It is generally accepted that somatic mutation in the EGFR gene can serve as a biomarker for predicting the efficacy of treatment with epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitor (TKI) in patients with non-small cell lung cancer (NSCLC). Recently, in fact, randomized phase 3 clinical trials have clearly proven that the EGFR-TKIs erlotinib and gefitinib are effective for first-line treatment of advanced NSCLC harboring EGFR gene mutation (1-3). EGFR mutation has been shown to have statistically significant correlation with progression-free survival, but not with overall survival, probably because of crossover to alternative treatments (4). However, the situation is not that simple. The efficacy of EGFR-TKI has been less evident when used as a second-line treatment for NSCLC patients who have been previously treated with platinum-based combination chemotherapy (5). The INTEREST phase 3 trial compared gefitinib and docetaxel in previously treated NSCLC patients (6). Although the hazard ratio for survival was lower in patients with tumors carrying EGFR mutation than in those with wild-type EGFR, no statistically significant interaction was reportedly demonstrated (7). A recent and important study conducted in China by Bai and colleagues has attempted to address this issue (8).
Disappearance of EGFR mutation after chemotherapy
Bai et al. (8) investigated the possibility that EGFR mutation status might change after chemotherapy. They analyzed the presence of EGFR gene mutation in paired plasma samples from 264 patients with advanced (stages IIIb and IV) NSCLC before and after two cycles of platinum-based first-line chemotherapy. EGFR gene mutations were detected in 34.5% of samples (91 of 264) obtained before the treatment, but in only 23.1% of samples (61 of 264) obtained after the treatment. The difference was statistically significant (P<0.001). Importantly, the EGFR mutation status in 20.5% of the patients (54 of 264) was found to be switched from positive (mutant) to negative (wild-type) after chemotherapy. Furthermore, these patients had a better response rate than patients showing a reverse change of mutation status (P=0.037). More recently, Honda et al. (9) reported the similar disappearance of an activated EGFR mutation in a Japanese woman after extensive treatment with EGFR TKIs and cytotoxic drugs. Although the patient’s malignant pleural effusion and supraclavicular lymph node metastasis carried the same EGFR mutation (L747–T751 deletion in exon 19), no EGFR gene mutation was detected in pleural effusion obtained after the treatments. A pair of cell lines established from the effusion were confirmed to lack the EGFR gene mutation, and the pathological features of xenografts established from the cell lines resembled those of the original lymph node metastasis, indicating they were the same tumor. In the INTEREST trial mentioned above, the EGFR mutation status was determined from archived primary tumor samples (6). Therefore, there would have been a gap between the time points at which mutation status was determined and study entry. If we assume that first-line chemotherapy had changed the EGFR gene status, failure to detect any correlation between the original EGFR status and the efficacy of EGFR-TKI therapy would not be surprising. Therefore, the study reported by Bai et al. (8) has had a huge impact on oncological practice, and suggested that data obtained in clinical trials should be interpreted carefully.
Detection of EGFR mutation in plasma samples
It is often difficult to obtain biopsy samples sufficient for DNA sequencing from patients with unresectable NSCLC. However, currently available highly sensitive state-of-the-art methods have made it feasible to determine tumor genotypes using peripheral blood samples, thus providing a relatively non-invasive and repeatable source from which to obtain information on EGFR mutation status. However, discrepancy of EGFR mutation status between peripheral blood samples and matched tumor tissues has been reported. For example, Goto et al. (10) investigated EGFR gene mutation status based on circulating free DNA in serum versus tumor biopsy samples in Japanese patients who participated in the IPASS (IRESSA Pan-Asia Study) study. There was a significant association between EGFR gene status based on circulating free DNA and progression-free survival after first-line gefitinib treatment, but a high rate of false negativity (56.9%) was evident when the circulating free DNA was analyzed. In a previous report, Bai et al. (11) showed that the concordance of EGFR mutation status between 230 plasma samples and matched tumor tissue samples was 78%. This discrepancy could have accidentally led to underestimation of EGFR gene mutations in plasma samples obtained after chemotherapy. To eliminate this possibility, they carefully applied two methods: DHPLC (denaturing high-performance liquid chromatography) and ARMS (amplification refractory mutation system), but further confirmatory analysis will be needed.
