Tumor Biol. DOI 10.1007/s13277-015-3249-x
RESEARCH ARTICLE
Statins augment efficacy of EGFR-TKIs in patients with advanced-stage non-small cell lung cancer harbouring KRAS mutation Ondrej Fiala & Milos Pesek & Jindrich Finek & Marek Minarik & Lucie Benesova & Zbynek Bortlicek & Ondrej Topolcan
Received: 9 December 2014 / Accepted: 10 February 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) represent novel effective agents approved for the treatment of patients with advanced-stage NSCLC. KRAS mutations have been reported as a negative prognostic and predictive factor in patients with NSCLC treated with EGFR-TKIs. Several studies have recently shown that statins can block tumour
O. Fiala (*) : J. Finek Department of Oncology and Radiotherapy, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, alej Svobody 80, 304 60 Pilsen, Czech Republic e-mail: [email protected] O. Fiala Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Prague, Czech Republic M. Pesek Department of Pneumology, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, Prague, Czech Republic
cell growth, invasion and metastatic potential. We analysed clinical data of 67 patients with locally advanced (IIIB) or metastatic stage (IV) NSCLC harbouring Kirsten rat sarcoma viral oncogene (KRAS) mutation treated with erlotinib or gefitinib. Twelve patients were treated with combination of EGFR-TKI and statin and 55 patients were treated with EGFR-TKI alone. Comparison of patients’ survival (progression-free survival (PFS) and overall survival (OS)) according to the treatment used was performed using the Gehan-Wilcoxon test. The median of PFS and OS for patients treated with EGFR-TKI alone was 1.0 and 5.4 months compared to 2.0 and 14.0 months for patients treated with combination of EGFR-TKI and statin (p=0.025, p=0.130). In conclusion, the study results suggest significant improvement of PFS for patients treated with combination of statin and EGFR-TKI, and the difference in OS was not significant. Keywords Statin . KRAS . Lung cancer . NSCLC . EGFR-TKI . Erlotinib . Gefitinib
M. Minarik : L. Benesova Center for Applied Genomics of Solid Tumours, Genomac Research Institute, Prague, Czech Republic M. Minarik Department of Analytical Chemistry, Faculty of Sciences, Charles University in Prague, Prague, Czech Republic Z. Bortlicek Institute of Biostatistics and Analysis, Faculty of Medicine, Masaryk University, Brno, Czech Republic O. Topolcan Department of Nuclear Medicine, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, Prague, Czech Republic
Introduction Lung cancer is a leading cause of cancer-related deaths worldwide [1]. Non-small cell lung cancer constitutes more than 80 % of all lung carcinomas [2]. Tyrosine kinase inhibitors (TKIs) directed at epidermal growth factor receptor (EGFR) represent one of novel effective agents approved for the treatment of patients with advancedstage NSCLC. The Kirsten rat sarcoma viral oncogene (KRAS) is a member of human RAS oncogene family
Tumor Biol.
producing a self-inactivating signal transducer. Activation of EGFR initiates phosphorylation of tyrosine residues leading to activation of several signalling pathways including RAS-RAF-MEK kinase pathway [3]. This activation is usually transient as a result of an intrinsic GTPase activity of a KRAS protein. KRAS gene mutations eliminate an intrinsic GTPase activity resulting in constitutively active KRAS protein triggering downstream signalling pathways [4, 5]. Among patients with NSCLC, KRAS mutations are found in 15 to 25 % of cases [6, 7]. The KRAS mutations have been widely reported as a negative prognostic factor and also as a biomarker predicting resistance to EGFR-TKIs, especially the predominant type G12C [8–12]. Thus, the treatment of patients with advanced-stage NSCLC harbouring KRAS mutation is still a challenging topic in the field of thoracic oncology. Several experimental studies have recently shown that statins can directly block tumour cell growth, invasion and metastatic potential both in vivo and in vitro [13–15]. In the present work, we focused on the role of statins in patients with advanced-stage NSCLC harbouring KRAS mutation treated with EGFR-TKIs.
