1 Modern Pathology 2012 Vol: 25(3):347-369. DOI: 10.1038/modpathol.2011.215

Molecular pathology of lung cancer: key to personalized medicine

The majority of lung adenocarcinoma patients with epidermal growth factor receptor- (EGFR) mutated or EML4–ALK rearrangement-positive tumors are sensitive to tyrosine kinase inhibitors. Both primary and acquired resistance in a significant number of those patients to these therapies remains a major clinical problem. The specific molecular mechanisms associated with tyrosine kinase inhibitor resistance are not fully understood. Clinicopathological observations suggest that molecular alterations involving so-called ‘driver mutations’ could be used as markers that aid in the selection of patients most likely to benefit from targeted therapies. In this review, we summarize recent developments involving the specific molecular mechanisms and markers that have been associated with primary and acquired resistance to EGFR-targeted therapy in lung adenocarcinomas. Understanding these mechanisms may provide new treatment avenues and improve current treatment algorithms.

Figure 1.
The EGFR-signaling pathway. EGFR is composed of an extracellular domain, a transmembrane lipophilic segment, and an intracellular region containing tyrosine kinase domains that occupy exons 18–24. The binding of ligands to EGFR results in autophosphorylation of key tyrosine residues in the tyrosine kinase domain and activates tyrosine kinase activity, which further activates the downstream PIK3CA/AKT1/MTOR and RAS/RAF1/MAP2K1 pathways. The aberrant signaling influences several key aspects including cell proliferation, apoptosis, migration, survival, and more complex processes such as angiogenesis. Figure 2.
Frequency of mutations in exons 18–21 of the EGFR gene and the association with responsiveness to EGFR targeted therapy. The EGFR located in chromosome 7p11.2 contains 28 exons. Exons 18–21 in the tyrosine kinase region of the EGFR gene are scaled up; a detailed list of EGFR mutations in these exons associated with sensitivity (green) or resistance (orange) to EGFR TKI.6, 12, 67, 68, 69, 70, 71, 80, 81, 82, 83, 84, 195 The frequency of the mutations is labeled to the side of the color-coded bars. The most prevalent EGFR mutations are in-frame deletions of exon 19 (45%), followed by L858R substitution in exon 21 (41%). Exon 18 mutations (G719A/C/S) account for ~5% of the overall mutations. The exon 19 deletions, L858R in exon 21, G719A/C/S in exon 18, the L861Q and L861R in exon 21, are mutations that predict the probability of benefit from EGFR TKI therapy of adenocarcinomas. The insertion mutations in exon 20 (D770_N771 (insNPG), D770_N771 (insSVQ), D770_N771 (insG)) are the second most common and are associated with EGFR TKI therapy resistance. D761Y in exon 19 is also associated with resistance to EGFR TKI although it occurs in low frequency. *T790M mutation represents ~1% of primary resistance but over 50% of acquired resistance in adenocarcinomas. **There are more than 20 exon 19 deletion forms in the lung adenocarcinomas, with the most common ones including delE746-A750, delL747-T751linsS, and delL747–P753insS. Figure 3.
Frequency of major driver mutations in signaling molecules in lung adenocarcinomas. About 64% of all adenocarcinoma cases harbor somatic driver mutations. According to the National Cancer Institute Lung Cancer Mutation Consortium data,79 ~23% of lung adenocarcinomas harbor EGFR mutations. The EGFR mutation status of the cancer is associated with its responsiveness or resistance to EGFR TKI therapy. KRAS mutations are more frequently found in adenocarcinomas (25%), which are mutually exclusive with EGFR mutations. Mutations in KRAS have been proposed as one of the mechanisms of primary resistance to gefitinib and erlotinib therapy. A subset of adenocarcinoma cases harbors a transforming fusion gene, EML4–ALK (6%), which mainly involves adenocarcinoma from non-smokers with wild-type EGFR and KRAS mutations. The mutation frequency of BRAF is 3%, PIK3CA 3%, MET amplifications 2%, ERBB2(Her2/neu) 1%, MAP2K1 0.4%, and NRAS 0.2%. Each of the molecular alterations has a role in the signal pathways, activating important cell functions, including cell proliferation and survival. Approximately 36.4% of lung adenocarcinomas do not harbor currently detectable mutations. Figure 4.
