Here, we have performed in-depth genetic and histological analyses on cancers that acquired resistance to EGFR inhibitors. We observed both known molecular mechanisms of acquired resistance and also several genotypic and phenotypic changes that we believe broaden the conceptual model of acquired drug resistance. Notably, we observed a surprisingly high frequency of conversion of NSCLC to SCLC, marked EGFR amplification in a subset of cases with the T790M EGFR mutation, the development of PIK3CA mutations, EMT, and the loss of genetic resistance mechanisms in the absence of continuous TKI treatment. These findings provide new insights into our understanding of drug resistance and emphasize the need to perform tumor biopsies after the development of resistance to identify the best treatment options for patients.
The development of drug resistance that invariably occurs after about 12 months of initiating treatment (4
) has spurred efforts to understand the biology underlying resistance and to identify therapeutic strategies to overcome or prevent it. These laboratory studies have primarily focused on exposing EGFR
-mutant, TKI-sensitive cell lines to EGFR TKIs until resistance develops. They have identified several resistance mechanisms, two of which—EGFR
mutation T790M (6
) and MET
)—have been validated in the clinic. Other acquired resistance mechanisms identified by studying the development of resistance to EGFR TKIs in vitro include loss of PTEN (27
) and activation of the insulin growth factor receptor (in cell lines addicted to wild-type EGFR
). However, these resistance mechanisms have not yet been validated in the clinic. Activation of MET
by hepatocyte growth factor (HGF) has been shown to drive resistance to EGFR TKIs, but these experiments were performed by adding exogenous HGF or HGF-secreting tumor-derived fibroblasts (14
), not by selecting cells after chronic exposure to TKIs. Analyses of resistant specimens support, but do not prove, that HGF may be a resistance mechanism in patients. To date, the various EGFR TKI resistance mechanisms share the same underlying concept: They enable the cancer cell to maintain its intracellular growth signaling pathways, especially the phosphatidylinositol 3-kinase (PI3K)–AKT pathway, in the presence of the EGFR TKI (32
In our cohort of patients with EGFR
mutation–positive NSCLC and acquired EGFR TKI resistance, we observed known mechanisms of resistance, the EGFR
T790M mutation and MET
amplification. Forty-nine percent developed the T790M mutation, consistent with the previously reported incidence of this mutation in patients with acquired resistance (8
). A subset of these patients also developed pronounced EGFR
amplification, and it appears that the T790M allele is selectively amplified. To the best of our knowledge, amplification of EGFR
T790M has not been previously appreciated in TKI-resistant specimens of NSCLC tumors. Balak et al.
) reported one patient with about twofold increase in EGFR
copy number in a drug-resistant specimen, but that case did not harbor the T790M mutation in EGFR
. Despite the promising activity of newer, irreversible EGFR inhibitors in patients with EGFR
), their efficacy has been minimal in patients with acquired resistance to gefitinib and erlotinib (42
). The recent report by Ercan and colleagues (17
) that amplified T790M mutations may promote resistance to irreversible EGFR inhibitors suggests that these patients may not respond to the current irreversible EGFR inhibitors and should be directed to other potential therapeutic strategies such as combined PI3K and MEK (mitogen-activated or extracellular signal–regulated protein kinase kinase) inhibition (44
), newer, more potent T790M–specific EGFR inhibitors (45
), or combinations of anti-EGFR therapies (46
). In addition, we observed that a subset of the T790M patients also acquired additional mutations, including two (11% of the T790M cohort) with acquired mutations in β-catenin
. To our knowledge, β-catenin has not been postulated as an EGFR TKI resistance mechanism. Anecdotally, in our clinic, we have three patients with concurrent EGFR
mutations at baseline, all of whom responded well to erlotinib without evidence of early-onset resistance.
amplification was identified in only two (5%) patients, which is less than the 15 to 20% frequency reported by our group and others (8
). We cannot easily explain this lower than expected frequency. Possible contributing reasons include the lack of sufficient tissue for MET
testing in two patients in the “unknown mechanism” category, the fairly conservative (high) threshold used for designating amplification used by our pathologists, and the sample size of our cohort. In addition, we failed to identify any acquired genetic resistance mechanism in several cases. Although we were unable to test for all potential resistance mechanisms because of tissue exhaustion and inadequate reagents, it does seem likely that further analyses with more sophisticated techniques such as deep sequencing will lead to the identification of new mechanisms of resistance to EGFR TKIs.
