The Landscape of EGFR Pathways and Personalized Management of Non-small-cell Lung Cancer

Liang Cheng; Shaobo Zhang; Riley Alexander; Yongxue Yao; Gregory T MacLennan; Chong-xian Pan; Jiaoti Huang; Mingsheng Wang; Rodolfo Montironi; Antonio Lopez-Beltran


Future Oncol. 2011;7(4):519-541. 

In This Article

EGFR-targeted Therapy & Mechanisms of Resistance

There are two major approaches for inhibiting EGFR signaling: to prevent ligand binding to the extracellular domain with a monoclonal antibody and to inhibit the intracellular TK activity with a small molecule. Use of the latter approach was the first method to be attempted clinically.[131] The EGFR TKIs are reversible competitive inhibitors of the TK domain of EGFR that bind to its ATP-binding site. Somatic activating mutations of the EGFR gene, increased gene copy number and certain clinical and pathological features have been associated with dramatic tumor responses and favorable clinical outcomes with these agents in patients with NSCLC. The majority of these patients inevitably acquire resistance to EGFR TKIs. Recent data indicate that a secondary mutation, such as T790M, expression of HGF, PTEN and/or early growth response-1 and changes in the epithelial-to-mesenchymal transition, were associated with EGFR TKI resistance. Uramoto et al. found that strong expression of HGF was detected in six out of eight specimens with the T790M mutation.[132] Three out of eight cases (38%) demonstrated a loss of PTEN in samples with the T790M mutation. A loss of early growth response-1 was detected in two out of seven cases (29%), including one tumor without PTEN. Four out of seven cases (57%) demonstrated positive expression of phosphorylated Akt. A change in the epithelial-to-mesenchymal transition status between pre- and post-treatment was observed in four out of nine cases (44%). These results suggest that alterations in gene or protein expression can account for all mechanisms by which tumors acquire resistance to EGFR TKIs.[132] This phenomenon suggests the existence of complicated relationships between acquired resistance-related genes.

Somatic activating mutations in EGFR are identified in a subset of NSCLC that responds to TKIs. As noted previously, acquisition of drug resistance has been linked to a specific secondary somatic mutation, EGFR T790M. Bell et al. described a family in which multiple members developed NSCLC associated with germline EGFR mutations of T790M.[25] These observations implicate altered EGFR signaling as a culprit in the genetic susceptibility to lung cancer in families with an increased incidence of NSCLC.

We propose an algorithm for molecular testing for patients with NSCLC (Figure 4). A stepwise approach, based on the frequency of specific mutations, is used to assess lung cancer patients for specific findings, which will allow proper therapeutic stratification for targeted therapy.

Figure 4.

Suggested algorithm for molecular testing for patients with non-small-cell lung cancer. A stepwise approach is used to test lung cancer patients according to the known frequencies of various mutations. Small-cell lung cancers are excluded from testing. NSCLC, which accounts for approximately 85% of all lung cancers, is tested for the presence of EGFR mutations. A positive test, found in approximately 20% of Caucasians and 40% of East Asians, predicts an 80% probability of response to EGFR TKI therapy. Nonmutated EGFR is found in approximately 80% of Caucasians and 60% of East Asians. These patients are further tested for EML4–ALK mutations. EML4ALK mutations are found in only 3% of patients with NSCLC, but the mutation predicts a 53% probability of response to targeted therapy. Cases lacking EML4–ALK mutations may undergo additional testing.
EGFR: EGF receptor; Mu: Mutation; NSCLC: Non-small-cell lung cancer; SCC: Small-cell carcinoma; TKI: Tyrosine kinase inhibitor.

EGFR-targeted Therapy Approaches

Monoclonal Antibodies Monoclonal antibodies, such as cetuximab and panitumumab, are either chimeric mouse–human or fully humanized antibodies targeting the extracellular domain of EGFR and thereby inhibiting the binding of activating ligands to the receptor. This class of treatment inhibits ligand-dependent activation of EGFR and inhibits the downstream pathways, which cause cell cycle progression, cell growth and angiogenesis (Figure 3A). In addition, binding of the antibody initiates EGFR internalization and degradation, which leads to signal termination.[108,133] Fully humanized antibodies such as panitumumab, have a high affinity for EGFR and a longer half-life.[134] Although EGFR is frequently expressed in patients with NSCLC, the clinical efficacy of treatment with anti-EGFR antibodies is limited to only a subset of patients.