Patients and methods Patients and treatment We analysed clinical data of 67 patients with cytologically or histologically confirmed locally advanced (IIIB) or metastatic stage (IV) NSCLC harbouring KRAS mutation treated with erlotinib or gefitinib. Patients were treated between years 2003 and 2013. We compared outcome of two groups of patients. The first group involved 55 patients treated with EGFR-TKI alone. The second group involved 12 patients treated with combination of EGFR-TKI and statin. Both erlotinib and gefitinib were administered orally at the standard approved doses of 150 and 250 mg daily, respectively. The treatment was continued until disease progression or development of intolerable toxic effects. Dose interruption or reduction was permitted in the event of treatment-related toxicity. Atorvastatin and simvastatin were administered orally at a standard dose of 20 mg daily. Data source The clinical registry TULUNG (http://tulung. registry.cz/), in which Faculty Hospital Pilsen is participating since its creation, is a non-interventional post-registration database of epidemiological and clinical data of patients with advanced-stage NSCLC treated with targeted therapies in the Czech Republic. The registry contains anonymized individual patient data including demographic parameters, initial staging and disease characteristics, baseline patient information at the start of targeted therapy, as well as data on survival and adverse
events which is updated at least twice a year. Data on statins therapy is not recorded in this registry, so we extracted these values from hospital information systemic and merged it to registry data. The protocol was approved by the independent ethics committee of the University Hospital Pilsen and complied with the International Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical Practice guidelines, the Declaration of Helsinki and local laws. Clinical monitoring and statistics The treatment was prospectively monitored and the clinical course of patients was continuously assessed at specific time points. Clinical follow-up controls including physical examination, plain chest X-ray and routine laboratory tests were performed every 3–4 weeks; computed tomography (CT) or positron emission tomography—(PET)-CT—was performed after 2 or 3 months of the treatment. The objective tumour response was assessed by investigators using Response Evaluation Criteria in Solid Tumours (RECIST) [16]. Progression-free survival (PFS) was determined from the date of erlotinib or gefitinib initiation until the date of the first documented progression or death. Overall survival (OS) was determined from the date of erlotinib or gefitinib initiation until the date of death. Statistics Standard summary statistics were used to describe the sample data set. The significance of differences in baseline characteristics according to statins therapy was assessed using Fisher’s exact test or Mann-Whitney test. PFS and OS were estimated using Kaplan-Meier method and all point estimates were accompanied by 95 % confidence intervals. Statistical significance of the differences in Kaplan-Meier estimates was assessed using the GehanWilcoxon test. As a level of statistical significance, alpha=0.05 was used. KRAS and EGFR mutation analysis The tumour specimens acquired during initial bronchoscopy examination were evaluated by a senior cytologist using standard Giemsa staining. In a few cases, a tumour biopsy was processed into formalin-fixed paraffin-embedded (FFPE) histological sections. The cytology slides or, eventually, the FFPE sections were submitted for molecular genetic testing, which included detection of somatic mutations in exon 1 of KRAS gene and mutations in exon 19 (deletion) and 21 (L858R) of EGFR gene. If necessary, tumour cells were carefully selected and removed from the samples by laser microdissection using a P.A.L.M. microlaser instrument (Carl Zeiss MicroImaging GmbH, Jena, Germany). The microdissected cells were collected directly into the polymerase chain reaction (PCR) buffer and processed without a special DNA extraction step. In all other cases, the DNA
Tumor Biol.
was extracted from tissue cells by a standard spin column procedure using JetQuick Tissue DNA Isolation Kit (Genomed GmbH, Loehne, Germany). Mutations were tested by GenoScan mutation detection kits (Genomac International, Prague, Czech Republic) utilizing a denaturing capillary electrophoresis (DCE) technique on an ABI PRISM 3100 16-capillary genetic analyzer (Applied Biosystems, Foster City, CA, USA). Detected mutations were confirmed by Sanger DNA sequencing using a BigDye v 3.0 chemistry (Applied Biosystems, Foster City, CA, USA). In rare cases, where the overall fraction of mutated DNA was below the 20 % threshold for DNA sequencing, mutation was identified indirectly after forming only a homoduplex fragment with a given known mutation reference standard.
are summarized in Table 1. There were no statistically significant differences in baseline patients’ characteristics between compared groups (Table 1). Relation between statin use and survival The median PFS for patients treated with EGFR-TKI without statin was 1.0 compared to 2.0 months for patients treated with combination of EGFR-TKI and statin (Fig. 1a), and the difference was statistically significant (p=0.025). The median OS for patients treated with EGFR-TKI without statin was 5.4 compared to 14.0 months for patients treated with combination of EGFR-TKI and statin (Fig. 1b), and the difference was not statistically significant (p=0.130). The PFS and OS data are summarized in Table 2.
