Abstract
Background: Erlotinib is a tyrosine kinase inhibitor targeting epidermal growth factor receptor (EGFR); it is used in the treatment of advanced non-small cell lung cancer (NSCLC). We focused on the role of serum concentration of erlotinib and its association with outcome and toxicity in patients with advanced NSCLC harbouring the wild-type EGFR gene or squamous histology. Patients and Methods: Clinical data of 122 patients were analyzed. Serum samples were collected within four weeks after the initiation of treatment. Results: There was no significant association of erlotinib concentration with PFS nor OS (p=0.352 and p=0.6393). Significant associations of erlotinib concentration with grade of skin rash and diarrhoea (p<0.0001 and p<0.0001) were found. Skin rash and diarrhoea were significantly associated with PFS (p=0.0338 and p=0.0001) and OS (p=0.0064 and p=0.0353). Conclusion: Erlotinib concentration was not associated with outcome. Erlotinib concentration was associated with occurrence and severity of skin rash and diarrhoea; the outcome was associated with erlotinib toxicity.
Lung cancer is one of the leading causes of cancer-related mortality in developed countries and non-small cell lung cancer (NSCLC) represents the most common histological type of lung cancer (1-3). Despite the significant progress reached in the treatment of NSCLC, the prognosis of advanced-stage patients remains poor. Several new effective agents, including erlotinib, have been recently approved for the treatment of advanced-stage NSCLC patients. Erlotinib is a low-molecular weight inhibitor targeting epidermal growth factor receptor (EGFR) tyrosine kinase (TKI). Efficacy and safety of erlotinib in patients with advanced NSCLC has been demonstrated in previous randomised phase III clinical trials (4-7).
Activating EGFR gene mutations, predominantly exon 19 deletions or the point mutation in exon 21 termed L858R, represent a strong predictive factor of good response to EGFR-TKIs (8, 9). On the other hand, the efficacy of EGFR-TKIs, particularly of erlotinib, in the predominant patient group with NSCLC harbouring wild-type EGFR gene or those with squamous histology is low (10-12). Although it has been previously established that the magnitude of the pharmacological effect of erlotinib in vitro is concentration-dependent, the data on the role of erlotinib serum concentration in patients treated in the real clinical practice are very limited (13). In this study, we aimed at the role of serum concentration of erlotinib and its association with outcome and toxicity in patients with advanced-stage NSCLC harbouring wild-type EGFR gene or squamous histology.
Patients and Methods
Patients and treatment. We retrospectively analysed clinical data of 122 patients with cytologically- or histologically-confirmed locally-advanced (IIIB) or metastatic-stage (IV) NSCLC harbouring wild-type EGFR gene, who were treated with erlotinib. Patients were treated between years 2012 and 2016. Erlotinib was administered orally at the standard approved dose of 150 mg daily. 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 were performed every 3-4 weeks; computed tomography (CT) or positron emission tomography - (PET)-CT was performed after two or three months of the treatment. Treatment response was assessed using the Response Evaluation Criteria In Solid Tumors version 1.1 and toxicity using the National Cancer Institute Common Terminology Criteria for Adverse Events Version 4.0 (14, 15).
