutations and patient’s outcomes. The treatment response was assessed evaluating kinetics of tumor growth as well as with RECIST criteria 1.1 [22,23]. 2. Patients and methods 2.1. Study population Consecutive patients with advanced NSCLC treated at Gustave Roussy between June 2009 and June 2012 were retrospectively reviewed. All patients with a determination of KRAS and EGFR status and treated with a platinum-based chemotherapy (CT) in the 1st line setting were included in this analysis. Patients with unknown mutational traits or with actionable alterations, when known, were excluded (i.e. PI3K, HER2, BRAF, FGFR4, ERBB4, PTEN, NRAS, and STK11 mutations; HER2, FGFR1, and MET amplification; ALK translocation). A baseline CT-scan (S0 ) and at least two further CT-scan or one before and one after S0 were deemed necessary for TGR evaluation. Patient data were collected from medical records including sex, age, smoking status, cardiovascular comorbidities, histological type performance status (PS), stage (TNM) at diagnosis and at 1st line, number and site of metastasis. The 7th lung cancer TNM edition has been used for staging at diagnosis and at 1st line. The platinum-based chemotherapy used for patients with a stage II–IIIA at diagnosis who relapsed within the following 12 months has been defined as first line therapy as well for patients with a stage IIIB relapsed within the subsequent 6 months after the first administration of therapy. 2.2. Mutations detection To identify EGFR, KRAS, BRAF, PI3K, HER2, FGFR4, ERBB4, PTEN, NRAS and STK11 mutations we used the Sanger sequencing approach. To detect ALK translocations and HER2, FGFR1 and MET amplifications we utilized the FISH (fluorescent in situ hybridization) method. 2.3. Tumor growth rate Tumor growth (GR) is calculated with GR = Log10 (Vt /V0 )/t where Vt is the tumor volume (V) at time t and V0 is the tumor volume at baseline (V = (D3 )/2 where D is the tumor size). Tumor growth rate (TGR) is defined as an increase in tumor volume during one month, expressed in percentage, obtained by the following formula: TGR = 100 (exp(GR) − 1) [21]. We defined four different periods: pre-treatment period (time between the last tumor evaluation before baseline and at baseline, n = 41), experimental period 1 (time between the first CT-scan evaluation after baseline and at baseline, n = 63), experimental period 2 and 3 for the second and third CT-scan after baseline (n = 56 and n = 39, respectively). TGR is calculated for each period and for each patient with CT-scan available. 2.4. Statistical analysis Objective response rate (ORR) was assessed according to RECIST 1.1 criteria; progression-free survival (PFS) was calculated from the start of the first line of platinum-based chemotherapy until the first evidence of disease progression or death whatever the cause or last follow-up, and overall survival (OS) from the same
Fig. 1. Flow chart.
starting date until death from any cause or last follow-up. Patients’ characteristics were compared between MUT and WT groups using Chi-square tests. Median follow-up was computed using inverted Kaplan–Meier method. The prognostic role of KRAS in patients treated by platinum-based CT was assessed using univariate and multivariate analysis. Survival rates were estimated by Kaplan–Meier method and univariate and multivariate analysis conducted by Cox regression model. Covariates with a P-value inferior or equal to 0.10 in univariate analysis were included in the multivariate model as well as variables with unbalance between KRAS MUT and WT groups. Survival rates and hazard ratio were reported with their 95% confidence interval (CI). To study tumor response, univariate and multivariate analyses were performed using Chi-square tests and logistic models. TGR difference between the two groups during different periods is compared with Kruskal–Wallis test. TGR difference between the pre-treatment period and the experimental periods is compared with Wilcoxon signed rank test. To study the impact of the type of mutations on radiological responses, PFS and OS, the rare types of mutation were excluded. 