Targeted therapy in non-small cell lung cancer

EGFR

The ErbB family of receptors includes the four members: ErbB-1 (EGFR), ErbB-2 (HER-2) which is relevant especially in breast cancer treatment, ErbB-3 and ErbB-4. The extracellular component is responsible for ligand binding; whereas the intracellular component consists of the tyrosine kinase, which is responsible for signal transduction.

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Figure 1

Epidermal growth factor receptor (EGFR), the intracellular tyrosine kinase (TKI) and the following intracellular pathways.

After a ligand (e.g. epidermal growth factor, transforming growth factor-α) binds to the extracellular receptor, a dimerisation results in the activation of the receptor. The signal is passed along various downstream pathways. The most relevant seem to be the RAS/RAF/MEK/MAPK pathway, the PI3K/AKT pathway and the STAT3/STAT5 pathway [8]. These pathways lead to changes in gene transcription and alterations in the cell cycle, resulting in increased cell proliferation and angiogenesis, inhibition of apoptosis and changes in their capacity for migration, adhesion and invasion. As one would expect, changes in these parameters and properties have an impact on the behaviour of the cancer.

There are various mutations that have been discovered in the EGFR gene. Some of these mutations lead to an increased activity of the tyrosine kinase and subsequent sensitivity to targeted therapy whereas others increase cell resistance to treatment with tyrosine kinase inhibitors (TKIs).

VEGF receptor

After ligand binding, proliferation and migration of endothelial cells are activated, leading to increased blood supply of tumour cells and contributing to cancer cell survival [9].

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Figure 2

Vascular endothelial growth factor (VEGF) can bind to the appropriate receptor leading to vascular proliferation.

EML4-ALK

⇓]This oncogene is created by an inversion on chromosome 2p. In this mutation the N-terminal end of EML4 fuses with the intracellular domain of ALK. Breaking points in EML4 vary, whereas they seem to be constant in ALK, several fusion variants have been discovered. The fusion leads to a constitutive activation of a chimeric tyrosine kinase leading to increased cell proliferation [10, 11].

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Figure 3

The fusion of the EML4 and ALK genes leads to a fusion oncogene.

PCR and DNA sequencing

Mutations within a gene may lead to a change in the functioning of the resulting protein. An identified gene is amplified (multiplied) by PCR before it can be sequenced. With DNA sequencing specific variants (mutations) of genes may be identified. Depending on the gene and the mutation these may lead to reduced or even increased function.

Sequencing nucleotide by nucleotide has been made possible by new technologies. With increased capacities and capabilities, sequencing has become available outside research settings.

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Figure 5

With the help of DNA sequencing, variations can be identified.

With gene sequencing, various changes in the DNA sequence can be identified; these include insertions, deletions or point mutations. The changes in sequence lead to changes in transcription to amino acids, thereby changing the form of a protein.

The advantage of gene sequencing is that specific mutations can be identified. Currently, many EGFR mutations have been identified. Many of those have shown to be associated with good responses to EGFR inhibiting drugs (e.g. deletion in exon 19 or point mutation in exon 21 L858R), some however have shown to be associated with resistance (e.g. exon 20 T790M).

Kirsten rat sarcoma viral oncogene homologue (KRAS); activating KRAS mutation have demonstrated to be associated with resistance to EGFR inhibitors. KRAS and EGFR mutations are considered to be mutually exclusive in lung cancer patients [12]. In practice, however, both tests are usually performed for control.

FISH

FISH is a technique that allows the visualisation of specific chromosome nucleic acid sequences (genes) within a cellular preparation. Specifically, FISH involves the precise annealing of a single-stranded, fluorophore-labelled DNA probe to complementary target sequences. After preparation, the DNA of the tissue on slides is denatured to single-strands and hybridised with DNA probes. Following hybridisation, the unbound probe is removed by a series of washes. The flurophore-labelled probes, now bound to the cellular DNA, can be visualised in fluorescence microscopy.