Discussion Results Patient characteristics The study included 67 patients harbouring KRAS-mutated and wild-type EGFR gene. Fifty-five patients were treated with EGFR-TKI without statins and 12 patients were treated with combination of EGFR-TKI and statin including 7 (58.3 %) patients treated with atorvastatin and five (41.7 %) patients treated with simvastatin. The baseline patients’ characteristics
Table 1
The mevalonate pathway produces various end products including cholesterol, dolichol, ubiquinone, isopentenyladenine, geranylgeranyl pyrophosphate and farnesyl pyrophosphate [17]. These end products play a critical role in many biological processes in both normal and cancer cells. Cancer cells are highly dependent on the sustained availability of the end products of the mevalonate pathway [13]. Geranylgeranyl pyrophosphate and farnesyl pyrophosphate
Baseline patients’ characteristics
Sex, n (%) Age (years) KRAS mutation, n (%) Smoking, n (%)
Histology, n (%)
Stage, n (%) ECOG PS, n (%)
Therapy, n (%) Line of therapy, n (%)
Female Male Median (min–max) G12C Other Current smoker Former smoker Never smoker Adenocarcinoma Squamous Not otherwise specified IIIB IV PS 0 PS 1 PS 2 Gefitinib Erlotinib 1st line 2nd line 3rd or 4th line
EGFR-TKI + statin (N=12)
EGFR-TKI alone (N=55)
p value
3 (25.0) 9 (75.0) 64 years (42–76) 6 (50.0) 6 (50.0) 6 (50.0) 4 (33.3) 2 (16.7) 11 (91.7) 1 (8.3) 0 (0) 1 (8.3) 11 (91.7) 0 (0) 8 (66.7) 4 (33.3) 1 (8.3) 11 (91.7) 3 (25.0) 4 (33.3) 5 (41.7)
25 (45.5) 30 (54.5) 61 years (28–81) 27 (49.1) 28 (50.9) 37 (67.3) 13 (23.6) 5 (9.1) 40 (72.7) 12 (21.8) 3 (5.5) 16 (29.1) 39 (70.9) 1 (1.8) 38 (69.1) 16 (29.1) 19 (34.5) 36 (65.5) 8 (14.5) 35 (63.6) 12 (21.8)
0.333 0.600 0.999 0.439
0.570
0.270 0.999
0.091 0.113
Tumor Biol. Fig. 1 Comparison of progression-free (a) and overall (b) survival between patients treated with EGFR-TKI in combination with statin and EGFR-TKI alone
are required for post-translational modifications of a wide variety of proteins including the RAS family that play a critical role in transducing EGFR signals as mentioned above [18]. Blockade of the conversion of 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) to mevalonate by the inhibition of HMGCoA reductase using statins results in decreased levels of mevalonate and its downstream metabolites, which may influence several cellular processes including EGFR signalling. However, recently published results of randomized phase II study comparing gefitinib plus simvastatin with gefitinib alone in an unselected population of NSCLC patients failed to demonstrate influence of statins on the efficacy of EGFRTKIs [19]; there are several findings suggesting that the effect of statins could be limited to patients harbouring KRAS mutation. Chen et al. and Park et al. have recently published interesting results showing that statins overcome resistance to gefitinib in KRAS-mutated NSCLC cells [20, 21]. The study published by Chen et al. clearly revealed that the inhibition of the PI3K/AKT and MEK/ERK pathways by atorvastatin in KRAS mutant NSCLC cells correlates with disruption of the KRAS/RAF and KRAS/PI3K complexes [20]. Based on the given findings of the previous experimental studies, we conducted this retrospective study focused on the effect of statins in patients with advanced-stage NSCLC harbouring KRAS mutation treated with EGFR-TKIs in conditions of a common clinical practice. We observed significantly longer PFS for patients treated with combination of statin and EGFR-TKI Table 2 Summary of progression-free and overall survival data Median PFS (95 % CI) 3-month PFS (95 % CI) 6-month PFS (95 % CI) Median OS (95 % CI) 6-month OS (95 % CI) 12-month OS (95 % CI)
compared to those treated with EGFR-TKI alone (2.0 vs. 1.0 month, p=0.025). PFS for patients treated with combination of statin and EGFR-TKI was comparable to a similar cohort of patients harbouring wild-type KRAS and wild-type EGFR genes reported in our previous studies [12, 22]. Although the difference in OS did not reach statistical significance, there was a visible trend towards longer OS for those treated with combination of statin and EGFR-TKI (14.0 vs. 5.4 months, p=0.130). The treatment in both groups was tolerated well and no cases of severe toxicity- or treatmentrelated deaths were recorded. The most common adverse events (AEs) were skin rash and diarrhoea. The incidence of AEs was comparable between both groups (data not shown), which is in agreement with results of several previous clinical trials [23–25]. Our study results suggest that combination of statins and EGFR-TKIs may represent a promising therapeutic approach for patients with NSCLC harbouring KRAS mutation. Moreover, the nonoverlapping spectrum of toxicities of these agents should be mentioned. The principal limitations of our study are relatively small number of patients included and its retrospective design. In conclusion, the present study results suggest significant improvement of PFS for patients treated with combination of statin and EGFR-TKI, and the difference in OS was not significant. To our knowledge, this is the first study focused on the effect of statins in selected patients with advanced-stage NSCLC harbouring KRAS
EGFR-TKI + statin (N=12)
EGFR-TKI alone (N=55)
p value
2.0 months (1.8–2.2) 33.3 % (6.7–60.0) 16.7 % (0.1–37.8) 14.0 months (0.1–29.6) 75.0 % (50.5–99.5) 57.1 % (28.5–85.8)
1.0 month (0.8–1.3) 13.0 % (4.0–22.0) 5.6 % (0.1–11.7) 5.4 months (2.8–8.0) 47.0 % (33.4–60.5) 32.2 % (19.2–45.2)
0.025
0.130
Tumor Biol.
