Predictive and prognostic factors in second- and third-line erlotinib treatment in NSCLC patients with known status of the EGFR gene
- Authors:
- Published online on: June 19, 2013 https://doi.org/10.3892/or.2013.2553
- Pages: 1463-1472
Abstract
Introduction
Lung cancer is the most common malignancy in humans. It is also the most common cause of cancer-related mortality among both men and women in most developed countries. Approximately 85% of cases involve non-small cell lung cancer (NSCLC), which generally has the histological structure of squamous cell carcinoma or adenocarcinoma (1,2). Surgery, chemotherapy and radiotherapy remain the standard of treatment in NSCLC. Despite the development of various methods of treatment, NSCLC is still associated with very poor prognosis. Only 10–15% of patients survive 5 years from diagnosis. Metastatic NSCLC is fatal in 100% of cases (2).
Progress in understanding the biology of cancer leads to personalization of therapy and introduction of drugs aimed at blocking defective metabolic pathways of cancer cells. Erlotinib is an example of a drug with a molecular mechanism of action, which is currently used in the treatment of patients with NSCLC. It is a reversible small molecule tyrosine kinase inhibitor of epidermal growth factor receptor (TKI EGFR). EGFR abnormalities such as strong EGFR protein expression, high polysomy or amplification and EGFR gene mutations are relatively common in NSCLC. Kinase inhibition stops cancerous cells in the G1 phase of the cell cycle and induces apoptosis, which leads to a decrease of proliferation and reduces the capacity for invasion and metastasis (3).
The aim of the present study was to determine the prognostic and predictive factors in second- and third-line erlotinib therapy in NSCLC patients with known status of EGFR gene mutation.
Materials and methods
Patient characteristics
This study presents results of research that was conducted in a group of 71 patients (36 men and 35 women) with inoperable, locally advanced and metastatic NSCLC treated with second- or third-line erlotinib in the period between 2008 and 2011. The study group consisted of patients aged 42–84 years (mean age 60.9±9.6), the majority (n=59, 83.1%) current or ex-smokers. Adenocarcinoma was diagnosed in 57% of tumors. Qualification for erlotinib was based on radiologically confirmed progression after first-line chemotherapy in patients with documented first-line response, without rapid deterioration of performance status and weight loss. The clinical characteristics of the study group are presented in Table I.
Criteria for assessing the effects of treatment (treatment response, disease stabilization or progression) were based mainly on the analysis of images from spiral computed tomography (CT) of the chest and sites suspected of metastases and were consistent with the RECIST 1.1 guidelines. The criteria for anemia and non-hematological complications were based on the Common Toxicity Criteria (CTC) v.6.0 guidelines. Patient performance status was assessed according to the ECOG-WHO score.
Examination of EGFR gene abnormalities
Patients were qualified for treatment without taking molecular factors into consideration; molecular tests were carried out retrospectively after completion of treatment. EGFR expression was determined by immunohistochemistry (IHC) using monoclonal antibody from Dako (Denmark). The fluorescence in situ hybridization (FISH) method was used to assess amplification of the EGFR gene using molecular probes for Abbott Molecular (USA). The presence of exon 19 deletions was examined using the PCR technique and the amplified PCR product fragment length analysis. In order to evaluate the L858R substitutions in exon 21, the ASP-PCR (allele-specific PCR) technique was used along with two pairs of PCR primers. PCR primers were marked with Cy5 fluorochrome. The results were analyzed on an ALF Express II sequencer. The direct sequencing by the Sanger method for EGFR gene mutation detection was also used.
Statistical analysis
For statistical analysis we used the Chi-square test to compare the quantity of patients with different response to treatment and survival, depending on the prevalence of selected predictive and prognostic factors. This test was also used to compare the number of patients with and without EGFR gene mutation in different subgroups of age, gender, histopathological diagnosis and smoking status. To compare the probability of survival and progression between the groups with different clinical and molecular factors, the Kaplan-Meier method was used. Cox regression model with ‘step by step’ selection was used to determine the influence of clinical and molecular factors on overall survival (OS) of patients treated with erlotinib.
Results
Estimation of EGFR gene abnormalities
Seventeen activating mutations were detected in the EGFR gene (24%), including 12 deletions in exon 19 (70.6% of all identified mutations including three rare deletions) and 5 L858R substitutions in exon 21 (19.4% of all identified mutations). Amplification of the EGFR gene in the tumor cells was observed in 29 patients (40.8%, a positive FISH test), and normal number of copies of the EGFR gene in 10 patients (14.1%, a negative FISH test). In 32 patients (45.1%) FISH testing either gave unreliable results or could not be performed due to the limited histological material. EGFR gene activating mutations occurred with similar frequency in the groups of patients with or without EGFR gene amplification detected by FISH, and in groups of patients with both presence and absence of EGFR expression on the cell surface detected by IHC.