In addition to these two well-described mechanisms of TKI resistance, we observed acquired PIK3CA
mutations in two patients. To our knowledge, this represents the first documentation of PIK3CA
mutations leading to drug resistance in cancer patients. This finding is supported by our previous laboratory findings that introduction of a PIK3CA
mutation in EGFR
-mutant HCC827 cells confers resistance to gefitinib (15
). This has important therapeutic implications because there are several ongoing early-phase clinical trials combining EGFR and PI3K pathway inhibitors that are attractive targeted therapy strategies to overcome this mode of resistance. We also hypothesize that patients who have EGFR
mutations in the original primary tumor (baseline) might experience an abbreviated duration of benefit from EGFR TKI therapy compared with patients lacking PIK3CA
mutations, and could be considered for enrollment in a first-line clinical trial combining an EGFR and PI3K inhibitor. Indeed, we have observed two patients with EGFR
mutations at baseline who both responded to first-line erlotinib therapy, but the responses lasted only 5 and 7 months. Neither of these cases is included in this cohort of patients who received repeat biopsies; one underwent a repeat biopsy but the tissue was nondiagnostic, and the other was not offered a repeat biopsy.
Perhaps, one of the more surprising findings from our study is the observation that 5 (14%) of the 37 patients experienced a fundamental histology transformation from NSCLC to SCLC at the time of TKI resistance. The original EGFR mutation was maintained in all five patients, disputing the rare possibility that these patients developed a second primary cancer. One patient also acquired a PIK3CA mutation in the SCLC specimen, but none of the patients demonstrated EGFR T790M or MET amplification. The pre- and posttreatment tissues were subjected to neuroendocrine immunohistochemical analyses including staining for synaptophysin, chromogranin, and/or CD56. Although the posttreatment (SCLC) specimens were all positive for neuroendocrine markers, most consistently synaptophysin, the pretreatment (NSCLC) samples were uniformly negative for neuroendocrine markers. We speculate that the high frequency of recognizing this unusual histological phenomenon may have been partly because of the implementation of thorough pathological evaluation of drug-resistant specimens as part of routine clinical care. These findings directly affected patient care decisions, and four of the five patients received SCLC chemotherapy regimens with a response obtained in three patients. This unequivocally suggests that the posttreatment biopsies provided useful clinical information in addition to research information, and that repeat biopsies at the time that clinical resistance to EGFR TKIs develops can directly benefit patients. The transition from NSCLC to SCLC appears to be specific for resistance to EGFR TKIs. We observed no evidence of SCLC in 10 cases of EGFR wild-type chemotherapy-resistant NSCLC and in 69 resected stage III lung cancers, where the patients had received chemotherapy and radiation.
Previous case reports have described patients with biopsy-proven SCLC and EGFR
). The individual cases reported by Zakowski et al.
) and by Morinaga et al.
) are most similar to our patients, and each describes a never-smoking female that presented with EGFR
-mutant metastatic adenocarcinoma that transformed into SCLC after developing resistance. Okamoto et al.
) describe a never-smoking female diagnosed with CD56-positive advanced SCLC harboring an exon 19 deletion in EGFR
, who had a good partial response to first-line gefitinib. Fukui et al.
) identified 6 patients with combined NSCLC-SCLC histology from a cohort of 64 SCLC patients undergoing surgical resection; one was a never-smoking female with an L858R EGFR
mutation in both the SCLC and adenocarcinoma components. The final report is a case series arising from an analysis of 122 Asian patients with SCLC or mixed histology tumors that were screened for EGFR
mutations, of which 5 (4%) samples were found to be mutation-positive (3 L858R, 1 exon 19 deletion, and 1 G719A) including a never-smoker and 4 smokers with tobacco histories ranging from 3 to 68 pack-years (51
). In this series, only one patient had a pretreatment adenocarcinoma that transformed into a combined SCLC-adenocarcinoma after developing clinical resistance to an EGFR TKI. The other four patients had EGFR
-mutant SCLC or mixed histology tumors at baseline.
The biological underpinnings of the SCLC transformation are unknown and are of great interest. The finding that the same EGFR
-mutant cancer can manifest both as an adenocarcinoma and as a SCLC hints at the existence of a pluripotent population of EGFR
-mutant cancer cells or cancer stem cells (52
) that are the source of resistance. The cause of the phenotypic switch to SCLC and concordant development of resistance remain to be determined. Perhaps, these patients developed drug resistance through a genetic or epigenetic event that concurrently led to a shift in phenotypic appearance. One of the marked molecular differences between NSCLC and SCLC is that most SCLCs exhibit loss of expression of the retinoblastoma protein (54
), a tumor suppressor. We attempted to determine whether the resistant specimens had loss of retinoblastoma protein expression by immunohistochemistry, but staining was not of sufficient quality for interpretation.
