Abstract
Background/Aim: Tumor biomarkers are used for diagnostics and follow-up monitoring of patients with non-small cell lung cancer (NSCLC). We focused on the predictive role of neuron-specific enolase (NSE) and thimidine kinase (TK) in patients with advanced-stage NSCLC treated with epidermal growth factor tyrosine kinase inhibitors (EGFR-TKIs). Patients and Methods: In a total of 163 patients with advanced-stage (IIIB or IV) NSCLC treated with EGFR-TKIs (erlotinib or gefitinib), pre-treatment levels of NSE and TK were measured. Results: We observed significantly shorter progression-free (PFS) and overall survival (OS) in patients with high NSE levels (p=0.002; p=0.003) and also in those with high TK levels (p=0.026; p=0.020). The multivariate Cox proportional hazards model confirmed that high NSE is a strong independent predictive factor for short PFS (hazard ratio; HR=2.36; p=0.003). Conclusion: High pre-treatment serum levels of NSE is an independent biomarker predicting poor outcome of patients with NSCLC treated with EGFR-TKIs.
Lung cancer is the most common cause of cancer-related deaths worldwide (1). Non-small cell lung cancer (NSCLC) is the most frequent histological type of lung cancer, representing approximately 85% of cases (2). Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are novel effective agents used for the treatment of locally-advanced or metastatic-stage NSCLC. Erlotinib and gefitinib are orally-administered low-molecular weight EGFR-TKIs. Randomised phase III clinical trials demonstrated efficacy and safety of erlotinib and gefitinib in the treatment of patients with advanced-stage NSCLC (3-7). The aim of our study was to evaluate the predictive role of pretreatment serum levels of neuron specific enolase (NSE) and thimidine kinase (TK) in patients with advanced-stage NSCLC treated with EGFR-TKIs.
Patients and Methods
Patients and treatment. We analysed clinical data of 163 patients with cytologically or histologically confirmed locally-advanced (IIIB) or metastatic stage (IV) NSCLC treated with erlotinib or gefitinib. Patients were treated between 2003 and 2013. Both erlotinib and gefitinib were administered orally at the standard approved doses of 150 mg 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.
Clinical monitoring. 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 was performed every 3-4 weeks; computed tomography (CT) or positron emission tomography - (PET)-CT was performed after two or three months of treatment. Progression-free survival (PFS) was determined from the date of erlotinib or gefitinib initiation until the date of first documented progression or death. Overall survival (OS) was determined from the date of erlotinib or gefitinib initiation until the date of death.
Tumor marker measurement. Serum samples for measurement of tumor markers were collected within one month before EGFR-TKI treatment. Serum levels of NSE were measured using a immunoradiometric titration method (IRMA) (Beckman-Immunotech, city, county, USA). Serum levels of TK were measured using radioenzymatic assay (REA) (Beckman-Immunotech). The measurement was performed in the Central Immunoanalytic Laboratory at the Department of Nuclear Medicine, using the following cut-off values: NSE: 12.5 μg/l and TK: 8 IU/l Statistical analysis. Standard summary statistics were used to describe the sample data set. PFS and OS were calculated using the Kaplan Meier method and all point estimates were accompanied by 95% confidence intervals (CI). Statistical significance of the differences in Kaplan-Meier estimates was assessed using the log-rank test. Multivariate Cox proportional hazards model (hazard ratio; HR) was used to evaluate influence of all potential predictive and prognostic factors on the survival measures p=0.05 was used as a level of statistical significance.
Baseline patient characteristics.
EGFR mutation analysis. The tumor specimens acquired during initial bronschoscopy were evaluated by a senior cytologist using standard Giemsa staining. In a few cases, a tumor 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 EGFR genes. If necessary, tumor 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 was extracted from tissue cells by a standard spin-column procedure using the JetQuick Tissue DNA Issolation Kit (Genomed GmbH, Loehne, Germany). Mutations in exons 19 and 21 of the EGFR gene were tested by the 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). 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.
Results
Patients' characteristics. The study included 163 patients. The median age was 64 years (range 28-88 years). Ninety-nine (60.7%) patients were male, 66 (40.5%) had a positive smoking history, 85 (52.1%) had adenocarcinoma, 138 (84.7%) had stage IV disease at EGFR-TKI treatment initiation, 90 (55.2%) had an Eastern Cooperative Oncology Group (ECOG) performance status (PS) 0 or 1 and 118 (72.4%) patients had received at least one previous chemotherapy regimen. One hundred and forty-seven (90.2%) patients were treated with erlotinib and 16 (9.8%) patients were treated with gefitinib. Ninety-three patients were tested for activating EGFR mutation, 77 (82.8%) of them were wild-type EGFR and 16 (17.2%) were EGFR mutation-positive. The baseline patients' characteristics are summarized in Table I.
Pre-treatment levels of NSE and TK. Before the beginning of EGFR-TKI treatment, a high serum level of NSE (≥12.5 μg/l) was measured in 18 (12.0%) patients and a low serum level of NSE (<12.5 μg/l) was measured in 132 (88.0%) patients; a high serum level of TK (≥8 U/I) was measured in 98 (60.1%) patients and a low serum level of TK (<8 U/I) was measured in 65 (39.9%) patients.
