1.INTRODUCTION 1.1 Background
3. PRIMARY & SECONDARY RESISTANCE TO FIRST-GENERATION EGFR-TKIs
Table 2. First-generation EGFR-TKIs versus chemotherapy as first-line treatment for NSCLC patients with activating EGFR mutations
Trial Patients features Treatment ORR
Median PFS/TTP (months)
Median OS (months) Gefitinib
(ref #61,#68) 261 patients EGFR-mutation positive Gefitinib or carbo+pac 71.2 (gef)
47.3 (CT) 9.5 (gef)
6.3 (CT) 21.6 (gef) 21.9 (CT) WJTOG3405
172 patients EGFR-mutation positive Gefitinib or cis+doc 62.1 (gef) 32.2 (CT)
9.2 (gef) 6.3 (CT)
30.9 (gef) NA NEJSG002
(ref #72) 230 patients EGFR-mutation positive Gefitinib or carbo+pac 73.7 (gef)
30.7 (CT) 10.8 (gef)
5.4 (CT) 30.5 (gef) 23.6 (CT) First-SIGNAL
313 Korean never-smokers with stage IIIB or IV lung adenocarcinoma
Gefitinib or cis+gem 55.4 (gef) 46.0 (CT)
5.8 (gef ) 6.4 (CT)
22.3 (gef) 22.9 (CT) Erlotinib
OPTIMAL (ref #73)
165 patients EGFR-mutation positive Erlotinib or carbo+gem
83.0 (erl) 36.0 (CT)
13.1 (erl) 4.6 (CT)
NA NA EURTAC
(ref #74) 174 patients EGFR-mutation positive Erlotinib or carbo/cis +
doc/gem 58.0 (erl)
15.0 (CT) 9.7 (erl)
5.2 (CT) 19.3 (erl) 19.5 (CT) Carbo, carboplatin; cis, cisplatin; CT, chemotherapy; doc, docetaxel; erl, erlotinib; gef, gefinib; gem, gemcitabine; NA, not available; pac, paclitaxel; pl, placebo.
Moreover, given the reduced toxicity, the improved quality of life and the rapid symptoms relieve with single agent TKI when compared to chemotherapy, the use of first-generation EGFR-TKIs has become the new standard of care for the upfront treatment of EGFR mutation-positive NSCLC patients. However, the recent South Korean randomized phase III trial First-SIGNAL comparing gefitinib to first-line chemotherapy in 309 Korean never-smokers with lung adenocarcinoma, failed to demonstrate any OS advantage (22.3 vs.
22.9 months, respectively; HR = 0.932; CI: 0.716-1.213; P = 0.604) derived from TKI treatment in this selected population  (Table 2).
In contrast, the Chinese study INFORM, comparing gefitinib to BSC as maintenance therapy in unselected NSCLC patients, reported a significantly longer PFS in the TKI treated arm with respect to placebo (4.8 vs. 2.6 months, respectively; HR = 0.42; P < 0.0001). Of note, consistent with other trials, EGFR-mutation positive patients took advantage the most from gefitinib maintenance treatment (median PFS: 16.6 vs. 2.7 months; HR = 0.16), but no difference in term of OS was reported . On the other hand, maintenance therapy with erlotinib in the SATURN study achieved significantly improved PFS and OS among the overall patients. Interestingly, erlotinib was able to improve PFS in both EGFR mutated (HR = 0.10; CI: 0.04-0.25; P < 0.0001) and wild-type (HR = 0.78; CI: 0.63-0.96; P = 0.0185) patients . Taken together, the results from INFORM and SATURN trials suggest that EGFR wild-type patients may benefit from EGFR-TKI maintenance therapy on the basis of the specific EGFR-TKI used.
Even though a large number of trials firmly established the predictive role of EGFR mutations as biomarkers of response to gefitinib or erlotinib, a small group of wild-type EGFR NSCLC patients also significantly benefits from the treatment with the EGFR-TKIs . These findings suggest the existence of additional mechanisms involved in sensitivity or resistance to these agents in EGFR mutation-negative patients.
of patients significantly benefits from EGFR inhibition while most of the tumors show primary resistance.
However, despite the initial promising response to gefitinib or erlotinib as first-line therapy EGFR-TKIs in the overwhelming majority of NSCLC patients harboring sensitising EGFR mutations [78,79], most of these patients inevitably relapse due to the emergence of acquired resistance. The challenge of tumor drug resistance therefore represents a pervasive barrier that confounds the ultimate goal of cure or long-term control of advanced/metastatic cancer.