Current molecular tests and options for targeted therapies. Adenocarcinomas and adenosquamous carcinomas have a relatively high incidence of EGFR mutations or EML4–ALK rearrangement. Patients with such tumors could potentially benefit from targeted therapies using EGFR TKI and ALK TKI. EGFRvIII is associated with a small subset of squamous cell carcinomas, but the rationale for the therapy targeted to this mutation has not yet been established. Large cell and sarcomatoid carcinomas are not considered suitable tumors for the EGFR TKI therapy although a recent article reported 28% EGFR mutations from a group of 32 sarcomatoid lung cancers. Figure 5.
Schematic of EML4–ALK rearrangement, its detection by FISH, and its downstream effects. Both EML4 and ALK genes are located on the short arm of chromosome 2. The EML4–ALK rearrangement results from a chromosomal inversion, t(2;5) (a). Green and orange bars represent DNA probes corresponding to the 5′ and 3′ fragments of the ALK gene. The EML4–ALK fusion gene is mainly found in adenocarcinomas that arise in non-smokers with wild-type EGFR and KRAS. The EML4–ALK fusion protein activates canonical signaling pathways, including STAT3, RAS/MAP2K1, and PIK3CA/AKT1 cascades, which further affect cell cycle regulation, cell proliferation, neovascularization, and cell survival. At least nine variants have been identified. FISH detection of ELM4–ALK uses break-apart technology, which detects the adjacently located EML4 and ALK genes in wild-type signals (overlapping green-red) (b), and break-apart signals (separated green-red in one set of green-red) caused by chromosomal inversion (c). Figure 6.
Mechanism of constitutive activation of EGFR results from EGFR mutation and strategies of anti-EGFR therapy. (a) EGFR mutations provoke autophosphorylation of key tyrosine residues (P) in the tyrosine kinase domain, thus activating tyrosine kinase activity constitutively and initiating downstream effectors. (b) Two strategies are used for inhibiting EGFR signaling: humanized antibodies and small molecule TKIs. The antibodies inhibit the ligand-dependent activation of EGFR by blocking the ligand-binding site and preventing EGFR from activation. In contrast, TKIs block the magnesium-adenosine triphosphate-binding pocket of the intracellular tyrosine kinase domain, further inhibiting autophosphorylation. This inhibition disrupts tyrosine kinase activity and abrogates intracellular downstream signaling. Figure 7.
Suggested algorithm for molecular testing for patients with lung adenocarcinoma. The algorithm defines the rationale in selecting patients who could benefit from EGFR and EML4–ALKA targeted therapy. Adenocarcinoma cases are subjected to testing for EGFR mutations. The EGFR mutation-positive cases (25%) are further divided into responsive and resistant groups according to their mutation profiles. A responsive mutation predicts a response rate of 91% and a resistant mutation predicts a response rate of 9%. The presence of wild-type EGFR characterizes about 75% of the adenocarcinomas, and predicts the likelihood of non-responsive to EGFR TKI. Tumors with wild-type EGFR are further tested for EML4–ALK rearrangement. Although EML4–ALK rearrangement is found in only 3% of patients with lung adenocarcinoma, its presence predicts a 53% probability of response to targeted therapy. Figure 8.
Alternative algorithm for molecular testing for patients with lung adenocarcinomas. Approximately 25% of lung adenocarcinomas harbor KRAS mutations, which predict non-response to EGFR TKI therapy. Of the remaining KRAS-negative lung adenocarcinomas, ~20% harbor EGFR mutations, which are associated with responsiveness to EGFR TKI therapy. EGFR mutation-negative cases may benefit from additional testing for the EML4–ALK rearrangement, which will be helpful in selecting patients potentially eligible for ALK targeted therapy.
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