Relation between NSE and TK levels and survival. The median PFS and OS for patients with high NSE was 1.1 and 3.7 compared to 2.6 and 11.6 months for patients with low NSE (p=0.002 and p=0.003) (Figure 1A, B). The median PFS and OS for patients with high TK was 2.1 and 8.5 compared to 2.9 and 17.4 months for patients with low TK (p=0.026 and p=0.020) (Figure 1C, D). The PFS and OS data are summarized in Table II. The multivariate Cox proportional hazards model revealed that the EGFR mutation status (HR=0.31; CI: 0.16-0.61; p=0.001) and pre-treatment levels of NSE (HR=2.36; CI: 1.34-4.17; p=0.003) were significant independent predictive factors for PFS, whereas EGFR mutation status (HR=0.40; 0.18-0.90; p=0.028) and PS (HR=1.89; 1.20-2.97; p=0.006) were significant independent predictive factors for OS (Table III).
Kaplan-Meier plots showing progression-free (PFS) and overall (OS) survival according to pretreatment levels of NSE (A, B) and TK (C, D).
Discussion
Considerable progress in the field of molecular biology led to identification of several biomarkers predicting for treatment efficacy of EGFR-TKIs. The presence of activating EGFR mutations (predominantly exon 19 deletions or a point-mutation in exon 21 termed L858R) is currently the strongest predictor of a good treatment response (8-12) to EGFR-TKIs and patients are selected for first-line treatment according to the presence of activating EGFR mutation. On the other hand the majority of NSCLC patients harbor wild-type EGFR gene and moreover there is still a large proportion of patients in whom it is not feasible to acquire an adequate tissue for EGFR mutation analysis. Therefore, new predictive tools are required. Serum tumor markers NSE and TK have been shown to be promising candidates for the improvement of diagnosis, histological differentiation and staging of lung cancer.
NSE is a glycolytic enzyme largely expressed in neuroendocrine tumors, particularly in small-cell lung cancer (SCLC) (13). Neuroendocrine markers are not commonly expressed in NSCLC, except for a subset of 10-20% of cases with neuroendocrine differentiation (14). In our study, we observed high NSE serum levels in 12% of patients which is consistent with the commonly reported rate of neuroendocrine differentiation in NSCLC (14). Pujol et al. have reported that high NSE levels is a negative prognostic factor in a large study including 621 patients (all stages) and similar results were reported by others (15-17). In our study, we observed significantly shorter PFS (1.1 vs. 2.6 months; p=0.002) and also significantly shorter OS (3.7 vs. 11.6 months; p=0.003) for patients with high NSE levels compared to those with low NSE levels. The multivariate Cox proportional hazards model confirmed that high NSE is a strong independent predictive factor for short PFS (HR=2.36; CI: 1.34-4.17; p=0.003), but not for OS (HR=1.90; CI: 0.95-3.80; p=0.071). It has been suggested that the expression of neuroendocrine markers could predict for better response to chemotherapy in NSCLC patients (18-20), although that question still seems to be controversial. On the other hand, very little is known about the relation between neuroendocrine markers, particularly NSE, and response to EGFR-TKIs. Wang et al. have recently reported that NSE mRNA expression was inversely correlated with sensitivity to gefitinib in NSCLC patients (21). However, in our study we used routine laboratory assessment of NSE serum levels and our results are in agreement with those reported by Wang et al. According to the results of our study, we suggest that high pretreatment NSE levels predicts de novo resistance to EGFR-TKIs in patients with advanced-stage NSCLC.
Progression-free (PFS) and overall (OS) survival data according to pretreatment levels of NSE and TK.
TK is an enzyme present in most cells, indicating their proliferative characteristics. It has two isoforms, TK I and TK II, different in chemical structure and biological function. TK I is the one most important, commonly used for detection and estimation of prognosis in cancer. TK I appears during cell dividion in the G1 and S phase while it is absent in resting cells (22). High TK levels have been previously reported as a negative prognostic factor for both SCLC and NSCLC by several authors (23, 24). The relation between TK levels and response to EGFR-TKIs is still unclear. However we observed significantly shorter PFS (2.1 vs. 2.9 months; p=0.026) and also significantly shorter OS (8.5 vs. 17.4 months; p=0.020) for patients with high TK levels compared to those with low TK levels. The multivariate Cox proportional hazards model did not confirm that high TK is an independent predictive factor for short PFS (HR=1.07; CI: 0.73-1.56; p=0.738) nor for OS (HR=1.24, CI: 0.78-1.98; p=0.356). The results of our study indicate no relation between pretreatment TK levels and response to the treatment with EGFR-TKIs. A high TK level is a negative prognostic factor, even if not independent. The principal limitations of our study are its retrospective design and relatively small number of patients included.
In conclusion, the results of the present study clearly showed that a high pre-treatment level of NSE, as a marker of neuroendocrine differentiation, predicts for poor outcome of patients with advanced-stage NSCLC treated with EGFR-TKIs. Thus, we suggest that the pre-treatment NSE level is a cheap and easily measurable independent predictive biomarker, feasible for the use in the routine clinical practice. This is the first study to show a negative predictive role of high pre-treatment serum NSE levels in NSCLC patients treated with EGFR-TKIs. Further studies should be performed to confirm these results. We hope that our findings could have a valuable impact on the treatment of patients with advanced-stage NSCLC in the future.
Multivariate Cox proportional hazards model.
- Received May 18, 2014.
- Revision received July 7, 2014.
- Accepted July 8, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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