The incidence of mutations was slightly higher among women (n=12, 34.3%) than among men (n=5, 13.9%) and did not depend on age. EGFR activating mutations were significantly more frequent in non-smoking patients (n=8, 66.7%) than in former or current smokers (n=9, 15.3%). The number of mutations in patients with adenocarcinoma and non-adenocarcinomas did not differ significantly. The incidence of grade 3–4 rash after erlotinib treatment was not significantly associated with the presence of EGFR gene activating mutations. It should be noted, however, that rash as a result of TKI EGFR therapy occurred slightly more often in patients with EGFR mutations (Table II), as well as in patients with EGFR gene amplification.
Although the response to first-line chemotherapy with platinum compounds was slightly more frequent in patients with EGFR mutations, the length of response and the time from diagnosis to start of erlotinib did not depend on the presence of the mutation. There was no significant relationship between the presence of EGFR mutation and loss of body mass.
Estimation of response to erlotinib treatment
An objective response to erlotinib treatment in the form of partial response (PR) occurred in only 5 patients (7%), including 3 second-line and 2 third-line patients. Duration of remission in relation to clinical characteristics of patients were: i) 4 months (observation cut-off) in a smoking (30 pack-years) 53-year-old woman with large-cell carcinoma and a deletion in exon 19 of the EGFR gene; ii) 8 months in a smoking (31 pack-years) 52-year-old man with large-cell carcinoma and mutation in exon 21 of the EGFR gene; iii) 9 months in a smoking (10 pack-years) 46-year-old man with adenocarcinoma and a deletion in exon 19 of the EGFR gene; iv) 16 months in a non-smoking 60-year-old woman with adenosquamous cell carcinoma and deletion in exon 19 of the EGFR gene and EGFR gene amplification (84% of tumor cells with abnormal number of EGFR gene copies); v) 29 months (observation cut-off) in a non-smoking 74-year-old man with adenocarcinoma and a deletion in exon 19 of the EGFR gene. It should be noted that in all cases of PR as a result of erlotinib treatment activating mutation in the EGFR gene was present in tumor cells, and that all patients with PR suffered a rash of varying severity.
Stable disease (SD) during treatment with erlotinib in second- or third-line occurred in 24 patients (33.8%), including 9 patients with activating EGFR mutations (37.5% of all patients with SD). Three patients with rare deletions in exon 19 of the EGFR gene achieved SD lasting 8 (ΔL747–P753), 8 (ΔE746–S752) and 4 months (ΔE746–P753).
Progressive disease (PD) during erlotinib treatment occurred in 42 patients (59.2%), of which only 3 had EGFR activating mutations (7.1% of all patients with PD); two cases with L858R substitution and one patient with deletion in exon 19.
Absence of mutation in the EGFR gene was the strongest independent factor increasing the percentage of early progression in patients treated with erlotinib (P=0.0002, χ2=13.76). Other factors that increased the incidence of progression during erlotinib therapy were: short response to first-line chemotherapy, high serum LDH level and the absence of a rash associated with erlotinib treatment. Notably, the progression or stabilization of disease did not depended on gender, age, smoking status (there were only 12 non-smokers in the group), pathological diagnosis and amplification of EGFR gene. Patients with poorer performance status, weight loss and anemia had only slightly decreased response rate compared to patients without these adverse prognostic factors (Table III).
 | Table IIIClinical and molecular factors influencing the risk of early progression in patients treated with erlotinib. |
The median progression-free survival (PFS) for the entire group of patients treated with erlotinib was 1.5 months. The study, however, had a considerable weakness resulting from the large number of incomplete observations (n=41). Observations in this group were carried out at least until the end of erlotinib therapy. Median PFS was significantly longer in patients with EGFR activating mutations (HR=0.309; 95% CI, 0.19–0.503) (Fig. 1) and in patients with rash associated with erlotinib (Fig. 2) as compared to patients without those factors. However, the median PFS was not the same in all carriers of EGFR gene mutation and was longer in patients with deletions in exon 19 compared to patients with the L858R substitution in exon 21. Performance status, weight loss and LDH levels had weaker but significant effects on PFS in patients treated with erlotinib. Median PFS was not dependent on the degree of amplification of the EGFR gene, gender, pathology and was only slightly longer in non-smokers (Fig. 3).