Tyrosine kinase inhibitors Tyrosine kinase inhibitors are synthetic small molecules that block the magnesium–ATP-binding pocket of the intracellular TK domain.[108] Several TKIs, such as gefitinib and erlotinib, are specific for EGFR, whereas others inhibit other receptors in addition to EGFR, such as HER2 and VEGF receptor 2. TKIs block ligand-induced receptor autophosphorylation by binding to the TK domain and disrupting TK activity, thereby abrogating intracellular downstream signaling (Figure 3B & Table 1).

Mechanisms of Resistance to EGFR-targeted Therapy

Acquired Resistance Caused by a Secondary Mutation Although EGFR mutations are associated with enhanced sensitivity to gefitinib and erlotinib, not all tumors that have activating mutations are associated with an enhanced response. The efficacy of EGFR TKIs is limited owing to either primary or acquired resistance after therapy. Most patients who initially respond to gefitinib and erlotinib eventually become resistant and experience progressive disease.

It is known that four mutations result in TKI drug sensitivity: point mutations in exon 18 (G719A/C) and exon 21 (L858R and L861Q), as well as in-frame deletions in exon 19, which eliminate four amino acids – leucine, arginine, glutamic acid and alanine – downstream of the lysine residue at position 745.[67,69–72] However, insertion mutations of exon 20 at D770–N771 were associated with EGFR TKI resistance.[60,135] This observation was confirmed in an in vitro model in which insertion mutations in exon 20 rendered transformed cells less responsive to EGFR TKIs compared with the sensitizing mutations of exons 19 and 21.[135]

Two established mechanisms of acquired resistance consist of additional mutations in the EGFR gene acquired during the course of treatment that change the protein coding sequence and amplification of other oncogene signaling pathways.[85,136–138]

Kobayashi et al. reported a gefitinib-resistant advanced NSCLC patient who had a relapse after 2 years of complete remission due to treatment with gefitinib.[86] The DNA sequence of EGFR at relapse revealed the presence of a second point mutation, resulting in a T790M mutation of EGFR. Structural modeling and biochemical studies showed that this second mutation led to gefitinib resistance.[86] The same mutation was confirmed by Pao et al. through molecular analysis of EGFR in patients with acquired resistance to gefitinib or erlotinib.[139] The gefitinib-resistant cases contain the same secondary mutation (T790M) in the kinase domain.[22] Codon 790 of EGFR is considered to be the 'gatekeeper' residue, which is an important determinant of inhibitor specificity in the ATP-binding pocket of EGFR.[108] Substitution of this residue in EGFR with a bulky methionine may cause resistance by steric interference with the binding of TKIs, including gefitinib and erlotinib.[86,139,140] This mutation may confer a survival advantage to the tumor and is probably selected for while the patient is receiving anti-EGFR TKI treatment.[25,84] These findings have led to the development of irreversible EGFR TKIs in an effort to effectively target this mechanism of resistance.[140]

The role of oncogenic activation of EGFR downstream effectors, such as KRAS, BRAF, PIK3CA and PTEN, in response to therapy is discussed extensively in a series of studies.[47,53] The RAS–MAPK and PI3K–AKT pathways are major signaling networks linking EGFR activation to cell proliferation and survival.[141,142] Mutations in these downstream effectors of EGFR signaling could lead to resistance to EGFR inhibitors.[136–138] The discovery of molecular aberrations, such as MET kinase amplification or mutations of EML4–ALK fusion, which causes constitutive activation of RASRAFMEK, has provided further insight and validation into factors limiting the therapeutic efficacy of EGFR inhibitors.[46,143,144]

KRAS Mutations KRAS plays a key role in the EGFR signaling network. The KRAS proto-oncogene encodes KRAS G-protein, which plays a critical role in the RASMAPK signaling pathway downstream of many growth factor receptors, including EGFR.

One of the most important discoveries for the clinical management of colorectal carcinoma has been the association of mutations in KRAS and the efficacy of monoclonal antibodies targeting EGFR, such as panitumumab and cetuximab. Some tumors harbor somatic mutations in exon 2 of KRAS that compromise the hydrolysis of RAS-bound GTP to GDP, resulting in constitutive activation of the RAS pathway.[145] In the presence of a KRAS mutation, EGFR pathway activation cannot be significantly inhibited by cetuximab or panitumumab, which acts upstream of the KRAS protein, diminishing the efficacy of the agents.