3. Results 3.1. Patient characteristics Among a total of 340 patients with NSCLC included from June 1, 2009 to June 30, 2012, 108 assessable patients were eligible for this analysis, 39 in MUT group (mutated KRAS and wild-type EGFR) and 69 WT group (wild-type KRAS and wild-type EGFR) (Fig. 1). Patient baseline demographics and disease characteristics are summarized in Table 1. The groups were balanced by sex, age, smoking status, PS and TNM. There was a trend for more adenocarcinomas in the MUT group (P = 0.10). Cardiovascular comorbidities were more frequent in WT group (P = 0.001). At baseline there was an excess of brain (P = 0.01) and liver (P = 0.04) metastases in the MUT group compared to the WT group (Table 1). A higher percentage of MUT patients has been treated with platinum/pemetrexed in the 1st line (72% vs. 49% in WT group; P = 0.02). In MUT group, 36% of patients received only one line of chemotherapy, 28% received only two lines, and 33% more than two lines, compared with 22, 22 and 55% in the WT group, respectively (Table 2). Moreover, in WT group a greater percentage of patients (30% vs. 16%) received a bi-chemotherapy at second line of treatment. 3.2. Responses and disease control The 1st line ORR of the MUT and WT groups, respectively 21 and 39%, were not statistically significant (P = 0.056). However, MUT
M. Macerelli et al. / Lung Cancer 83 (2014) 383–388 Table 1 Patient baseline demographics and disease characteristics. KRAS mutation (N = 39), N (%) Sex 26 (67) Male 13 (33) Female Age 59 (45–78) Median (range) 28 (72) <65 ≥65 11 (28) Smoking history Never smoker 1 (3) Ex smoker 29 (76) Current smoker 8 (21) Cardiovascular comorbidity 7 (18) Yes No 32 (82) Histological type Adenocarcinoma 33 (85) Squamous cell carcinoma 3 (8) Other 3 (8) Performance status at first-line 0–1 32 (82) 2–3 7 (18) TNM at diagnosis II/IIa/IIb/IIIa 4 (10) IIIb/IV 35 (90) TNM at first-line 2 (5) IIIb 37 (95) IV Number of organ with metastasis 0–1 11 (28) 2 16 (41) 12 (31) ≥3 Adrenal gland metastasis 11 (28) Yes No 28 (72) Bone metastasis 18 (46) Yes No 21 (54) Brain metastasis Yes 13 (33) No 26 (67) Liver metastasis Yes 8 (21) No 31 (79) Lung metastasis Yes 12 (31) 27 (69) No Other metastasis 17 (44) Yes 22 (56) No
KRAS wild type (N = 69), N (%)
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Table 2 Treatment received according to KRAS status. Chi2 test 1.00
46 (67) 23 (33) 0.40 61 (34–85) 44 (64) 25 (36) 0.25 8 (12) 50 (72) 11 (16) 0.001 34 (49) 35 (51) 0.10 46 (67) 7 (10) 16 (23) 0.64 59 (86) 10 (14) 0.24 13 (19) 56 (81) 0.85 3 (4) 66 (96) 0.18 31 (45) 25 (36) 13 (19) 0.45 15 (22) 54 (78) 0.57 28 (41) 41 (59) 0.01 9 (13) 60 (87) 0.04 5 (7) 64 (93) 0.49 17 (25) 52 (75) 0.32 37 (54) 32 (46)
patients had lower DCR than WT patients (76% vs. 91%; P = 0.03). This difference was also observed in the 2nd line setting but not significant (46% vs. 61%). The TGR did not confirm the difference in DCR between the two groups (Suppl. Fig. 1) in the first line setting during the experimental period 1 (P = 0.08). Platinum/pemetrexed drug association did not reveal significant differences in objective response and disease control rates within the two groups in the 1st and 2nd line (Suppl. Table 1). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan. 2013.12.013. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan. 2013.12.013. 3.3. Survival outcomes With a median follow-up not reached but superior to 24 months, 78 deaths and 107 events for PFS were observed. In univariate
Number of lines 1 2 ≥3 Data missinga First-line treatment Bi-CT Platinum–pemetrexed Platinum–vinorelbine Platimum–gemcitabine Platimum–paclitaxel CT with bevacizumab Platinum–paclitaxel–bevacizumab Platinum–gemcitabine–bevacizumab Platinum–pemetrexed–bevacizumab CT with target therapyb Second-line treatment Mono-CT Paclitaxel Pemetrexed Docetaxel Gemcitabine Bi-CT Platinum–paclitaxel Platinum–pemetrexed Platinum–taxotere Platinum–gemcitabine Others Target therapy EGFR inhibitors MEK inhibitor Others CT with bevacizumab Carboplatin/paclitaxel/beva Paclitaxel/beva Pemetrexed/beva
MUT group (N = 39), number of patients (%)
WT group (N = 69), number of patients (%)
14 (36) 11 (28) 13 (33) 1 (3)
15 (22) 15 (22) 38 (55) 1 (1)
32 (82) 25 (64) 2 (5) 3 (7) 2 (5) 4 (10) 2 (50) – 2 (50) 3 (7) (N = 25) 13 (52) 7 (54) 1 (8) 5 (38) – 4 (16) 2 (50) 1 (25) – – 1 (25)c 3 (12) 2 (66) 1 (33) – 5 (20) 3 (60) 2 (40) –
59 (85) 33 (48) 13 (19) 6 (9) 7 (10) 9 (13) 7 (78) – 2 (22) 1 (1) (N = 53) 15 (28) 5 (33) 7 (47) 2 (13) 1 (7) 16 (30) 8 (50) 4 (25) 1 (6) 1 (6) 2 (13)d 15 (28) 12 (80) – 3 (20)e 7 (13) 3 (43) 3 (43) 1 (14)
CT, chemotherapy. a These patients received at least one line. b CT administered within clinical trials: 2 patients (MUT and WT): platinum–pemetrexed–cetuximab; 1 (MUT): platinum–paclitaxel–PI3K inhibitor; 1 (MUT): platinum–taxotere–antiangiogenic agent (ombrabulin). c Platinum–vinorelbine. d 1 patient: platinum–etoposide; 1 patient: cisplatin–adriamicine–cyclophosphamide. e Drugs administered within clinical trials: 1 patient: Aurora kinases inhibitor; 1 patient: cMET inhibitor (tivantinib)/erlotinib; 1 patient: platinum–paclitaxel–PI3K inhibitor.