3.1. Primary Resistance and EGFR Status
De novo resistance includes patients who are initially refractory to EGFR-TKI treatment. The IPASS trial reported for wild-type EGFR patients of Asian origin an objective RR of 2% and reduced TTP with TKI treatment as compared with chemotherapy . These results suggest that wild-type EGFR is a negative biomarker for the response to TKI treatment. Moreover, other genetic alterations than activating EGFR mutations, such as insertions (D770_N771insNPG, D770_N771insSVQ, and D770_N771insG) or the single point mutation T790M in exon 20 of EGFR, have been correlated with primary resistance to TKIs (Table 3).
Table 3. Summary of the mechanisms of resistance to EGFR-TKIs in NSCLC
Primary Resistance Secondary Resistance
aberrations - D770_N771 (ins NPG) - D770_N771 (ins SVQ) - D770_N771 (ins G) - D770_N771 (ins VDSVDNP) - S768_V769 (ins VAS) - T790M
- L861R - L862V
aberrations - T790M - L474S - D761Y - T854A
- altered trafficking
mechanisms - ins in exon 20 HER2 - K-RAS mut
- PI3KCA mut - loss of PTEN - BRAF mut
- overexpression of MAPKs - overexpression of ABCG2 - overexpression of IGF-1R - overexpression of Bcl-2 - overexpression of angiogenesis regulators
- overexpression of SRC-3 - ALK translocations - CSCs
- MET amplification
mechanisms - MET amplification - overexpression or hyperactivation of IGF-1R - constitutive association of IRS-1 with PI3K
- overexpression of ABCG2 - PI3KCA mut
- SCLC transformation - EMT
ins, insertion; mut, mutation.
These alterations, which preclude the interaction of gefitinib or erlotinib with their target, occur in almost 5% of NSCLCs [59,80]. Of note, somatic insertions in HER2 exon 20 have also been detected in NSCLC and were similarly associated with absence of sensitivity to EGFR-TKIs . However, some studies reported the existence of T790M at a low frequency within the tumor cells before TKI treatment [82-85]. Since this mutation is also correlated with secondary resistance, drug selection pressure of pre-existing kinase domain mutations has been proposed as explanation for the presence of T790M as dominant clone after TKI
treatment. A few cases of germline transmission of T790M have been described and this mutation has been recognised as initiating genetic event, but studies to date failed to demonstrate growth advantage as well as altered signalling conferred by T790M-EGFR compared with the wild-type receptor. However, given the limited sensitivity of detection methods, the question whether T790M is primary rather than acquired is still unanswered.
Despite the low incidence of these mutations, various studies aimed to overcome resistance conferred by exon 20 aberrations of EGFR and HER2 were conducted. In these studies, different strategies, including the use of EGFR-HER2 irreversible inhibitors as well as heat shock protein-90 (HSP-90) inhibitors, were tested [86, 87]. Few other aberrations, such as S784F, S768_V769insVAS, D770_N771insVDSVDNP, L861R and L862V, have been associated with EGFR-TKIs resistance in NSCLC patients . However, for many of the rare EGFR mutations, the effect on responsiveness to TKIs treatment remains unknown.
3.2. Other Mechanisms of Primary Resistance
Mutations in genes other than EGFR and its family members have also been shown to play an important role in conferring primary resistance to EGFR-TKIs (Table 3). The best described examples of such resistance are mutations of the K-RAS oncoprotein which have been correlated with de novo resistance to TKIs in different tumor types including NSCLC [63,88,89]. K-RAS is a member of the RAS family of oncogenes and gene mutations have been reported to be related to the development of many cancers .
K-RAS aberrations have been detected in 20-30% of NSCLC patients with >90% localised in codons 12 and 13. Such alterations are responsible for the constitutive activation of RAS signaling, which promotes cell proliferation and reduction of apoptosis through the activation of EGFR downstream pathways such as PI3K and MAPKs . Of note, K-RAS aberrations have been more frequently observed in adenocarcinomas from elderly and heavy smokers patients who have been identified as groups unlikely to benefit from EGFR-TKIs therapy [92,93]. Although mutations in K-RAS and EGFR are almost mutually exclusive, there are rare instances where they coexist. Interestingly, mutations of the catalytic region of PI3K (PIK3CA), exons 9 and 20, in cis- with EGFR mutations have been reported in NSCLC patients who have never been treated with EGFR-TKIs [94,95]. Other uncommon markers for de novo resistance, such as loss of PTEN, BRAF mutations, overexpression of MAPKs, ATP-binding cassette sub-family G member 2 (ABCG2), insulin-like growth factor-1 receptor (IGF-1R), Bcl-2, and angiogenesis regulators, as well as SRC-3 have recently been observed [96,97]. Furthermore, ALK translocations, which have been found to be mutually exclusive with EGFR or KRAS mutations, have also been associated with primary resistance to gefitinib or erlotinib treatment in advanced NSCLC patients .
