Abstract
Background/Aim: Patients with non-small cell lung cancer (NSCLC) harboring epidermal growth factor receptor (EGFR) gene mutations or echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) rearrangement often have a better prognosis when they are treated with specific inhibitors than when treated with cytotoxic agents. However, the associations between gene mutations and cytotoxic chemosensitivity are still unclear. The objective of the present study was to identify which clinicopathological factors, including genetic mutations, influence chemosensitivity, determined using the succinate dehydrogenase inhibition (SDI) test in patients with NSCLC. Materials and Methods: The chemosensitivity of tumor tissues from 96 patients with NSCLC who underwent surgical resection was evaluated using the SDI test. Results: In patients with adenocarcinoma, tumors with EGFR gene mutations were significantly more sensitive to 5-fluorouracil (5-FU) than tumors without EGFR gene mutations (p<0.0149). Conclusion: Our data suggest that patients with adenocarcinoma harboring EGFR gene mutations may be susceptible to 5-FU.
- Non-small cell lung cancer
- anaplastic lymphoma kinase
- epidermal growth factor receptor
- chemosensitivity
Mutations in the epidermal growth factor receptor (EGFR) gene and echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) rearrangements are acknowledged as specific biomarkers for the efficacy of EGFR tyrosine kinase inhibitors (TKIs) and ALK inhibitors. The discovery of inhibitors targeting specific EGFR mutations and EML4-ALK rearrangement has dramatically changed the conventional treatment of NSCLC. Patients with NSCLC harboring those mutations often have a better prognosis when they are treated with specific inhibitors than when they are treated with cytotoxic agents (1, 2). However, administration of EGFR-TKIs may promote the emergence of resistance to EGFR-TKIs, ultimately leading to tumor relapse. Although several drugs have been developed to target acquired resistance to EGFR-TKIs (3), platinum-based doublet chemotherapy or other cytotoxic chemotherapeutic agents are still used as first-line systemic chemotherapy in patients with advanced NSCLC or following postoperative recurrence and in patients with EGFR mutations.
Insufficient studies have examined the association between driver genetic mutations and chemosensitivity. Some studies have revealed that EGFR gene mutations could be an effective biomarker for cytotoxic agents (4-6). Moreover, several recent studies have shown that the EML4-ALK fusion gene might reduce thymidine synthase expression, increasing the sensitivity of EML4-ALK rearrangement-positive carcinoma to pemetrexed (7, 8). Although Yoshimasu et al. (9) reported an association between EGFR mutations and response to docetaxel, the association between these somatic gene mutations and cytotoxic chemosensitivity is unclear. This is because of the difficulty of conducting in vitro studies to demonstrate the efficacy of cytotoxic agents under conditions in which the driver genes are inhibited (i.e., “loss of function”); inhibiting these mutations directly promotes apoptosis and cell death.
The succinate dehydrogenase inhibition (SDI) test is an ex vivo method that has been used for clinical chemosensitivity testing and the screening of anticancer drugs because of is simplicity and cost-effectiveness (10, 11). Therefore, we applied the SDI test to fresh samples obtained during surgery to evaluate the chemosensitivity of the NSCLC tissue samples and determine statistical associations between gene mutations and cytotoxic chemosensitivity. Our objective was to identify clinicopathological factors, including genetic mutations, that were associated with chemosensitivity using the SDI test in patients with NSCLC.
Patients and Methods
Patients and sample collection. In this retrospective study, we enrolled 96 patients with NSCLC who underwent surgical resection at the Kyushu University Hospital between April 2004 and March 2013 and who had sufficient tumor volume not to prevent precise pathological diagnosis in sampling the tumor for the SDI test. At the time of surgery, all patients signed board informed consent forms covering the procedures and the use of their medical records for research purposes.