Estimation of OS of erlotinib-treated patients
Median OS was 10 months. OS in patients treated with erlotinib was partially influenced by factors other than those affecting the PFS. Median OS was only insignificantly longer in patients with EGFR activating mutations than in patients without such mutations. Prolonged median OS applied only to patients with deletions in exon 19 of the EGFR gene, whereas patients with L858R substitution had median OS similar to that of patients without mutations in this gene (Fig. 4). Median OS was significantly longer in patients with treatment-related rash compared to patients without this adverse effect (Fig. 5). Median OS was not affected by smoking status, gender, age, pathology and EGFR gene amplification. Significant shortening of OS was associated with typical negative prognostic factors, such as poor performance status, significant loss of body mass, lack of response to first-line chemotherapy, short duration of first-line response, short follow-up time since diagnosis and LDH high serum levels.
OS and PFS are affected by multiple factors in Cox regression model
Multivariate analysis using Cox logistic regression confirmed that the strongest factors increasing risk of progression in patients treated with erlotinib are the absence of EGFR gene activating mutations (risk nearly 6-fold higher) and absence of treatment-related rash (risk increase 4.5-fold). These factors are closely related to the mechanism of action of TKI EGFR. The value of other factors that affect the risk of progression is associated with their traditional prognostic role, affecting the course of the disease (Table IV).
| Table IVFactors affecting progression-free survival of patients treated with erlotinib in multivariate Cox logistic regression. |
On the other hand, prognostic factors have a decisive influence on the risk of early mortality in patients treated with erlotinib. This risk is multiplied in patients with poor performance status, short response to first-line chemotherapy and short time from diagnosis to start of erlotinib. Predictive factors affecting the effectiveness of TKI EGFR also affect the risk of mortality. Although the absence of EGFR gene mutation increases the risk by only 2.8-fold with low level of significance (P=0.04), the absence of a rash associated with erlotinib treatment has significant effect on the risk of mortality (Table V).
| Table VFactors affecting the overall survival in patients treated with erlotinib in multivariate Cox logistic regression. |
Discussion
Erlotinib was introduced for the treatment of patients with NSCLC, according to the recommendations of the FDA, on November 18, 2004 (4). The efficacy and safety of the drug was confirmed in a randomized clinical double-blind, placebo-controlled trial (BR.21). The study included 731 patients with locally advanced or metastatic NSCLC in whom standard chemotherapy had failed. Median OS in patients treated with erlotinib was 6.7 months compared to 4.7 months in the placebo group. Regardless of the presence of molecular factors, erlotinib prolonged PFS and improved quality of life compared to the best supportive care (BSC), but the objective response occurred in only 8.9% of patients (5).
Our study does not indicate that the median OS and PFS depend significantly on gender, age, tumor pathology or smoking status. Results differ from those commonly found in literature but our group was dominated by smokers or ex-smokers. Shepherd et al(5) in the BR.21 study demonstrated that the median OS is higher in non-smokers vs. past or active smokers (12.3 vs. 5.5 months, P<0.006), women vs. men (8.4 vs. 5.7 months, P=0.76) and in patients diagnosed with adenocarcinoma vs. squamous (7.8 vs. 5.6 months, P=0.97). The TRIBUTE study demonstrated results of treatment in non-smoking patients. OS was significantly longer (by 12.4 months) in patients who were treated with erlotinib compared to placebo (22.5 vs. 10.1 months; HR, 0.49; 95% CI, 0.28–0.85; P=0.01). It should be noted that the above mentioned group of patients was dominated by younger people (average age 58 vs. 64 years), women (60 vs. 37%), and patients diagnosed with adenocarcinoma (82 vs. 58%) (6).
There is a clinically confirmed correlation between the occurrence of rash and response to erlotinib treatment, which is also demonstrated in this study. Petrelli et al compared the relationship between the occurrence of a rash and the results of TKI treatment based on 24 trials. They found that the presence of a rash is an important independent predictive factor for treatment with EGFR TKI, affecting OS (HR, 0.30; P<0.00001) and the risk of disease progression (HR, 0.50; P<0.00001). In addition, objective response to treatment was significantly higher in patients who experienced cutaneous adverse effects of treatment (42% compared to 7% in patients without rash) (7). Tendency for cutaneous adverse effect complications in the case of erlotinib is most likely associated with EGFR gene polymorphism.