Erlotinib concentration measurement. Serum samples were collected and the measurement was performed one month after the initiation of erlotinib treatment. The samples were collected approximately four hours after erlotinib administration. The total serum concentration of erlotinib in venous blood was assessed by an analytical method consisting of liquid-liquid extraction followed by sensitive ion-pairing reverse-phase chromatography with UV detection (332 nm). This method is a modified method of L. Faivre (16). The chromatographic separation was carried out on a 250×4.6 mm I.D. LC-ABZ + column (5 μm particle size) with guard column LC-ABZ + (50×4.6 mm I.D., 40 μm particle size). The column and the guard column were supplied by Supelco (Bellefonte, PA, USA). The chromatography was performed on a component system of Spectra Physics (San Jose, CA, USA). This system consisted of: Model SP 8770 pump, Model UV 2000 detector, Model SP 8775 autosampler, and Chrom-Jet integrator. All models were connected with system LabNet. 4-aminoacetophenone (PACP) was used as an internal standard. The mobile phase was 0.5% triethylamine, 1.5% acetic acid, 35% acetonitrile and 63% water (v/v/v/v). The flow rate was 1.2 ml/min. The temperature of the column and guard column was maintained at 35°C. Under the conditions described the retention times of erlotinib and PACP were 9.5 and 4.3 min, respectively. The extractions were processed the following way. A 50 μl volume of the internal standard solution (3.2 μg PACP/ml 2-propanol) were measured into a 10 ml clear, dry glass centrifuge tube to which 200 μl serum, 100 μl phosphate buffer (0.75M, pH=10) and 1 ml of tert-butyl-methylaether were added. The tube was vortex-mixed for 30 seconds. The mixture was centrifuged for 10 min at 3,000 × g. The upper (organic) layer was transferred to a clean, dry glass conical tube and evaporated to dryness (50°C). The residue was reconstituted with 100 μl 2-propanol and 25 μl were injected into the chromatographic column. All statistical evaluations were processed by statistical packet Statgraphics. The intra/inter-day reproducibility of the calibrations curves were studied at seven different serum concentrations. The calibration curves were linear in the concentration range of 0.05-3.2 μg/ml serum. Mean RSD was 4.8±1.9 % (n=72). The limit of detection was about 0.01 μg/ml serum. The quantification limit was about 0.025 μg/ml serum. The recovery from serum was expressed as the percentage ratio of the peak height of erlotinib and PACP in the serum sample to that in the standard solution (the recovery for erlotinib and PACP was 84±4% and 69±3%, respectively). Stability tests were performed in the calibration point 0.8 μg/ml serum (n=4). Erlotinib was stable in frozen serum (−18°C) for at least one month (0.79±0.3 μg/ml serum, RSD=3.8%). Erlotinib was stable over the test three freeze-frozen cycle (0.79±0.35 μg/ml serum, RSD=4.4%). Erlotinib was stable in 2-propanol extracts over the test 24 h storage of the sample before the chromatography analysis (0.8±0.25 μg/ml serum, RSD=3.1%). We tested interferences of other drugs. The doses of drugs were administrated in the therapeutic ranges. The following drugs were tested: Essentiale F, Lactobacilus, Dithiaden, V-Fend, Berodual, Ambrobene, Foradil, Spiriva, Erdomed, Augmentin, Klacid. These drugs are frequently applied to patients with lung cancer. None of these drugs interfered with the analysis of erlotinib. No interferences with the metabolites of erlotinib were found. The measurement was performed in the central biochemical laboratory at the Department of Clinical Biochemistry and Haematology, University Hospital Pilsen, using the predefined therapeutic range (0.8-1.25 μg/ml).
EGFR mutation analysis. The tumour specimens acquired during initial bronschoscopy examination were evaluated by a senior cytologist using standard Giemsa staining. In a few cases, the 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 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 micro-dissected 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 JetQuick Tissue DNA Issolation 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 analyser (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.
Statistics. Standard frequency tables and descriptive statistics were used to characterize the sample data set. Association between erlotinib serum concentration and toxic effects was tested using Spearman's correlation. PFS and OS were estimated using the Kaplan-Meier method and all point estimates were accompanied by 95% confidence intervals. Progression-free survival (PFS) was defined from the date of erlotinib treatment initiation until the date of first documented progression or death. Overall survival (OS) was defined from the date of erlotinib initiation until the date of death due to any cause. Patients who had not progressed or died were censored at the date of last follow-up. The relationship between erlotinib concentration and PFS and OS was assessed using univariable Cox proportional hazards model as well as Gehan-Wilcoxon test for three-sample comparison after patient stratification according to erlotinib concentration. Differences in PFS and OS in groups with and without toxic effects were tested for statistical significance using the log-rank test. The level of statistical significance was set at α=0.05 and all reported p-values are two-tailed.