An activating mutation of KRAS is present in 15–30% of NSCLC cases[26,146,147] and accounts for approximately 35–45% of TKI-nonresponsive cases.[148] Approximately 30% of lung adenocarcinomas contain activating KRAS mutations, which are associated with resistance to EGFR TKIs.[22] It is noteworthy that the presence of a KRAS mutation is common in NSCLC, but the occurrences of KRAS and EGFR mutations seem to be mutually exclusive.[4,27,149–152]EGFR and KRAS mutations are rarely if ever detected in the same tumor, suggesting that they may perform functionally equivalent roles in lung tumorigenesis.[58,153] However, there is growing evidence that coexistence of EGFR and KRAS mutations is possible,[27,151,154] although the frequency is low. Due to the limited number of cases, it is difficult to obtain conclusive results; however, the available data suggests a negative association between EGFR/KRAS mutation and EGFR TKI responsiveness.[27,151,154]

It remains unclear whether assessment of KRAS mutation status will prove to be clinically useful with regard to anti-EGFR therapy.[50] Although an association between the presence of a KRAS mutation and lack of response to EGFR TKIs has been observed, it remains indeterminate whether this association is clinically relevant with respect to progression-free and overall survival. Investigations of KRAS mutation status as a negative predictor of outcome after anti-EGFR therapy have been undertaken, but small sample sizes due to low prevalence of KRAS mutations have limited the power of such studies. Some investigators have reported that KRAS mutation is a negative predictor of response to anti-EGFR monoclonal antibodies and also an important mechanism of resistance to TKIs in NSCLC.[26] Unlike colorectal cancer, KRAS mutations do not seem to identify patients who do not benefit from anti-EGFR monoclonal antibodies in NSCLC.

KRAS mutations are almost exclusively detected in codons 12 and 13 of exon 2, which may result in EGFR-independent intracellular signal transduction activation. In a study by Eberhard et al., EGFR exons 18–21 and KRAS exon 2 mutations were investigated via sequencing in tumors of 274 patients.[27]KRAS mutations were present in 21% of tumors, which were associated with significantly decreased TTP and survival in patients treated with erlotinib plus chemotherapy. Others have reported that KRAS mutation status did not impact EGFR TKI therapy.[28] In a study that included 223 chemotherapy-naive patients with advanced NSCLC treated with erlotinib or gefitinib monotherapy, EGFR mutations were associated with a 67% response rate. Wild-type EGFR was associated with poorer outcomes, regardless of KRAS mutation status.[28]

A study by Wang et al. utilizing PCR-restriction fragment length polymorphism analysis investigated the KRAS mutations in codons 12 and 13 in 273 NSCLC cases.[155] Of the 120 patients who received EGFR TKI therapy, only 5.3% (one out of 19) of the patients with a KRAS mutation demonstrated a response compared with a 29.7% response rate for patients lacking a KRAS mutation.[152] Furthermore, the median progression-free survival time of patients with a KRAS mutation was 2.5 months compared with 8.8 months for patients with wild-type KRAS.

A meta-analysis by Linardou et al. provided empirical evidence that somatic mutations of the KRAS oncogene are highly specific negative predictors of response to single-agent EGFR TKIs in advanced NSCLC.[148] Among 17 publications, 165 out of 1008 (16%) NSCLC patients presented with KRAS mutations. The presence of KRAS mutations was significantly associated with an absence of response to TKIs in these patients.

Having an intact KRAS is necessary, but not sufficient, to derive benefit from EGFR inhibition, and additional mechanisms of resistance to EGFR inhibitors exist.

KRAS testing scenarios in the management of NSCLC are summarized in Figure 5. The incidence of KRAS mutations in NSCLC is reportedly up to 20%.[156] In the subset of tumors with KRAS mutations, less than 3% also contain an EGFR mutation, and the remaining 97% of tumors with KRAS mutations have wild-type EGFR. Both KRAS/EGFR double mutations and wild-type EGFR are associated with nonresponsiveness to EGFR-targeted therapy.

Figure 5.

KRAS testing scenarios in the management of non-small-cell lung cancer. NSCLC accounts for approximately 85% of all lung cancers. The incidence of KRAS mutations in NSCLC is reportedly up to 20%. In the subset of patients with KRAS mutation, less than 3% of the tumors also contain an EGFR mutation; the remaining 97% have wild-type EGFR. Both KRAS/EGFR double mutations and wild-type EGFR are associated with nonresponsiveness to EGFR-targeted therapy. Of the 80% of NSCLCs that do not have KRAS mutations, approximately 20% harbor EGFR mutations, which are associated with an 80% likelihood of response to EGFR TKI therapy.
EGFR: EGF receptor; Mu: Mutation; NSCLC: Non-small-cell lung cancer; TKI: Tyrosine kinase inhibitor.