SDI test. The chemosensitivities of fresh surgical specimens to cisplatin, pemetrexed, 5-fluorouracil (5-FU), gemcitabine, irinotecan, paclitaxel and gefitinib were determined using the SDI test. The SDI test was performed as previously described (11, 12). Briefly, the SDI test is based on the cellular succinate dehydrogenase activity as determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT). The tissue specimens were digested to obtain single-cell suspensions and incubated for 72 h with the specified drugs. The formazan formed from MTT was extracted with dimethyl sulfoxide and cell viability was determined by the absorbance of the formazan ,which was measured at 540 nm using a spectrophotometer (Labsystems Multiskan JX; Thermo Bioanalysis, Helsinki, Finland).
Detection of EGFR mutations and EML4-ALK rearrangements. Gene mutations were determined in all NSCLC samples obtained in this study. A pathologist grossly dissected the tumor specimens to enrich the samples with as many tumor cells as possible. Genomic DNA was extracted and purified from formalin-fixed paraffin-embedded tissue samples. EGFR gene mutations were determined using the peptide nucleic acid-locked nucleic acid (PNA-LNA) polymerase chain reaction (PCR) clamp method (13) or a PCR-Invader assay (14). EML4-ALK rearrangements were detected by fluorescence in situ hybridization (FISH), immunohistochemistry or reverse-transcription PCR (RT-PCR) assays as previously described (15).
Statistical analyses. For the chemosensitivity analysis, we used one-sided unpaired Student's t-tests for comparisons between patients with or without mutations at a significance level of 5%. All analyses were performed using the JMP 11.0.0 software (SAS Institute Inc., Cary, NC, USA).
Results
Patients' characteristics. The key characteristics and clinical outcomes of the patients are presented in Table I. The median patient age was 67 years (range=37-85 years). Most of the patients had Stage IA tumors. Eighty-two patients had adenocarcinoma and 14 patients had squamous cell carcinoma. Classical activating EGFR gene mutations were detected in 39 patients (40.6%). Immunohistochemistry revealed abnormal ALK protein expression in 9 patients (9.4%). Patients with squamous cell carcinoma had no EGFR mutations. None of the patients harbored both EGFR mutations and EML4-ALK rearrangements.
Patients' characteristics.
Chemosensitivity to anticancer drugs according to tumor histology. Results of the SDI chemosensitivity test, according to tumor histology, are shown in Figure 1. Adenocarcinoma was more sensitive to pemetrexed than was squamous cell carcinoma (p=0.0007). By contrast, squamous cell carcinoma showed significantly greater sensitivity to cisplatin, gemcitabine, irinotecan and paclitaxel than did adenocarcinoma (p=0.0246, 0.0358, 0.0072 and 0.0035, respectively). The sensitivity to 5-FU was not significantly different between adenocarcinoma and squamous cell carcinoma.
Chemosensitivity to anticancer drugs according to the somatic gene mutation status. The results of the SDI test in patients with adenocarcinoma are shown in Figure 2 for EGFR mutations and Figure 3 for EML4-ALK rearrangement. Patients with EGFR gene mutations were significantly more sensitive to 5-FU than patients without EGFR gene mutations (p=0.0149). By contrast, there were no significant differences in the chemosensitivities to cisplatin (p=0.1336), pemetrexed (p=0.7428), gemcitabine (p=0.0975), irinotecan (p=0.0548) and paclitaxel (p=0.2621) between patients with or without EGFR gene mutations.
Distributions of the survival rate of cancer cells exposed to a panel of anticancer drugs in the succinate dehydrogenase inhibition test according to the histological type of non-small cell lung cancer. Ad, Adenocarcinoma; Sq, squamous cell carcinoma.
Regarding the ALK gene, we found no significant difference in the sensitivity to cytotoxic chemotherapy between patients with or without EML4-ALK rearrangement, because of the small number of patients with EML4-ALK rearrangement.
We also determined the sensitivity to gefitinib. Adenocarcinomas with EGFR gene mutations were more sensitive to gefitinib (p=0.0101) than adenocarcinomas with wild-type EGFR (data not shown).