mutation treated with EGFR-TKIs. A randomized prospective clinical trial should be conducted to confirm our results in the future. Acknowledgments The authors would like to thank all patients voluntarily taking part in this study. This work was supported by the grant no. 9087 of the Czech Ministry of Health and by the project CZ.1.05/2.1.00/ 03.0076 from European Regional Development Fund. Conflicts of interest None
References 1. Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol. 2001;2(9):533–43. 2. Brambilla E, Travis WD, Colby TV, Corrin B, Shimosato Y. The new World Health Organization classification of lung tumours. Eur Respir J. 2001;18:1059–68. 3. Molina JR, Adjei AA. The Ras/Raf/MAPK pathway. J Thorac Oncol. 2006;1:7–9. 4. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer. 2003;3:459–65. 5. Siddiqui AD, Piperdi B. KRAS mutation in colon cancer: a marker of resistance to EGFR-I therapy. Ann Surg Oncol. 2010;17:1168–76. 6. Lopez-Chavez A, Carter CA, Giaccone G. The role of KRAS mutations in resistance to EGFR inhibition in the treatment of cancer. Curr Opin Investig Drugs. 2009;10:1305–14. 7. Ding L, Getz G, Wheeler DA, Mardis ER, McLellan MD, Cibulskis K, et al. Somatic mutations affect key pathways in lung adenocarcinoma. Nature. 2008;455:1069–75. 8. Marchetti A, Milella M, Felicioni L, Cappuzzo F, Irtelli L, Del Grammastro M, et al. Clinical implications of KRAS mutations in lung cancer patients treated with tyrosine kinase inhibitors: an important role for mutations in minor clones. Neoplasia. 2009;11:1084–92. 9. Eberhard DA, Johnson BE, Amler LC, Goddard AD, Heldens SL, Herbst RS, et al. Mutations in the epidermal growth factor receptor and in KRAS are predictive and prognostic indicators in patients with non-small-cell lung cancer treated with chemotherapy alone and in combination with erlotinib. J Clin Oncol. 2005;23:5900–9. 10. Bonanno L, Schiavon M, Nardo G, Bertorelle R, Bonaldi L, Galligioni A, et al. Prognostic and predictive implications of EGFR mutations, EGFR copy number and KRAS mutations in advanced stage lung adenocarcinoma. Anticancer Res. 2010;30:5121–8. 11. Liu HP, Isaac Wu HD, Chang JW, Wu YC, Yang HY, Chen YT, et al. Prognostic implications of epidermal growth factor receptor and KRAS gene mutations and epidermal growth factor
receptor gene copy numbers in patients with surgically resectable non-small cell lung cancer in Taiwan. J Thorac Oncol. 2010;5:1175–84. 12. Fiala O, Pesek M, Finek J, Benesova L, Belsanova B, Minarik M. The dominant role of G12C over other KRAS mutation types in the negative prediction of efficacy of epidermal growth factor receptor tyrosine kinase inhibitors in non-small cell lung cancer. Cancer Genet. 2013;206:26–31. 13. Chan KK, Oza AM, Siu LL. The statins as anticancer agents. Clin Cancer Res. 2003;9(1):10–9. 14. Keyomarsi K, Sandoval L, Band V, et al. Synchronization of tumor and normal cells from G1 to multiple cell cycles by lovastatin. Cancer Res. 2003;9:10–9. 15. Wong WW, Dimitroulakos J, Minden MD, Penn LZ. HMG-CoA reductase inhibitors and the malignant cell: the statin family of drugs as triggers of tumor-specific apoptosis. Leukemia. 2002;16:508–19. 16. Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, et al. New guidelines to evaluate the response to treatment in solid tumours. 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. 17. Goldstein JL, Brown MS. Regulation of the mevalonate pathway. Nature. 1990;343:425–30. 18. Pruitt K, Der CJ. Ras and Rho regulation of the cell cycle and oncogenesis. Cancer Lett. 2001;171:1–10. 19. Han JY, Lee SH, Yoo NJ, Hyung LS, Moon YJ, Yun T, et al. A randomized phase II study of gefitinib plus simvastatin versus gefitinib alone in previously treated patients with advanced non-small cell lung cancer. Clin Cancer Res. 2011;17:1553–60. 20. Chen J, Bi H, Hou J, Zhang X, Zhang C, Yue L, et al. Atorvastatin overcomes gefitinib resistance in KRAS mutant human non-small cell lung carcinoma cells. Cell Death Dis. 2013;4:e814. 