BRAF Mutation Both KRAS and BRAF are part of the signaling cascade for EGFR family proteins.[157] The BRAF protein is a serine/threonine protein kinase that is activated by KRAS in a GTP-dependent manner.[158] The major BRAF functions are believed to be mediated by phosphorylation and thus activation of the MAPK, MEK1 and MEK2 pathways. Mutant BRAF proteins have elevated kinase activity and can transform NIH3T3 cells.[159] KRAS function is not required for the growth of cancer cell lines with the BRAF mutation.

BRAF mutations are found in a mutually exclusive pattern with KRAS mutations, suggesting that these genetic events activate a set of common downstream effectors of transformation. The histologic phenotype of BRAF mutant adenocarcinomas has been analyzed by Yousem et al., who investigated 222 adenocarcinomas of the lung without KRAS and EGFR mutations.[160] Of these, ten adenocarcinomas with the BRAF-V600E mutation were identified. BRAF mutations were reported more frequently in micropapillary lung adenocarcinoma.[161] De Oliveira Duarte Achcar and colleagues analyzed the clinical and molecular profile of 15 primary micropapillary adenocarcinomas and found BRAF mutations in three cases (20%). The BRAF-V600E mutation-bearing tumors had a slight female predilection (6:4 female:male). The elderly patients were found to have a greater than expected incidence of intralobar satellite nodules and N2 node involvement.[161] The adenocarcinomas were largely of mixed type, with a high incidence of papillary (80%) and lepidic growth (50%). However, due to the relatively small sample size, it is yet to be determined whether BRAF mutant tumors represent a distinct subset of lung adenocarcinoma.[162]

Mutations in BRAF have been shown to impair responsiveness to panitumumab or cetuximab in patients with colorectal carcinomas. This initial retrospective work was performed on a cohort of 132 patients.[163] The results showed that none of the patients who experienced a response displayed BRAF mutations, whereas 11 of 79 nonresponders (14.0%) carried a BRAF-V600E allele.

Nevertheless, BRAF mutations are rarely detected in NSCLC when compared with KRAS mutations.[162,164,165] A total of 80% of the reported mutations are located within the kinase domain of BRAF.[159] Brose et al. identified activating BRAF mutations in five out of 292 cases (1.7%) of NSCLC. Among these mutations, three were found in exon 11 and two in exon 15.[164] It has been proposed that mutations in BRAF, a downstream signaling molecule of EGFR, predict clinical response to EGFR inhibitors, but this has yet to be validated in a larger number of cases.[157] Notably, a single substitution of glutamic acid for valine at codon 600 (V600E) accounts for approximately 90% of the BRAF missense mutations found in human tumors.[159] Considering BRAF is a serine/threonine kinase that is commonly activated by a somatic point mutation in human cancer, it may provide new therapeutic opportunities in a subset of NSCLC.

ALK Rearrangement ALK encodes a TK receptor found in a number of fusion proteins consisting of the intracellular kinase domain of ALK and the amino terminal portions of different genes.[166,167] A subset of NSCLC cases harbor a transforming fusion gene, EML4–ALK, within the genome. To date, seven gene fusion variants have been reported in NSCLCs. All involve the intracellular TK domain of ALK starting at a portion encoded by exon 20.[168] This fusion is formed as the result of a small inversion within the short arm of chromosome 2 that joins EML4 to ALK, inv(2)(p21;p23), which encodes an activated TK protein.[169,170] Several other transforming EML4–ALK fusion gene variants have also been identified, involving various EML4 exons and ALK.[171,172]ALK can also fuse with some other rare fusion partners, such as KIF5B and TFG.[173]

Activated ALK is involved in the inhibition of apoptosis and the promotion of cellular proliferation through activation of downstream PI3K–AKT and MAPK signaling pathways.[174] The EML4–ALK fusion is a rare abnormality detected in approximately 6% of patients with NSCLC.[144,169,175] Fusion of the EML4–ALK gene and its associated EML4–ALK product may lead to constitutive activation of the RAS–RAF–MEK–MAPK pathway.[47] In addition, two other less frequent ALK fusions in lung cancer have been reported.[176]