Discussion
The results of our study allowed us to reach three major conclusions. First, adenocarcinomas harboring EGFR mutations were sensitive to 5-FU and, as would be expected, gefitinib. Second, NSCLC with EML4-ALK rearrangement(s) was not sensitive to any of the anticancer drugs applied in the SDI test. Third, adenocarcinoma was sensitive to pemetrexed, whereas squamous cell carcinoma was sensitive to cisplatin, gemcitabine, irinotecan and paclitaxel.
The application of screening tests, such as the SDI test, to surgically-resected raw samples is especially important considering the increasing reliance on individualized treatment of NSCLC. Of course, there are several arguments for and against using the SDI test in this setting. Until recently, chemosensitivity tests were performed before starting chemotherapy to determine which drugs were likely to be more effective (16). However, the SDI test has not been widely used for testing the chemosensitivity of lung cancer in clinical settings because the results of the SDI test are not always consistent with the response to effective drugs. Our data indicate that adenocarcinomas are more sensitive to pemetrexed than is squamous cell carcinoma, similar to the results of the JMDB trial (17). Our results also suggest that tumors with EGFR gene mutations are sensitive to gefitinib; these findings are also consistent with those of previous clinical trials (1). Our results provide evidence supporting the validity and accuracy of applying the SDI test to tumor samples from patients with NSCLC.
Distributions of the survival rate of cancer cells exposed to a panel of anticancer drugs in the succinate dehydrogenase inhibition test applied to adenocarcinomas with or without epidermal growth factor receptor (EGFR) gene mutations. +, EGFR mutation-positive; −, EGFR mutation-negative.
We consider that the SDI test is a useful means for investigating the association between chemosensitivity and driver oncogenes as possible tumor biomarkers. It has been suggested that 22% of the driver oncogenes in lung adenocarcinoma have not yet been identified (18) giving space to the identification of novel oncogenes in the near future by referencing the oncogenic mutation status to SDI data.
Perhaps, the most important finding of this study is that patients with adenocarcinoma harboring EGFR gene mutations may be sensitive to 5-FU. Suehisa et al. reported that EGFR wild-type cells are more responsive to 5-FU than EGFR mutant cells (19). Moreover, Mochinaga et al. reported that high dihydropyrimidine dehydrogenase expression was associated with EGFR gene mutations in adenocarcinoma, which suggests that tumors with EGFR mutations are resistant to 5-FU. However, these findings are still debatable because all of the studies were retrospective and performed at single institutions. Therefore, although multi-Institutional prospective studies are needed to determine the underlying mechanisms, the current results imply that the EGFR gene status might be a predictive biomarker for the response to 5-FU in patients with adenocarcinoma.
There are a few limitations to our study. First, the number of patients with ALK rearrangements might be too small to reach a firm conclusion. Second, the SDI test has a weakness in that the dissociated cells always contain normal cells, including fibroblasts and endothelial cells. Other assay systems, including the culture–drug sensitivity test (20) and the histoculture drug response assay (21), might overcome the weakness associated with the SDI test and provide valuable data regarding the chemosensitivity of NSCLC.
Distributions of the survival rate of cancer cells exposed to a panel of anticancer drugs in the succinate dehydrogenase inhibition test applied to adenocarcinoma with or without echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) rearrangement. +, EML4-ALK rearrangement-positive; −, EML4-ALK rearrangement-negative.
In conclusion, this was the first study to determine the association between the chemosensitivity of NSCLC harboring somatic mutations in an ex vivo setting. In particular, we found that EGFR gene mutations might be a predictive biomarker for the response to 5-FU in patients with adenocarcinoma. The SDI test might be useful to determine the sensitivity to anticancer drugs in NSCLC with driver gene mutations and, thus, its use should be reevaluated in this setting. Although the SDI test is a classical assay, it has yet to be fully-evaluated in the era of individualized chemotherapy.
Acknowledgements
The Authors thank Kyoko Miyamoto (Center for Cellular and Molecular Medicine, Kyushu University Hospital) for valuable comments and suggestions regarding the SDI test.
Footnotes
-
Conflicts of Interest
The Authors declare no conflicts of interest.
- Received November 7, 2014.
- Revision received November 16, 2014.
- Accepted November 25, 2014.
- Copyright© 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.