21. Park IH, Kim JY, Jung JI, Han JY. Lovastatin overcomes gefitinib resistance in human non-small cell lung cancer cells with K-Ras mutations. Invest New Drugs. 2010;28:791–9. 22. Fiala O, Pesek M, Finek J, Krejci J, Ricar J, Bortlicek Z, et al. Skin rash as useful marker of erlotinib efficacy in NSCLC and its impact on clinical practice. Neoplasma. 2013;60:26–32. 23. Thibault A, Samid D, Tompkins AC, Figg WD, Cooper MR, Hohl RJ, et al. Phase I study of lovastatin, an inhibitor of the mevalonate pathway, in patients with cancer. Clin Cancer Res. 1996;2:483–91. 24. Arteaga CL, Johnson DH. Tyrosine kinase inhibitors-ZD1839 (Iressa). Curr Opin Oncol. 2001;13:491–8. 25. Albanell J, Rojo F, Averbuch S, Feyereislova A, Mascaro JM, Herbst R, et al. Pharmacodynamic studies of the epidermal growth factor receptor inhibitor ZD1839 in skin from cancer patients: histopathologic and molecular consequences of receptor inhibition. J Clin Oncol. 2002;20:110–24.
RESEARCH ARTICLE
Statins augment efficacy of EGFR-TKIs in patients with advanced-stage non-small cell lung cancer harbouring KRAS mutation Ondrej Fiala & Milos Pesek & Jindrich Finek & Marek Minarik & Lucie Benesova & Zbynek Bortlicek & Ondrej Topolcan
Received: 9 December 2014 / Accepted: 10 February 2015 # International Society of Oncology and BioMarkers (ISOBM) 2015
Abstract Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) represent novel effective agents approved for the treatment of patients with advanced-stage NSCLC. KRAS mutations have been reported as a negative prognostic and predictive factor in patients with NSCLC treated with EGFR-TKIs. Several studies have recently shown that statins can block tumour
O. Fiala (*) : J. Finek Department of Oncology and Radiotherapy, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, alej Svobody 80, 304 60 Pilsen, Czech Republic e-mail: [email protected] O. Fiala Biomedical Center, Faculty of Medicine in Pilsen, Charles University in Prague, Prague, Czech Republic M. Pesek Department of Pneumology, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, Prague, Czech Republic
cell growth, invasion and metastatic potential. We analysed clinical data of 67 patients with locally advanced (IIIB) or metastatic stage (IV) NSCLC harbouring Kirsten rat sarcoma viral oncogene (KRAS) mutation treated with erlotinib or gefitinib. Twelve patients were treated with combination of EGFR-TKI and statin and 55 patients were treated with EGFR-TKI alone. Comparison of patients’ survival (progression-free survival (PFS) and overall survival (OS)) according to the treatment used was performed using the Gehan-Wilcoxon test. The median of PFS and OS for patients treated with EGFR-TKI alone was 1.0 and 5.4 months compared to 2.0 and 14.0 months for patients treated with combination of EGFR-TKI and statin (p=0.025, p=0.130). In conclusion, the study results suggest significant improvement of PFS for patients treated with combination of statin and EGFR-TKI, and the difference in OS was not significant. Keywords Statin . KRAS . Lung cancer . NSCLC . EGFR-TKI . Erlotinib . Gefitinib
M. Minarik : L. Benesova Center for Applied Genomics of Solid Tumours, Genomac Research Institute, Prague, Czech Republic M. Minarik Department of Analytical Chemistry, Faculty of Sciences, Charles University in Prague, Prague, Czech Republic Z. Bortlicek Institute of Biostatistics and Analysis, Faculty of Medicine, Masaryk University, Brno, Czech Republic O. Topolcan Department of Nuclear Medicine, Medical School and Teaching Hospital in Pilsen, Charles University in Prague, Prague, Czech Republic
Introduction Lung cancer is a leading cause of cancer-related deaths worldwide [1]. Non-small cell lung cancer constitutes more than 80 % of all lung carcinomas [2]. Tyrosine kinase inhibitors (TKIs) directed at epidermal growth factor receptor (EGFR) represent one of novel effective agents approved for the treatment of patients with advancedstage NSCLC. The Kirsten rat sarcoma viral oncogene (KRAS) is a member of human RAS oncogene family
Tumor Biol.