In addition, we clearly observed the EMT in two cases of acquired TKI resistance. Neither case had another identified resistance mechanism, but more cases will be required to determine whether this mutual exclusivity can be generalized. Similarly, we observed an EMT in an EGFR
-mutant cell line rendered resistant to an EGFR inhibitor in vitro. Several groups have noted that cell lines undergoing EMT are intrinsically resistant to EGFR inhibitors (18
). However, those cancer models do not have EGFR
mutations and many have KRAS
mutations, so the relevance of those findings to acquired TKI resistance is less straightforward. Two case reports just published support our observation of an EMT in EGFR
-mutant NSCLC at the time of TKI resistance (57
). The molecular mechanisms connecting the resistance of the cancer cells to the mesenchymal phenotype remain unknown. However, the recent findings that KRAS
-mutant lung cancers with mesenchymal features are resistant to both KRAS knockdown (59
) and combined PI3K and MEK inhibition (60
) suggest that mesenchymal cells may have an intrinsic lack of sensitivity to the intracellular signaling pathway down-regulation that is normally the hallmark of sensitivity to EGFR TKIs.
Evidence from three patients with multiple biopsies over the course of their disease suggests that both tumor genotype and phenotype may evolve dynamically under the selective pressure of targeted therapies. Two patients developed T790M EGFR
mutations at the time of TKI resistance and subsequently lost evidence of that resistance mutation in the same anatomic tumor after a period free from TKI treatment. These patients both responded to a challenge with an EGFR inhibitor subsequent to losing the T790M mutation. The third patient underwent a SCLC transformation with acquisition of a PIK3CA
mutation at the time of resistance and, after a TKI-free interval, was found to have adenocarcinoma without a detectable PIK3CA
mutation. This cycle was repeated when, after a second response to erlotinib, the cancer ultimately developed resistance again and the biopsy of the resistant cancer again revealed the SCLC phenotype with the EGFR
L858R and PIK3CA
mutations. The mechanisms underlying these fluctuations remain to be proven, but it is tempting to speculate that the baseline heterogeneity of the cancers may contribute to these findings. Indeed, it is possible that substantial populations of “sensitive” cancer cells may remain dormant while subjected to TKI treatment, as recently suggested by laboratory studies (61
). Withdrawal of the TKI may permit their rapid expansion to a degree that overtakes the bulk of the tumor burden. Such a mechanism could also provide insight into the pronounced tumor flare that is often clinically observed when the TKI is removed from slowly progressing cancers (62
). Indeed, these findings confirm that even “genetic” mechanisms of resistance are potentially reversible. Therefore, a static diagnostic biopsy may be insufficient to guide therapeutic decisionmaking throughout the course of a patient’s disease. Moreover, all of our patients experienced a second response to erlotinib when their resistance mechanism was no longer detectable, suggesting that repeat biopsies can provide molecular guidance about the likely benefit of a second treatment regimen with EGFR TKI therapy.
The primary limitations of our study are its retrospective nature and the heterogeneity among practice patterns that led to patients undergoing repeat biopsies at various times during their disease (). Although all of these treatment variations could have affected the resistance mechanisms observed, the most direct confounder is likely to be whether the patient was “on” or “off” of the primary TKI at the time of biopsy. All of our patients except one were on TKI at the time of biopsy, or had been off drug treatment for ≤5 months (table S1
). Another limitation is that in many cases, because of safety and feasibility concerns or because of the predominant radiographic progression in one anatomic area over another, the repeat biopsies were obtained from different tumor locations compared to the original biopsies. Although distinct mechanisms of resistance in different anatomic locations within the same patient have been described (8
), we observed that the primary resistance mechanism was often consistent throughout different metastatic sites both in our autopsy cases and in patients with multiple sites biopsied over time. Larger studies will be helpful in further clarifying the impact of these variables.
In conclusion, this study provides further impetus for the utility of reassessing cancers after they acquire resistance to targeted therapies. As our study shows, there is tremendous heterogeneity in resistance mechanisms, each of which may require its own therapeutic strategy. A recent report suggests that cancers with various resistance mechanisms may have distinct prognoses (63
). Although invasive biopsies have associated risks, we did not encounter any significant complications. We anticipate that technologies to assess cancers via noninvasive measures such as circulating tumor cell analyses, plasma DNA analyses, or molecular radiology may eventually obviate the need for invasive procedures. The knowledge gained from our repeat biopsy program directly affected treatment decisions and outcomes, and we were better equipped to rationally treat patients (for example, those with SCLC) as their tumors evolved. Several patients in our cohort were enrolled in clinical trials specifically targeting T790M, MET
, or the PI3K
signaling pathway after biopsies of their drug-resistant tumors, and several had disease stabilization or response to those therapies. Indeed, it is becoming increasingly clear, from experiences with both chronic myelogenous leukemia treated with ABL kinase inhibitors and EGFR
-mutant lung cancers treated with EGFR kinase inhibitors, that the era of targeted therapies will mandate continual assessment of each cancer’s evolution over the course of treatment to determine how it became resistant to therapy and to identify the optimal strategies to prevent or overcome it.