Cancer stem cells (CSCs), together with their self-renewal, maintenance, and plasticity signaling pathways, such as TGF-α, Wnt, Notch, Hedgehog, PI3K/PTEN/mTOR, IGF-1R, histone demethylase and histone deacetylase (HDAC), have been also proposed as potential mechanisms of primary drug-resistance, including EGFR-TKIs [99,100]. Since CSCs seem to be less dependent on growth factor pathways than the abundant non-CSCs in a tumor, they may survive to drug inhibition leading to the failure of the therapy.
Indeed, acquisition of secondary mutations in this quiescent stem cell population might cause the emergence of secondary resistance.
3.3. Secondary Resistance and EGFR Status
The acquisition of resistance to kinase inhibitors treatment in clinical oncology is by now a well-documented phenomenon in multiple cancer types. As the major EGFR activating mutations improve the sensitivity of the tumor to anilinoquinazoline inhibitors stabilizing their interactions with crucial amino acids in the ATP-binding pocket , to date it seems that after an initial response to such agents all the patients eventually experience progressive disease . Given the rapid increasing number of patients who acquire resistance to gefitinib or erlotinib after their approval as first-line treatment in selected NSCLCs, a clinical definition of secondary resistance to these agents was established to unify treatments and investigate this subgroup of lung tumor .
One commonly described mechanism of drug resistance involves additional genetic alterations within the target oncogene itself. This mechanism was first described in patients with CML treated with imatinib.
However, while several secondary mutations have been associated with secondary resistance to imatinib treatment, only a few point mutations, such as T790M, L747S , D761Y  and T854A , have been correlated to resistance to gefitinib and erlotinib in NSCLC (Table 3). In particular, the EGFR-T790M secondary mutation of kinase domain is the best characterized and most frequent mechanism of acquired resistance to EGFR-TKIs accounting for approximately 50% of cases relapsed from prior targeting treatment [104,106-108]. The T790M mutation, often referred to as the “gatekeeper”, was initially thought to confer resistance to EGFR-TKIs due to the steric hindrance of the bulkier methionine residue [106,107]. However, this explanation is difficult to reconcile with the evidence that irreversible EGFR-TKIs structurally similar to gefitinib and erlotinib are able to overcome the resistance associated with this mutation, as discussed later.
Recently, Yun and colleagues have reported that T790M-mutant retains affinity to gefitinib (Kd 4.6 nM) similar to that observed for the L858R mutant (Kd 2.4 nM) . Interestingly, the acquisition of such mutation increases about 10-fold the ATP affinity with respect to the L858R-mutant receptor. The authors concluded that the increased ATP affinity, and not the sterical blocking binding, is the mechanism by which the T790M mutation confers drug resistance .
The T790M mutation has been associated with shorter PFS when compared to the wild-type receptor (7.7 vs. 16.5 months, respectively; P < 0.001) . However, this gatekeeper mutation has also been correlated with more indolent disease and better survival outcomes than other mechanisms of acquired resistance to TKIs . Moreover, discontinuing EGFR-TKI treatments and removing the selection pressure for T790M has been associated with mutation-independent tumor growth and loss of the secondary mutation .
3.4. Other Mechanisms of Acquired Resistance
In 2007, Engelman and colleagues first reported the amplification of the receptor for the hepatocyte growth factor (MET) as the second major mechanism of acquired resistance to EGFR-TKIs  (Table 3).
MET amplification has been detected in about 20% of patients who relapsed after gefitinib or erlotinib prior therapy, but only in 3% of untreated patients and cases of coexistence of the gatekeeper mutation with MET amplification have been described [113,114]. Furthermore, MET amplification was reported to sustain the HER3/PI3K/Akt antiapoptotic signaling pathway despite the presence of gefitinib in NSCLC cells [113-115].
Based on these findings, preclinical studies indicate the combination of EGFR and MET-TKIs as treatment
strategy for tumors harboring both EGFR mutation or MET amplification [115,116].
Several other mechanisms have been associated with EGFRTKIs acquired resistance, including expression and hyper-activation of the IGF-1R and constitutive association of IRS-1 with PI3K through loss of expression of IGF-binding proteins [117,118], altered EGFR trafficking , and expression of the ABCG2 drug efflux transporter . The alteration of PIK3CA has also been indicated as secondary resistance mechanism to first-generation EGFR-TKIs in vitro [112,121]. Phenotypic transformation of EGFR mutation-positive adenocarcinoma patients who “switched” to small-cell lung cancer (SCLC) and epithelial-to-mesenchymal transition (EMT) processes, identified as acquired vimentin and loss of E-cadherin expression, were also reported as mechanisms of secondary resistance to EGFR-TKIs by Sequist and colleagues . Recently, the activation of TGF-α/IL-6 signaling has been associated with erlotinib resistance because of its involvement in EMT . Of note, in approximately 30% of cases, the mechanisms underlying secondary resistance to EGFR-TKIs treatment are still unknown.