producing a self-inactivating signal transducer. Activation of EGFR initiates phosphorylation of tyrosine residues leading to activation of several signalling pathways including RAS-RAF-MEK kinase pathway [3]. This activation is usually transient as a result of an intrinsic GTPase activity of a KRAS protein. KRAS gene mutations eliminate an intrinsic GTPase activity resulting in constitutively active KRAS protein triggering downstream signalling pathways [4, 5]. Among patients with NSCLC, KRAS mutations are found in 15 to 25 % of cases [6, 7]. The KRAS mutations have been widely reported as a negative prognostic factor and also as a biomarker predicting resistance to EGFR-TKIs, especially the predominant type G12C [8–12]. Thus, the treatment of patients with advanced-stage NSCLC harbouring KRAS mutation is still a challenging topic in the field of thoracic oncology. Several experimental studies have recently shown that statins can directly block tumour cell growth, invasion and metastatic potential both in vivo and in vitro [13–15]. In the present work, we focused on the role of statins in patients with advanced-stage NSCLC harbouring KRAS mutation treated with EGFR-TKIs.
Patients and methods Patients and treatment We analysed clinical data of 67 patients with cytologically or histologically confirmed locally advanced (IIIB) or metastatic stage (IV) NSCLC harbouring KRAS mutation treated with erlotinib or gefitinib. Patients were treated between years 2003 and 2013. We compared outcome of two groups of patients. The first group involved 55 patients treated with EGFR-TKI alone. The second group involved 12 patients treated with combination of EGFR-TKI and statin. Both erlotinib and gefitinib were administered orally at the standard approved doses of 150 and 250 mg daily, respectively. The treatment was continued until disease progression or development of intolerable toxic effects. Dose interruption or reduction was permitted in the event of treatment-related toxicity. Atorvastatin and simvastatin were administered orally at a standard dose of 20 mg daily. Data source The clinical registry TULUNG (http://tulung. registry.cz/), in which Faculty Hospital Pilsen is participating since its creation, is a non-interventional post-registration database of epidemiological and clinical data of patients with advanced-stage NSCLC treated with targeted therapies in the Czech Republic. The registry contains anonymized individual patient data including demographic parameters, initial staging and disease characteristics, baseline patient information at the start of targeted therapy, as well as data on survival and adverse
events which is updated at least twice a year. Data on statins therapy is not recorded in this registry, so we extracted these values from hospital information systemic and merged it to registry data. The protocol was approved by the independent ethics committee of the University Hospital Pilsen and complied with the International Ethical Guidelines for Biomedical Research Involving Human Subjects, Good Clinical Practice guidelines, the Declaration of Helsinki and local laws. Clinical monitoring and statistics The treatment was prospectively monitored and the clinical course of patients was continuously assessed at specific time points. Clinical follow-up controls including physical examination, plain chest X-ray and routine laboratory tests were performed every 3–4 weeks; computed tomography (CT) or positron emission tomography—(PET)-CT—was performed after 2 or 3 months of the treatment. The objective tumour response was assessed by investigators using Response Evaluation Criteria in Solid Tumours (RECIST) [16]. Progression-free survival (PFS) was determined from the date of erlotinib or gefitinib initiation until the date of the first documented progression or death. Overall survival (OS) was determined from the date of erlotinib or gefitinib initiation until the date of death. Statistics Standard summary statistics were used to describe the sample data set. The significance of differences in baseline characteristics according to statins therapy was assessed using Fisher’s exact test or Mann-Whitney test. PFS and OS were estimated using Kaplan-Meier method and all point estimates were accompanied by 95 % confidence intervals. Statistical significance of the differences in Kaplan-Meier estimates was assessed using the GehanWilcoxon test. As a level of statistical significance, alpha=0.05 was used. KRAS and EGFR mutation analysis The tumour specimens acquired during initial bronchoscopy examination were evaluated by a senior cytologist using standard Giemsa staining. In a few cases, a tumour biopsy was processed into formalin-fixed paraffin-embedded (FFPE) histological sections. The cytology slides or, eventually, the FFPE sections were submitted for molecular genetic testing, which included detection of somatic mutations in exon 1 of KRAS gene and mutations in exon 19 (deletion) and 21 (L858R) of EGFR gene. If necessary, tumour cells were carefully selected and removed from the samples by laser microdissection using a P.A.L.M. microlaser instrument (Carl Zeiss MicroImaging GmbH, Jena, Germany). The microdissected cells were collected directly into the polymerase chain reaction (PCR) buffer and processed without a special DNA extraction step. In all other cases, the DNA
Tumor Biol.