analysis, the PFS was not significantly different (P = 0.79, HR = 1.06 [95% CI 0.71–1.57]) between the MUT (median: 4.9 [95% CI 2.6–7.1] months) and the WT group (6.0 [4.7–7.5] months) (Fig. 2A). The OS was not significantly different (P = 0.40, HR = 1.22 [0.77–1.94]) between the MUT (median: 10.3 [5.67–16.6] months) and WT group (13.2 [10.6–18.1] months) (Fig. 2B). In multivariate analyses, performance status >1 and more than 2 metastatic sites at baseline predicted a shorter PFS and OS, but KRAS was not associated to these endpoints (Suppl. Table 2). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.lungcan. 2013.12.013. 3.4. KRAS mutations Overall, the most common sites of KRAS mutations were found at codon 12 in 28 patients (72%) and at codon 13 in 7 patients (18%, Suppl. Table 3). Between these two subgroups (codon 12 vs. 13) no significant difference was found in ORR (0% vs. 26%; P = 0.13) and DCR (78% vs. 71%; P = 0.72). The median PFS were
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Fig. 2. Kaplan–Meier curves for progression-free survival (A) and overall survival (B) in patients with non-small cell lung cancer treated with platinum-based chemotherapy in first line. Curves are shown for patients harboring KRAS mutations vs. KRAS wild-type/EGFR wild-type tumors.
5.8 (95% CI 2.8–8.8) and 1.08 months (1.0–14.3) months, and the median OS were 11.7 (5.7–19.6) and 6.4 (4.4–16.6) months, respectively. Nonetheless, no significant difference in PFS and OS was found between common mutations at codons 12 and 13 (P = 0.61, HR = 0.79 [95% CI 0.31–1.99]; P = 0.37, HR = 1.52 [0.61–3.83]), and among these two subgroups and WT group (Fig. 3A and B). There was not significant variation of the metastasis localization according to mutated codons (12 vs. 13; data not shown). Among specific KRAS mutations observed in at least 2 patients, median OS ranged from 5 to 21 months (Suppl. Table 3). Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.lungcan.2013.12.013. 4. Discussion The prognostic and predictive role of KRAS tumor mutations has been debated for years. Some studies showed a poorer outcome for patients with KRAS mutant tumor, but other failed to confirm it [14,24–27]. The resistance to treatment with EGFR tyrosin-kinase inhibitors (TKIs) of tumor harboring KRAS mutations is also partially controversial, considering potential escape pathways downstream [28,29] and different response according to type of KRAS mutations (codon 12 vs. 13) [30]. In our study, patients harboring a KRAS mutation presented a worse DCR in 1st line platinum-based therapy.