Non-small-cell lung cancer cases harboring EML4–ALK are characteristically found to have wild-type EGFR, as well as wild-type KRAS.[177,178] In addition, patients with these tumors tend to be younger, have an advanced clinical stage at presentation, have never smoked and their tumors exhibit solid histology, often with a component of signet ring cells.[178,179]

Patients with this alteration demonstrated an extraordinary response to the MET–ALK inhibitor, PF-02341066, in a Phase I/II trial.[144] Ten out of 19 patients (53%) experienced objective responses. A total of 15 out of 19 patients (79%) demonstrated disease control at 8 weeks, lasting as long as 40 weeks in some. Only four patients demonstrated disease progression. Kwak et al. screened 1500 NSCLC patients and identified 82 patients with advanced ALK-positive disease.[180] The vast majority (96%) of these cancers were adenocarcinomas. A total of 94% of these patients had received at least one prior therapy. All of these patients were treated with crizotinib, an oral small-molecule ALK TKI. The disease control rate was 90%, which included a 57% response rate plus 33% with stable disease. Furthermore, Rodig et al. evaluated the incidence and the characteristics of ALK-rearranged lung adenocarcinomas within the western population to elucidate the optimal diagnostic modality to detect ALK rearrangements in routine clinical practice.[179] In their study, 358 lung adenocarcinomas were tested for ALK rearrangement by FISH and immunohistochemistry. ALK rearrangement again demonstrated an association with younger age, never having smoked, advanced clinical stage, and a solid histology with signet ring cells in some cases. Of note, none of the ALK-rearranged tumors harbored coexisting EGFR mutations. The results of this study demonstrate that ALK rearrangements are uncommon in the western population, but represent a distinct clinical entity with unique attributes and the possibility of distinct treatment options. For suspected cases, dual diagnostic testing with FISH and immunohistochemistry should be considered in order to accurately identify lung adenocarcinomas with ALK rearrangement.[179] A study by Zhang et al. involving 266 Chinese patients with NSCLC revealed approximately similar results.[178]

Shaw and colleagues investigated 141 NSCLC cases and found that 19 (13%) contained EML4ALK fusion, 31 (22%) demonstrated EGFR mutantations and 91 (65%) were wild-type for both.[144] Several studies provide consistent evidence that EML4–ALK and EGFR mutations are mutually exclusive.[144,169,177,181] However, Tiseo et al. reported a 48-year-old Caucasian man who had never smoked and who was diagnosed with NSCLC with a concomitant EGFR mutation and ALK translocation that was resistant to erlotinib therapy.[182] This may be representative of the nature of this subgroup of NSCLC.[154]

No EGFR mutations in the EML4–ALK cohort and no instances of ALK rearrangement in the EGFR cohort have been found.[144] Among patients with metastatic disease, EML4–ALK rearrangements were associated with resistance to EGFR TKI treatment.

Compared with the EGFR-mutated and wild-type EGFR/ALK cohorts, patients with EML4–ALK fusion gene-positive tumors were significantly younger and more likely to be male.[144] However, another study demonstrated that males and females are equally affected.[139] Patients with EML4–ALK-positive tumors are more likely to be never/light smokers. A total of 18 of the 19 EML4–ALK-positive tumors were adenocarcinomas, predominantly of the signet ring cell subtype.[144] EML4–ALK defines a molecular subset of NSCLC with distinct clinical characteristics. Specifically, patients who harbor this mutation do not benefit from EGFR TKIs and should be directed to trials investigating ALK-targeted agents.


  • Two classes of anti-EGFR agents – monoclonal anti-EGFR antibodies and small-molecule EGFR TKIs – are currently used in EGFR-targeted therapy.

  • Additional mutations in EGFR are an important mechanism of acquired resistance to EGFR-targeted therapy.

  • Some mutations, such as exon 20 D770–N771 and T790M, are associated with EGFR TKI resistance.

  • Oncogenic activation of EGFR downstream effectors, such as KRAS, BRAF, PIK3CA and deletion of PTEN, is associated with resistance to TKI therapy.

  • KRAS mutation accounts for approximately 35–45% of TKI-nonresponsive NSCLC patients.

  • KRAS and BRAF mutations, as well as ALK rearrangements, are mutually exclusive with EGFR mutations.

  • ALK rearrangements are more common in younger patients who have never smoked, and their tumors often exhibit solid architecture, signet ring cell histology and wild-type EGFR and KRAS.


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