was extracted from tissue cells by a standard spin column procedure using JetQuick Tissue DNA Isolation Kit (Genomed GmbH, Loehne, Germany). Mutations were tested by GenoScan mutation detection kits (Genomac International, Prague, Czech Republic) utilizing a denaturing capillary electrophoresis (DCE) technique on an ABI PRISM 3100 16-capillary genetic analyzer (Applied Biosystems, Foster City, CA, USA). Detected mutations were confirmed by Sanger DNA sequencing using a BigDye v 3.0 chemistry (Applied Biosystems, Foster City, CA, USA). In rare cases, where the overall fraction of mutated DNA was below the 20 % threshold for DNA sequencing, mutation was identified indirectly after forming only a homoduplex fragment with a given known mutation reference standard.
are summarized in Table 1. There were no statistically significant differences in baseline patients’ characteristics between compared groups (Table 1). Relation between statin use and survival The median PFS for patients treated with EGFR-TKI without statin was 1.0 compared to 2.0 months for patients treated with combination of EGFR-TKI and statin (Fig. 1a), and the difference was statistically significant (p=0.025). The median OS for patients treated with EGFR-TKI without statin was 5.4 compared to 14.0 months for patients treated with combination of EGFR-TKI and statin (Fig. 1b), and the difference was not statistically significant (p=0.130). The PFS and OS data are summarized in Table 2.
Discussion Results Patient characteristics The study included 67 patients harbouring KRAS-mutated and wild-type EGFR gene. Fifty-five patients were treated with EGFR-TKI without statins and 12 patients were treated with combination of EGFR-TKI and statin including 7 (58.3 %) patients treated with atorvastatin and five (41.7 %) patients treated with simvastatin. The baseline patients’ characteristics
Table 1
The mevalonate pathway produces various end products including cholesterol, dolichol, ubiquinone, isopentenyladenine, geranylgeranyl pyrophosphate and farnesyl pyrophosphate [17]. These end products play a critical role in many biological processes in both normal and cancer cells. Cancer cells are highly dependent on the sustained availability of the end products of the mevalonate pathway [13]. Geranylgeranyl pyrophosphate and farnesyl pyrophosphate
Baseline patients’ characteristics
Sex, n (%) Age (years) KRAS mutation, n (%) Smoking, n (%)
Histology, n (%)
Stage, n (%) ECOG PS, n (%)
Therapy, n (%) Line of therapy, n (%)
Female Male Median (min–max) G12C Other Current smoker Former smoker Never smoker Adenocarcinoma Squamous Not otherwise specified IIIB IV PS 0 PS 1 PS 2 Gefitinib Erlotinib 1st line 2nd line 3rd or 4th line
EGFR-TKI + statin (N=12)
EGFR-TKI alone (N=55)
p value
3 (25.0) 9 (75.0) 64 years (42–76) 6 (50.0) 6 (50.0) 6 (50.0) 4 (33.3) 2 (16.7) 11 (91.7) 1 (8.3) 0 (0) 1 (8.3) 11 (91.7) 0 (0) 8 (66.7) 4 (33.3) 1 (8.3) 11 (91.7) 3 (25.0) 4 (33.3) 5 (41.7)
25 (45.5) 30 (54.5) 61 years (28–81) 27 (49.1) 28 (50.9) 37 (67.3) 13 (23.6) 5 (9.1) 40 (72.7) 12 (21.8) 3 (5.5) 16 (29.1) 39 (70.9) 1 (1.8) 38 (69.1) 16 (29.1) 19 (34.5) 36 (65.5) 8 (14.5) 35 (63.6) 12 (21.8)
0.333 0.600 0.999 0.439
0.570
0.270 0.999
0.091 0.113
Tumor Biol. Fig. 1 Comparison of progression-free (a) and overall (b) survival between patients treated with EGFR-TKI in combination with statin and EGFR-TKI alone