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Figure 6

A complementary DNA is labelled with a fluorescent dye and allowed to bind to a specific sequence on the DNA.

To detect EML4-ALK fusion genes, fluorescence in situ hybridisation can be used. When hybridised with the appropriate labelled FISH probes, the 2p23 ALK region in its native state will be seen as two immediately adjacent or fused (overlapping) orange/green (yellow) signals. However, if a chromosome rearrangement at the 2p23 ALK breakpoint region has occurred (inversion), one red and one green signal separated by at least two signal diameters will be seen.

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Figure 7

The fluorescence in situ hybridisation image shows ALK gene (red represents the telomer side of the ALK gene, whereas green represents the centromere side). The white arrow shows the genes in the same location (wild-type), the blue arrows show separated signals after mutation (inversion) of the gene.

IHC

With specific dyes, antibodies to proteins can be labelled and can then be visualised under the microscope.

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Figure 8

Immunohistochemistry; with the help of labelled antibodies (Ab) the expression of receptors and proteins can be visualised.

Immunohistochemistry refers to the process of detecting proteins by binding labelled antibodies to these. Visualising an antibody–antigen interaction can be accomplished in a number of ways. In the most common instance, an antibody is conjugated to an enzyme, such as peroxidase, that can catalyse a colour-producing reaction.

IHC methods have been successfully used to evaluate the expression (over-expression) of the EGFR, which is of clinical relevance for EGFR antibody therapy.

EGFR inhibition

With the identification of receptors and pathways playing important roles in cancer, drugs have been developed to influence them. In the case of the EGFR, drugs have been developed to inhibit the transfer of the activating signal to intracellular pathways. These include agents which can block the receptor from outside the cell (antibodies) or drugs acting intracellularly (tyrosine kinase inhibitors (TKIs)).

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Figure 9

Antibodies and tyrosine kinase inhibitors (TKI) can inhibit signal transduction from the extracellular environment to intracellular pathways. Ab: antibody; EGFR: epidermal growth factor receptor.

VEGF inhibition

Antibodies specific for the VEGF Receptor can inhibit the downstream signalling leading to inhibition of angio- and vasculogenesis. The principle is analogous to the antibodies in EGFR inhibition. In addition, numerous small molecule tyrosine kinase inhibitors, including sunitib, sorafenib, vandetanib and others, have been explored.

EML4-ALK inhibition

The EML4-ALK fusion oncogene is responsible for cell proliferation. With the help of a targeted agent against the ALK intracellular tyrosine kinase, the function of the tyrosine kinase can be inhibited, thus blocking further downstream signalling. This is analogous to the functioning of the TKIs in the aforementioned EGFR example.

Resistance

Although the principles of treatment seem very clear and effective, the clinical activity is limited by the development of resistance to these agents. For EGFR tyrosine kinase inhibitors (erlotinib, gefitinib) secondary mutations or bypass activation of alternate pathways, which may lead to a downstream signalling, lead to resistance. These mechanisms have been specifically described and represent a significant proportion of resistance development [13, 14]. With other targeted treatments, similar mechanisms have been described, e.g. for crizotinib and the EML4-ALK fusion oncogene [10].

EGFR TKIs

Patients whose tumours harbour an activating EGFR mutation (e.g. deletion in exon 19 or point mutation in exon 21 (L858R)) derive significant benefit from the treatment with small molecule TKIs. The first trial to demonstrate, that the first-line use of a TKI is superior to chemotherapy in these patients was a subgroup analysis of the IPASS study which enrolled >1,200 Asian patients and compared carboplatin/paclitaxel with gefitinib in patients with adenocarcinoma who were light or never smokers [15]. Since this report, several prospective randomised phase-III studies in patients with EGFR mutations confirmed these findings, demonstrating a progression free survival of about 1 year and survival rates of up to 28 months [16–18]. Based on these results, EGFR TKIs (e.g. erlotinib, gefitinib) today represent the preferred initial systemic treatment option for patients with an activating EGFR mutation.

