Abstract
Background: Tumor biomarkers are used for predicting therapy effect and prognosis of patients with non-small cell lung cancer (NSCLC). We focused on their potential role in prediction of the efficacy of erlotinib. Patients and Methods: In a total of 144 patients with advanced-stage (IIIB or IV) NSCLC treated with erlotinib, pre-treatment levels of soluble carcinoembryonic antigen (CEA) and cytokeratin markers in serum were measured. Results: The median progression-free and overall survival for patients with a high level of carcinoembryonic antigen (CEA) was 1.9 and 8.6 vs. 2.9 and 16.1 months for patients with low CEA (p=0.046 and p=0.116). The respective medians for patients with a high level of cytokeratin-19 fragment were 1.9 and 6.1 vs. 3.4 and 23.8 months for patients with the low cytokeratin-19 fragment (p<0.001 and p<0.001). Conclusion: High pre-treatment serum levels of one or both biomarkers are associated with poor outcome of patients with NSCLC treated with erlotinib.
Non-small cell lung cancer (NSCLC) is the most frequent histological type of lung cancer (1), which is one of the most common human malignant diseases and the leading cause of cancer-related deaths worldwide (2). Targeted-treatment based on tyrozine kinase inhibitors (TKI) directed at epidermal growth factor receptor (EGFR) represents a novel effective tool in management of advanced-stage NSCLC. The aim of our study was to evaluate the predictive role of pre-treatment serum levels of carcinoembryonic antigen (CEA) and cytokeratin-19 fragments (CYFRA 21-1) in patients with advanced-stage NSCLC treated with erlotinib.
Patients and Methods
Patients' characteristics. The study included 144 patients. The median age was 64 years (range 28-84 years). 85 (59.0%) patients were male, 109 (75.7%) patients had a history of smoking, 73 (50.7%) patients had adenocarcinoma, 121 (84.0%) patients had stage IV disease, 77 (53.5%) patients had ECOG PS 0 or 1 and 113 (78.5%) patients had received at least one previous chemotherapy regimen. A total of 78 patients were tested for activating EGFR mutation, 70 of them were wild-type EGFR and eight were EGFR mutation-positive. The baseline patient characteristics are summarized in Table I.
Study design and treatment. We retrospectively analyzed clinical and laboratory data of patients with cytologically- or histologically-confirmed advanced-stage (stage IIIB or IV) NSCLC treated with erlotinib between 2006 and 2013 at the Department of Pneumology in Pilsen. Erlotinib was administered orally at the standard approved dose of 150 mg daily; dose interruption or reduction was permitted in the event of treatment-related toxicity. The treatment was continued until disease progression or development of intolerable toxic effects.
Clinical monitoring. The treatment was prospectively monitored and the clinical course of patients was continuously assessed at specific time points. Clinical follow-up examinations 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 two or three months of treatment with erlotinib. The objective tumor response was assessed by investigators in terms of complete response (CR), partial response (PR), stable disease (SD) and progressive disease (PD) using Response Evaluation Criteria in Solid Tumours (RECIST) (3). The disease control rate (DCR) was defined as the sum of CR, PR and SD. Progression-free survival (PFS) was determined from the date of erlotinib initiation until the date of first documented progression or death. Overall survival (OS) was determined from the date of erlotinib initiation until the date of death.
Tumor marker assessment. Serum samples for measurement of tumor markers were collected within one month before erlotinib treatment. Serum levels of CEA were measured using chemiluminiscent method on an DXI 800i analyzer (Beckman, USA). Serum levels of CYFRA 21-1 were measured using immunoradiometric titration method (IRMA) (Beckman-Immunotech, USA). The measurement was performed in Central Immunoanalytic Laboratory at Department of Nuclear Medicine, using following cut-off values: CEA: 3 μg/l and CYFRA 21-1: 2.5 μg/l.
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, DNA was extracted from tissue cells by a standard spin-column procedure using JetQuick Tissue DNA Issolation Kit (Genomed GmbH, Loehne, Germany). Mutations in exons 19 and 21 of the EGFR gene 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). 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.