With the new data on intracellular signaling pathways and their role in cancer development, molecular targets for small molecule TKI EGFR, including erlotinib, have been identified. Biomarkers which were initially analyzed included expression score of the EGFR extracellular domain and the gene number copies in tumor cells.
The increased number of EGFR gene copies was the first molecular predictive marker for TKI. There are no clear data on the impact of gene amplification on the effectiveness of gefitinib or erlotinib. Results of previous studies are controversial. Conclusions from the BR.21 study showed that patients with amplification or high polisomy of EGFR gene benefit more from treatment with erlotinib (8). Similar conclusions were presented by a group of Japanese researchers evaluating the efficacy of gefitinib in patients with EGFR gene amplification; they achieved statistically longer survival, higher objective response rate (ORR) and longer PFS compared to patients without gene amplification (P=0.014) (9). A number of phase III studies have been performed concerning erlotinib and gefitinib effectiveness in second- and third-line therapy of unselected NSCLC patients. However, molecular analysis in these studies was conducted retrospectively and on small groups of patients (Table VI).
| Table VIResults of clinical trials concerning erlotinib and gefitinib effectiveness in second- and third-line therapy examined at molecular level in NSCLC patients. |
The results of our analysis do not indicate EGFR gene amplification as a predictive marker for therapy. Median OS and PFS do not depend on the presence of gene amplification. Moreover, there was no correlation between EGFR gene amplification and the presence of activating mutation of EGFR gene. This may be associated with FISH analysis limitations, challenges with obtaining results of diagnostic value.
The results of first clinical trials, of low molecular weight, proved that there are populations of patients who are significantly more responsive to TKI EGFR (erlotinib and gefitinib) treatment. These are patients of predominantly Asian origin, diagnosed with adenocarcinoma, with therapy associated rash, women and never smokers. Subsequently, activating mutations in the EGFR gene responsible for this relationship were described (16–18). The presence of the mutation is currently the most important molecular factor for predicting response to TKI EGFR treatment.
The results of several phase III clinical trials with gefitinib (IPASS, JP 0056 - NEJ 002, WJTOG3405, First-Signal) in the group of patients with confirmed mutation in the EGFR gene, showed a significantly higher efficacy of gefitinib compared to chemotherapy (19–22). In the OPTIMAL study conducted among Asian patients with EGFR mutations, erlotinib therapy achieved a significant increase in response rate (83 vs. 36%) and PFS (13.1 vs. 4.6 months) (23). In the EURTAC study, which included Caucasian patients with EGFR gene mutations the results were: ORR = 58 vs. 15%; PFS = 9.7 vs. 5.2 months (24).
The presence of activating mutations in the EGFR gene was an important predictive factor for erlotinib therapy in our study group as well. Rosell et al(25) evaluated the incidence of activating mutations in the EGFR gene in 2105 Caucasian patients, of whom 350 (16.6%) showed a mutation. Erlotinib was used as a first-line therapy in 113 patients, and as a second- or third-line therapy in 104 patients. PFS and median OS in patients with EGFR activating mutations treated with TKI EGFR were similar regardless of line of therapy (PFS, 14 months; OS, 28 months in first-line therapy and 27 months in second- and third-line therapy). Patients who had a deletion in exon 19 achieved a higher ORR compared to patients with L858R substitution (P=0.001).
Although, there is a clear need for the use of TKI EGFR for second- and third-line therapy in patients with EGFR gene mutations, the use of these drugs in patients with undetected EGFR mutation remains problematic (26). In the present study, disease control was observed in 27.8% of patients with no detected EGFR mutation. On the other hand, early progression was described in 17.6% of patients with activated EGFR gene mutation. The reason for this may be underestimation of EGFR mutation or appearance of genetic abnormalities responsible for resistance to TKI EGFR. Therefore, careful analysis of clinical factors for qualification to molecular examination and TKI EGFR therapy as well as appropriate selection of molecular tests are required.
In conclusion, this study presents the results of second- and third-line erlotinib treatment in a group of 71 patients with advanced NSCLC. Objective response occurred in only 5 patients (7%); all had activating mutations of EGFR and developed rash during therapy. The influence of a number of clinical and molecular factors on the efficacy of erlotinib was assessed. We concluded that detection of EGFR gene mutation is not the only factor determining the effectiveness of erlotinib in second- or third-line therapy in advanced NSCLC patients. Evaluation of molecular and clinical predictive factors in individual patients is important both from a clinical and an economic point of view, assuming similar efficacy and favorable toxicity profile of erlotinib compared to docetaxel or pemetrexed in patients with relapsed NSCLC.
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