EGFR antibodies

Cetuximab is an inhibitory anti-EGFR antibody which interacts with domain III of the soluble extracellular region of EGFR, sterically preventing the receptor from adopting the extended conformation required for dimerisation and ultimately intracellular pathway activation. In the phase-III FLEX trial, cetuximab improved response rate and overall survival when added to cisplatin/vinorelbine in patients with advanced NSCLC expressing EGFR by immunohistochemistry. Response rates were statistically significantly improved from 29 to 36%; however, the median survival was only improved from 10.1 to 11.3 months [19]. As the survival benefit was relatively small, approval was denied by health authorities. Reports have shown that a high IHC-EGFR score (i.e. >200) can be used to identify about a third of patients in whom the addition of cetuximab to chemotherapy was associated with an increased benefit [19].

Anti-angiogenic treatment

Bevacizumab is a recombinant humanised monoclonal antibody that binds to and neutralises the biological activity of human VEGF [20]. This agent was the first targeted treatment showing a survival advantage in lung cancer [21]. Interestingly, the two randomised phase-III trials with bevacizumab did not show the same results. Although both trials demonstrated improved response rates and progression free survival with the addition of bevacizumab to a combination chemotherapy regimen with carboplatin/paclitaxel and cisplatin/gemcitabine, respectively, prolongation of overall survival (OS) has only been demonstrated for the carboplatin/paclitaxel/bevacizumab combination (OS: 12.3 versus 10.3 months; HR 0.80; p = 0.013) [21, 22]. The recently presented AVAPEARL study [23], which was investigating the role of maintenance pemetrexed and bevacizumab after an induction cisplatin/pemetrexed/bevacizumab phase, confirmed the activity of bevacizumab, also in combination with newer chemotherapy agents and demonstrated very promising preliminary survival times. As a result of these trials, bevacizumab combined with a platinum-based chemotherapy is a treatment option in patients with performance status 0–1 and non-squamous cell histology.

EML4-ALK gene rearrangement

The most common rearrangement of the ALK gene arises from an inversion in the short arm of chromosome 2 that creates a fusion between the 5′ portion of the EML4 gene and the 3′ portion of ALK. The incidence of the ALK fusion gene, which possesses potent oncogenic activity, is estimated to be 4% in NSCLC. Patients with ALK gene rearrangements have been reported to exhibit marked sensitivity to crizotinib, a small molecule inhibitor of ALK [24]. A recent retrospective review focusing on the impact of ALK rearrangement on OS in patients treated with crizotinib suggested improved survival compared with that of crizotinib-naïve controls [11]. An update of the pivotal phase-I trial with this agent reported on 119 mostly heavily pretreated patients. The median progression-free survival and the one- and two-year survival were 10 months, 70% and 55%, respectively, which by far exceeds anything previously observed [25]. Results from randomised crizotinib trials in pretreated or chemotherapy naïve are eagerly awaited.

Other mutations

In a recently published article the results of mutation testing in >1,000 patients with adenocarcimoma of the lung in North America were presented. The incidence of rarer mutations is 2% for BRAF and <2% for AKT1, HER2, MEK, NRAS and PIK3CA [26]. In addition, gene amplification of MET was also detected in <2%. The majority of these genetic alterations are considered to be oncogenic drivers and clinical investigations with corresponding experimental drugs are warranted and ongoing. KRAS mutations are present in approximately one out of five adenocarcinomas and drugs targeting several downstream signalling pathways of RAS such as PIK3ACA, ERK, MEK and mTOR or their combinations are currently being explored. While recent research has primarily focused on genetic subtypes of adenocarcinomas, potential targets for squamous cell carcinoma such as focal fibroblast growth factor receptor 1 (FGFR1) amplification (22%), DDR2 mutations (4%) and other mutations have been identified and results of early trials are eagerly awaited [27, 28].