Results
Patient characteristics. The cohort included 74 (60.7%) men and 48 (39.3%) women; 3 (2.5%) current-smokers, 85 (69.7%) former-smokers and 11 (9.0%) never-smokers; 60 (49.2%) patients with adenocarcinoma, 47 (38.5%) patients with squamous carcinoma, one patient with large-cell carcinoma (0.8%) and 14 (11.5%) patients with not otherwise specified (NOS) NSCLC; 101 (82.8%) patients with stage IV and 21 (17.2%) patients with stage IIIB; 9 (7.4%) patients with ECOG PS 0, 87 (71.3%) patients with ECOG PS 1 and 25 (20.5%) patients with ECOG PS 2; 64 (52.5%) patients treated in the second line, 56 (45.9%) patients treated in the third line and two (1.6%) patients treated the fourth line. After four weeks of erlotinib therapy, the median and average erlotinib serum level were 1.60 μg/ml and 1.64 μg/ml, respectively. The erlotinib level was below theraputic range (<0.8 μg/ml) in 16 (13.1%) patients, within theraputic range (0.8-1.25 μg/ml) in 36 (29.5%) patients and above theraputic range (>1.25 μg/ml) in 70 (57.4%) patients. The median PFS and OS for the whole cohort were 2.6 months (95% CI 2.1-3.1) and 13.1 months (95% CI 10.3-17.8), respectively. The baseline patient characteristics are summarized in Table I.
Association between erlotinib concentration and survival. Univariable Cox proportional hazards model did not indicate significant association between erlotinib level and PFS (p=0.7125) nor between erlotinib level and OS (p=0.2976). The median PFS and OS in patients with erlotinib concentration below the therapeutic range (<0.8 μg/ml) were 2.0 months (95% CI=1.8-3.2) and 8.9 months (95% CI=6.6-12.9) compared to 2.9 months (95% CI=2.1-4.0) and 11.4 months (95% CI=9.1-17.4) in those with erlotinib level within the therapeutic range (95% CI=0.8-1.25 μg/ml), and finally compared to 3.0 months (95% CI=2.0-3.7) and 16.0 months (95% CI 10.9-19.8) in patients with erlotinib level above the therapeutic range (>1.25 μg/ml), respectively. The differences in PFS and OS between the compared groups were not statistically significant (p=0.3520 and p=0.6393, respectively) (Table II and Figure 1).
Association between erlotinib concentration and toxicity. After four weeks of erlotinib therapy, skin rash and diarrhoea (all grades) occured in 98 (80.3%) and 55 (43.4%) patients, respectively. The grade of skin rash and diarrhoea were in strong correlation with with erlotinib serum levels (p<0.0001 and p<0.0001, respectively) (Table III and Figure 2).
Baseline characteristics of patients.
Association between erlotinib toxicity and survival. The median PFS and OS in patients with skin rash (all grades) were 3.0 months (95% CI=2.6-3.3) and 15.8 months (95% CI=11.3-19.5) compared to 2.0 months (95% CI=1.9-3.1) and 7.4 months (95% CI=4.0-10.9) in those without skin rash (p=0.0338 and p=0.0064, respectively). The median PFS and OS in patients with diarrhoea (all grades) were 3.9 months (95% CI=2.7-6.2) and 17.2 months (95% CI=11.0-25.9) compared to 2.2 months (95% CI=1.9-2.9) and 10.4 months (95% CI=8.8-14.3) in those without diarrhoea (p=0.0001 and p=0.0353, respectively) (Table IV and Figure 3).
Discussion
Erlotinib is currently used for the treatment of advanced-stage NSCLC in the first line for patients with tumours harbouring activating EGFR mutations and in the second or higher lines for those with tumours harbouring wild-type EGFR gene or squamous histology (in whom the EGFR mutations are rare and lack the predictive value). The efficacy of erlotinib, particularly in patients with tumours harbouring wild-type EGFR gene or squamous histology is low, reaching median PFS of 2-3 months. In the present study, we assessed the association of erlotinib serum concentration with outcome and the most common adverse effects represented by skin rash and diarrhoea. Erlotinib concentration was assessed after four weeks of the treatment. We focussed on the specific cohort of patients with tumours harbouring wild-type EGFR gene or squamous histology.
Progression-free and overall survival data according to erlotinib serum concentration.
Correlation of erlotinib serum concentration with grade of skin rash and diarrhoea.