EGFR mutation shift in tissue samples
Bai et al. compared the EGFR mutation status of paired initial diagnostic biopsy and surgically resected tumor samples from 63 NSCL patients who received two to four cycles of cisplatin-based neoadjuvant therapy (8). Consistent with the aforementioned analysis of plasma samples, EGFR mutations were detected in 34.9% (22 of 63) of patients before the neoadjuvant chemotherapy, but in only 19.0% (12 of 63) after the treatment. In 12 patients (19.0%) the EGFR status changed from mutant to wild type. Although the number of patients was smaller than that used for the plasma DNA analysis, these results clearly substantiated the shift of EGFR status after treatments with cytotoxic drugs.
Origin of cells with the wild-type EGFR gene in mutant tumors
The next question is the origin of different mutations from the same tumors. Although the growth of cancer cells has been considered clonal, there is now accumulating evidence for the presence of minor clones with different genetic profiles within the same tumor. To reveal intra-tumor heterogeneity, Bai et al. analyzed EGFR mutation status in more than 2,506 tumor foci microdissected from 79 tumors in patients with NSCLC who had undergone palliative surgery. Surprisingly, aproximately 38% of the tumors contained both EGFR-mutant and wild-type foci (8). Taniguchi et al. had earlier reported similar data (12). Although Yatabe et al. reported that heterogeneous distribution of EGFR mutations was extremely rare (13), the number of foci per person or the number of patients they examined were too small for statistical substantiation of their findings.
Emergence of drug-resistant tumor cells after treatment
It is often found that tumors initially showing a favorable response to drug treatment become refractory to the same drugs after continuation of the treatment. Intra-tumor genetic heterogeneity has been shown to be one of the major molecular mechanisms responsible for such acquired resistance of NCLC to EGFR-TKIs.
Emergence of the EGFR-TKI-resistant EGFR gene mutation T790M is one of the main causes leading to failure of EGFR-TKI treatments (14). A recent study using a highly sensitive method based on matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has revealed that minor populations of cells with T790M mutant alleles were already present in untreated tumors. Su et al. reported that T790M was detected in 25.2% (27 of 107) of untreated NSCLCs (15). T790M mutant cells are resistant to EGFR-TKIs, and are thought to become the prominent populations after EGFR-TKI treatment has eradicated the sensitive cells. Similar emergence of EGFR-TKI-resistant tumor cells with MET gene amplification has also been reported (16). We previously established a gefitinib-resistant subline from PC9 NSCLC cells bearing EGFR mutation (17). The gefitinib-resistant PC9 cells demonstrated increased Akt phosphorylation (not inhibitable by gefitinib), reduced expression of PTEN protein, and loss of the EGFR mutation. This result suggests that minor clones with different EGFR gene status may exist even in an established cancer cell line.
The EGFR gene status of a tumor changes as a result of chemotherapy. This concept is quite new and needs to be validated independently by other investigators. Cytotoxic drugs used for chemotherapy are highly mutagenic and may cause new mutations, but are unlikely to repair the mutated EGFR gene. The presence of genetic heterogeneity within an untreated NSCLC is now accepted. The shift of EGFR mutation status after chemotherapy is likely to result from the emergence of minor cell populations differing in EGFR gene status. However, this hypothesis requires caution because it is based on the assumption that the sensitivity of tumor cells with EGFR mutation to cytotoxic drugs is higher than that of cells bearing the wild-type EGFR gene. Further studies will be necessary to fully elucidate the molecular mechanisms behind this genetic shift.
Disclosure: The authors declare no conflict of interest.
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