Statistics. Standard frequency tables and descriptive statistics were used to characterize sample data set. The significance of differences between baseline characteristics, as well as treatment response, and level of tumor markers, was determined using the Fisher's exact test. PFS and OS were calculated 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 log-rank test. Univariate and multivariate Cox proportional hazards model was used to evaluate influence of all potential predictive and prognostic factors on the survival measures. Based on univariate results, variables with the greatest effect on survival measures (EGFR mutation status, stage and PS) were involved in multivariate Cox model to adjust results in terms of tumor markers. As a level of statistical significance, p=0.05 was used.
Results
CEA and CYFRA 21-1 levels before the treatment. Before the begining of erlotinib treatment, high CEA (≥3 μg/l) was measured in 99 (68.8%) patients, high CYFRA 21-1 (≥2.5 μg/l) was measured in 83 (57.6%) patients and high CEA and CYFRA 21-1 were measured in 59 (41%) patients. Adenocarcinoma histology was significantly associated with high CEA levels (p=0.019); PS 2 or 3 was significantly associated with high CYFRA 21-1 levels (p=0.002) and and stage IV was significantly associated with both high CEA (p=0.026) and high CYFRA 21-1 (p=0.021) (data not shown).
Baseline patients' characteristics.
Relation between CEA and CYFRA 21-1 levels and treatment efficacy. For patients with high CEA the DCR was 51.5% compared to 75.6% for patients with low CEA (p=0.01). For patients with high CYFRA 21-1 the DCR was 45.8% compared to 77.0% for patients with low CYFRA 21-1 (p<0.001). For patients with high CEA the median PFS and OS was 1.9 and 8.6 compared to 2.9 and 16.1 months for patiens with low CEA (p=0.046 and p=0.116) (Figure 1A, B). For patients with high CYFRA 21-1 the median PFS and OS was 1.9 and 6.1 compared to 3.4 and 23.8 months for patiens with low CYFRA 21-1 (p<0.001 and p<0.001) (Figure 1C, D). The univariate Cox proportional hazards model revealed that EGFR mutation status (HR=0.20, p=0.001), CEA (HR=1.44, p=0.049) and CYFRA 21-1 (HR=2.06, p<0.001) were significant factors for PFS, whereas EGFR mutation status (HR=0.25, p=0.019), stage (HR=3.36, p=0.004,); PS (HR=2.36, p<0.001) and CYFRA 21-1 (HR=3.73, p<0.001) were significant factors for OS (Table II). Finally the multivariate Cox proportional hazards model revealed that EGFR mutation status (HR=0.21, p=0.001), CEA (HR=1.72, p=0.007) and CYFRA 21-1 (HR=2.17, p<0.001) were independent predictive factors for PFS, whereas EGFR mutation status (HR=0.30, p=0.044), and CYFRA 21-1 (HR=2.74, p<0.001) were independent predictive factors for OS (Table III).
Kaplan Meier plots showing the comparison of progression-free survival (PFS) and overall survival (OS) according to CEA (A, B) and CYFRA 21-1 (C, D) levels.
Discussion
Erlotinib is a low-molecular EGFR-TKI approved for the treatment of patients with locally-advanced or metastatic NSCLC. It has been proven that EGFR-TKIs are highly efficient particularly in patients harboring activating EGFR mutations, occuring approximately in 5-20% of patients (4-7). Activating EGFR mutations are the strongest predictor of efficacy of EGFR-TKIs, however the majority of NSCLC patiens harbor a wild-type EGFR gene. Moreover there is still a large proportion of patients in whom it is not feasible to acquire an adequate tissue for EGFR mutation analysis. Hence looking for other predictive biomarkers is required. Both CEA and CYFRA 21-1 are serum tumor markers commonly used for diagnostics and follow-up monitoring of patients with NSCLC. In the present study we focused on their potential role in prediction of efficacy of erlotinib in patients with advanced-stage NSCLC.