are required for post-translational modifications of a wide variety of proteins including the RAS family that play a critical role in transducing EGFR signals as mentioned above [18]. Blockade of the conversion of 3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) to mevalonate by the inhibition of HMGCoA reductase using statins results in decreased levels of mevalonate and its downstream metabolites, which may influence several cellular processes including EGFR signalling. However, recently published results of randomized phase II study comparing gefitinib plus simvastatin with gefitinib alone in an unselected population of NSCLC patients failed to demonstrate influence of statins on the efficacy of EGFRTKIs [19]; there are several findings suggesting that the effect of statins could be limited to patients harbouring KRAS mutation. Chen et al. and Park et al. have recently published interesting results showing that statins overcome resistance to gefitinib in KRAS-mutated NSCLC cells [20, 21]. The study published by Chen et al. clearly revealed that the inhibition of the PI3K/AKT and MEK/ERK pathways by atorvastatin in KRAS mutant NSCLC cells correlates with disruption of the KRAS/RAF and KRAS/PI3K complexes [20]. Based on the given findings of the previous experimental studies, we conducted this retrospective study focused on the effect of statins in patients with advanced-stage NSCLC harbouring KRAS mutation treated with EGFR-TKIs in conditions of a common clinical practice. We observed significantly longer PFS for patients treated with combination of statin and EGFR-TKI Table 2 Summary of progression-free and overall survival data Median PFS (95 % CI) 3-month PFS (95 % CI) 6-month PFS (95 % CI) Median OS (95 % CI) 6-month OS (95 % CI) 12-month OS (95 % CI)
compared to those treated with EGFR-TKI alone (2.0 vs. 1.0 month, p=0.025). PFS for patients treated with combination of statin and EGFR-TKI was comparable to a similar cohort of patients harbouring wild-type KRAS and wild-type EGFR genes reported in our previous studies [12, 22]. Although the difference in OS did not reach statistical significance, there was a visible trend towards longer OS for those treated with combination of statin and EGFR-TKI (14.0 vs. 5.4 months, p=0.130). The treatment in both groups was tolerated well and no cases of severe toxicity- or treatmentrelated deaths were recorded. The most common adverse events (AEs) were skin rash and diarrhoea. The incidence of AEs was comparable between both groups (data not shown), which is in agreement with results of several previous clinical trials [23–25]. Our study results suggest that combination of statins and EGFR-TKIs may represent a promising therapeutic approach for patients with NSCLC harbouring KRAS mutation. Moreover, the nonoverlapping spectrum of toxicities of these agents should be mentioned. The principal limitations of our study are relatively small number of patients included and its retrospective design. In conclusion, the present study results suggest significant improvement of PFS for patients treated with combination of statin and EGFR-TKI, and the difference in OS was not significant. To our knowledge, this is the first study focused on the effect of statins in selected patients with advanced-stage NSCLC harbouring KRAS
EGFR-TKI + statin (N=12)
EGFR-TKI alone (N=55)
p value
2.0 months (1.8–2.2) 33.3 % (6.7–60.0) 16.7 % (0.1–37.8) 14.0 months (0.1–29.6) 75.0 % (50.5–99.5) 57.1 % (28.5–85.8)
1.0 month (0.8–1.3) 13.0 % (4.0–22.0) 5.6 % (0.1–11.7) 5.4 months (2.8–8.0) 47.0 % (33.4–60.5) 32.2 % (19.2–45.2)
0.025
0.130
Tumor Biol.
mutation treated with EGFR-TKIs. A randomized prospective clinical trial should be conducted to confirm our results in the future. Acknowledgments The authors would like to thank all patients voluntarily taking part in this study. This work was supported by the grant no. 9087 of the Czech Ministry of Health and by the project CZ.1.05/2.1.00/ 03.0076 from European Regional Development Fund. Conflicts of interest None
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