Erlotinib concentration within the predefined therapeutic range (0.8-1.25 μg/ml) was measured in 36 (29.5%) patients, above the therapeutic range in 70 (57.4%) patients and below the therapeutic range in 16 (13.1%) patients, respectively, while only in one (0.82%) patient was the concentration below 0.183 μg/ml, which has been estimated the minimal effective therapeutic level of erlotinib for tumours harbouring wild-type EGFR, as deduced from half maximal inhibitory concentration (IC50) in vitro after correction for plasma protein binding (13, 17). Although we observed a trend of longer PFS and OS in patients with serum erlotinib above the therapeutic range, especially in comparison with those with levels below the therapeutic range, the difference was not statistically significant (p=0.352 and p=0.6393, respectively). Our results suggest that the effect of erlotinib is associated with the specifics of tumour biology rather than with the drug exposure. This could be clearly demonstrated on the dramatic responses to erlotinib in patients with tumours harbouring activating EGFR mutations who are treated with a dose reduced to 25 mg daily (18, 19). We observed significant association of erlotinib serum level with the occurence and grade of skin rash and also diarrhoea (both p<0.0001). This is in agreement with the data obtained from population-based pharmacokinetic and exposure safety analyses conducted by Lu et al. and White-Konig et al. who reported positive correlation between erlotinib exposure and the grade of skin toxicity (20, 21). We observed significantly longer PFS and OS in patients with skin rash (p=0.0338 and p=0.0064, respectively) and also in those with diarrhoea (p=0.0001 and p=0.0353, respectively). These results confirmed the data showing the association between the outcome and erlotinib toxicity previously published by others (22-28). The evaluation of skin toxicity has been suggested as a method to predict the efficacy of EGFR-TKIs in patients with tumours harbouring wild-type EGFR gene. Accordingly, increasing of EGFR-TKI dosage until the occurrence of skin rash has recently been discussed as a promising management strategy called “dosing-to-rash” (29, 30). Even though this strategy seems interesting, its impact on the outcome of patients treated with erlotinib is not clear and the results of two phase II clinical trials conducted so far are conflicting. The study by Brahmer et al. shows improved survival in patients treated with dosing-to-rash strategy in the first line (31). On the contrary, the study conducted by Mita et al. failed to show the impact of the dosing-to-rash strategy in patients previously treated with chemotherapy, which is in agreement with our present results showing no direct impact of erlotinib serum levels on the treatment efficacy (32). Moreover, our study also included patients previously treated with chemotherapy, which is comparable to the cohort included in the study by Mita et al.
Progression-free (A) and overall (B) survival according to erlotinib serum concentration.
Correlation of erlotinib serum concentration with grade of skin rash and diarrhoea.
Progression-free and overall survival according to skin rash (A, B) and diarrhoea (C, D).
The principal limitations of the present study are the retrospective nature and relatively limited number of patients. The patients in whom the dose was reduced due to the toxicity were not excluded which could influence the analysis focused on the association between erlotinib concentration and outcome. However, the vast majority of patients primary resistant to erlotinib develop disease progression within the first month of the treatment that was not affected by the possible dose reduction after the measurement. Nevertheless, this is the largest study evaluating the role of erlotinib serum concentration in a homogenous cohort of patients with advanced NSCLC in conditions of the real-life clinical practice.
In conclusion, the results of the present study indicate no significant association of erlotinib serum concentration with PFS nor OS in patients with advanced-stage NSCLC harbouring wild-type EGFR gene or squamous histology. We observed significant association of erlotinib serum concentration with occurrence and severity of skin rash and diarrhoea. Our results confirmed the association between the treatment outcome and erlotinib toxicity.
Progression-free and overall survival data according to skin rash and diarrhoea.
Acknowledgements
This study was supported by the National Sustainability Program I (NPU I) Nr. LO1503 provided by the Ministry of Education Youth and Sports of the Czech Republic and by the grants of Ministry of Health of the Czech Republic - 17-30748A and Conceptual Development of Research Organization (Faculty Hospital in Pilsen - FNPl, 00669806).
Footnotes
Conflicts of Interest
OF received honoraria from Roche and GSK for consultations and lectures unrelated to this project. JF has received honoraria from Astra Zeneca, Roche and Novartis for consultations and lectures unrelated to this project. PH, MP, JR, PS, OS, MM, LB, AC, IN, RK and OT 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 August 4, 2017.
- Revision received September 26, 2017.
- Accepted September 29, 2017.
- Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