CEA is thought to play a role in cell-to-cell adhesion and has a dominant effect in blocking cell differentiation (8) and it cooperates with Myc and Bcl-2 in cellular tranformation (9). Additionally, it can also inhibit cell death induced by a loss of anchorage to the extracellular matrix (anoikis) (10). High CEA levels have been described as a significant marker of poor prognosis in patients with NSCLC regardless of treatment (11-13). In our study, we observed significantly lower DCR (51.5% vs. 75.6%; p=0.010) and shorter PFS (1.9 vs. 2.9 months; p=0.046) for patients with high CEA levels compared to those with low CEA levels. The multivariate Cox proportional hazards model confirmed that high CEA is an independent predictive factor for short PFS (HR=1.72, p=0.007). On the contrary, Okamoto et al. and Jung et al. have recently reported that high CEA levels predict a good outcome of advanced-stage NSCLC patients treated with EGFR-TKIs (14, 15). Okamoto et al. hypothesized that up-regulated expression of the anti-apoptotic protein CEA could be caused by the aberrant activation of mutated EGFR via activation of downstream molecules such as Akt and STAT3/5 which play crucial role in the anti-apoptotic patways (14). The hypothesis is in accordance with findings reported by Shoji et al. who has shown an association between high CEA and presence of activating EGFR mutations (16). In our study, no association with the presence of activating EGFR mutations was observed. The reason why the results of our study did not confirm the previously-published notion could be addressed to the different study populations, while both studies by Okamoto et al. and Jung et al. included Asians compared to our study including Caucasians. There is a notable difference in the presence of various driving mutations between Asians and Caucasians. Activating EGFR mutations predicting high efficacy of EGFR-TKIs (4-7) are frequently found in Asians whereas rarely in Caucasians (17). On the other hand, KRAS and BRAF mutations conferring resistance to EGFR-TKIs (18-20) are more frequently found in Caucasians whereas rarely in Asians (17, 21, 22). In accordance with the hypothesis by Okamoto mentioned above, we hypothesize that up-regulated expression of CEA also could be caused by the aberrant activation of several molecules downstream EGFR, such as KRAS and BRAF mutations, respectively. Thus high CEA in Caucasians could be associated predominantly with KRAS and BRAF mutations resulting in a poor outcome of patients treated with erlotinib.
Univariate Cox proportional hazards model.
Multivariate Cox proportional hazards model.
CYFRA 21-1, a fragment of cytokeratin subunit 19 has been previously extensively evaluated in the setting of NSCLC. Various studies has shown an unfavourable prognosis of patients with high CYFRA 21-1 levels regardless of treatment (23-25). In our study, we observed lower DCR (45.8% vs. 77.0%, p<0.001), shorter PFS (1.9 vs. 3.4 months, p<0.001) and also shorter OS (6.1 vs. 23.8 months, p<0.001) for patients with high CYFRA 21-1 levels compared to those with low CYFRA 21-1 levels. The results were confirmed by multivariate Cox proportional hazards model (PFS: HR=2.17, p<0.001; OS: HR=2.74, p<0.001). Our findings clearly confirmed results of several previously published studies showing negative predictive role of high CYFRA 21-1 levels in NSCLC patients treated with EGFR-TKIs (15, 26, 27).
In conclusion, this is the first study focusing on the predictive role of pre-treatment levels of CEA and CYFRA 21-1 in the Caucasian population. CEA and CYFRA 21-1 are commonly used serum tumor markers which are simple and easy to detect. We observed that high pre-treatment serum levels of CEA and/or CYFRA 21-1 are associated with poor outcome of NSCLC patients treated with erlotinib. Based on the present study results, one can say that pre-treatment serum levels of CEA and CYFRA 21-1 are feasible diagnostic and also predictive tools in patients with advanced-stage NSCLC. Futher research is required to elucidate the different predictive role of CEA between Asian and Caucasian populations.
Acknowledgements
The Authors would like to thank all patients voluntarily taking part in this study. This work was supported by grant no. 9087 of the Czech Ministry of Health.
Footnotes
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Conflicts of Interest
All Authors declare that they have no actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence this work.
- Received January 17, 2014.
- Revision received April 7, 2014.
- Accepted April 9, 2014.
- Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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