Nonetheless, no statistically significant differences were found in ORR, with a trend of worse responses in MUT group, consistent with previous studies in advanced NSCLC and in the adjuvant setting [31–34]. Significant difference in DCR did not translate in worse PFS and OS for MUT group as in these previous analyses [19]. Preclinical and clinical data on chemosensitivity of NSCLC cellular clones showed a different response to various antineoplastic drugs depending on the type of KRAS point mutations [35] (i.e. tumor cells with G12V mutant alleles may respond better to cisplatinum and with less sensitivity to pemetrexed unlike G12C mutant alleles [20]). In our study no significant difference was found among the various platinum-based treatments in ORR (19% vs. 27%; pemetrexed vs. others – paclitaxel, bevacizumab, vinorelbine, gemcitabine) and in DCR (94% vs. 89%). Kandar et al. showed that the efficacy of pemetrexed was superior in patients with specific KRAS subtypes such as those with G12C, G12A and G13A point mutations compared to G12V in 1st line and maintenance therapy for advanced NSCLC [35]. Patients treated with pemetrexed and with KRAS G12A, G12V and G13D mutations had a better survival (21, 12 and 12 months, respectively) compared to those with G12C and G13C mutations (both 6 months). Shepherd et al. demonstrated a deleterious impact of adjuvant CT for codon 13 KRAS mutant NSCLC compared to codon 12 and wild-type KRAS disease (HR = 5.78; 95% CI 2.06–16.2; P < .001) [18]. We found no statistically significant difference in response rate and survival between
Fig. 3. Kaplan–Meier curves for progression-free survival (A) and overall survival (B) for patients harboring codon 12 KRAS mutations (MUT) vs. codon 13 KRAS mutations (MUT) vs. wild-type (WT) patients. The P-values correspond to the comparison of the 3 curves by a log-rank test.
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KRAS mutations at codons 12 and 13, although there is a trend toward decreased OS for codon 13 mutations (Fig. 3B). Further, we found a more important tumor burden with a greater incidence of liver and brain metastases at baseline for MUT patients. Previous data did not show any difference between MUT and WT patients with respect to the presence of specific metastatic sites [36,37]. Likely, tumor biology guides not only the chemosensitivity but also metastatic localizations of mutant disease. A different behavior of KRAS mutant disease could also explain the larger number of MUT patients (23% vs. 16%; data not shown) who cannot receive even a second line therapy. Our study has some limitations. First, we used a retrospective design. Second, the power of this study is limited and our population has been selected from a large local database where all patients with stage IIIB and IV and treated with platinum-based CT has been included in order to obtain a comprehensive molecular profile. Thus, we removed for our analysis 32% of reviewed patients that had unknown molecular traits and this exclusion may have affected the final analysis of our data. We also excluded 16% of the patients because of known actionable alterations beyond KRAS mutation (Fig. 1) to avoid confounding effects related to the presence of other mutations and to compare as homogeneous and pure as possible groups. The small size of the MUT cohort does not allow to analyze the impact of the numerous different KRAS mutations. Treatment strategy was different between the two groups (type of regimen, number of lines) and might be a confounding factor for the outcome. In conclusion, this study did not show a different outcome for patient with KRAS mutated tumors respect to WT tumors. It suggests that KRAS phenotype might be a more aggressive pattern and that the different KRAS mutations might be linked to a different chemosensitivity and behavior. Conflict of interest statement None declared. Acknowledgements During this project, Marianna Macerelli was supported by a grant from the Italian Association of Thoracic Oncology (AIOT). This work was supported by the European Community’s Seventh Framework Programme (FP7/2007–2013) under Grant agreement number HEALTH-F2-2010-258677 – CURELUNG project. References [1] Jacoulet NP, Gainet M, Elleuch R. Third-line chemotherapy in advanced non-small cell lung cancer: identifying the candidates for routine. J Thorac Oncol 2009;4:1544–9. [2] Sekine HI, Horinouchi H, Nokihara H, Yamamoto N, Kubota K, Tamura T. Retrospective analysis of third-line and fourth-line chemotherapy for advanced non-small-cell lung cancer. Clin Lung Cancer 2012;13:39–43. [3] Langer CJ, Mok T, Postmus PE. Targeted agents in the third-/fourth-line treatment of patients with advanced (stage III/IV) non-small cell lung cancer (NSCLC). Cancer Treat Rev 2013;39:252–60. [4] Liu P, Morrison C, Wang L, Xiong D, Vedell P, Cui P, et al. Identification of somatic mutations in non-small cell lung carcinomas using whole-exome sequencing. Carcinogenesis 2012;33:1270–6. [5] Li T, Kung HJ, Mack PC, Gandara DR. Genotyping and genomic profiling of non-small-cell lung cancer: implications for current and future therapies. J Clin Oncol 2013;31:1039–49. [6] Hirsch FR, Jänne PA, Eberhardt WE, Cappuzzo F, Thatcher N, Pirker R, et al. Epidermal growth factor receptor inhibition in lung cancer: status 2012. J Thorac Oncol 2013;8:373–84. [7] Reungwetwattana T, Weroha SJ, Molina JR. Oncogenic pathways, molecularly targeted therapies, and highlighted clinical trials in non-small-cell lung cancer (NSCLC). Clin Lung Cancer 2012;13:252–66.
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