Introduction
Lung cancer is the most commonly diagnosed cancer in Canada, and it accounts for about 14% of all newly diagnosed cancers. 1 It is the leading cause of cancer-related mortality in both men and women worldwide, Reference Zamay, Zamay and Kolovskaya2 primarily due to the lack of early detection or outward signs and symptoms, thereby often progressing to advanced stages (e.g., stage IV) before it is diagnosed. The two main types of lung cancers are non-small-cell lung cancer (NSCLC) and small-cell lung cancer (SCLC), each with different histological characteristics, invasiveness, response to treatment options and risk factors. Reference Polanski, Chabowski, Jankowska-Polanska, Janczak and Rosinczuk3 The SCLC accounts for about 15–20%, whereas the NSCLC accounts for about 80–85% of all diagnosed lung cancers Reference Polanski, Chabowski, Jankowska-Polanska, Janczak and Rosinczuk3,Reference Li, Asmitananda and Gao4 and is further subtyped histologically into adenocarcinoma (40%), squamous cell carcinoma (25%) and large-cell carcinoma (15%). 1,Reference Polanski, Chabowski, Jankowska-Polanska, Janczak and Rosinczuk3 If lung cancers can be diagnosed in the early stages and moreover if clinicians can prospectively identify patients likely to respond to specific treatments, there would be a very high potential to increase patients’ survival. The American National Lung Screen Trial reported a 20% reduction in lung cancer mortality due to screening (for early detection) via low-dose computed tomography (CT) scans, though there was concern regarding the risk of radiation exposure from the low-dose CT scans. Reference Ostrowski, Marjanski and Rzyman5 And in recent years, several investigations have been conducted to identify cancer biomarkers for lung cancer risk assessment, early detection and diagnosis, the likelihood of identifying the group of patients who will benefit from a particular treatment and monitoring patient response to treatment.
Biomarkers are measurable compounds that can be indicative or predictive of the presence or pathology of some state of the body or abnormality such as disease or infection, Reference Greenberg and Lee6 and these objectively measured quantities are evaluated as an indicator of normal biological processes, pathogenic processes or pharmacological responses to a therapeutic intervention. 7 A biomarker can be used in screening for early detection (i.e., diagnostic biomarkers) or to monitor patient response to specific treatment options (i.e., predictive biomarkers) or informs about a likely cancer outcome (e.g., disease recurrence, disease progression, mortality) independent of the treatment received (i.e., prognostic biomarkers). Reference Goossens, Nakagawa, Sun and Hoshida8–Reference Ballman10 The concept of precision medicine from prevention to treatment techniques that take into account individual patient variability will depend on the development of effective clinical biomarkers that interrogate the key aberrant pathways potentially targetable with molecular targeted or immunologic therapies. Reference Goossens, Nakagawa, Sun and Hoshida8 Therefore, the goal of this paper is to review current clinical and emerging biomarkers used in risk assessment, screening for early detection and diagnosis, and for monitoring the treatment response of NSCLC.
Epidermal Growth Factor Receptor (EGFR)
The EGFR gene is located on the short arm of chromosome 7 at position 12 (7p12) and encodes the EGFR protein, which is a tyrosine kinase transmembrane receptor belonging to the human EGFR (HER/ERBB) family of proteins Reference Villalobos and Wistuba11–Reference Hodoglugil, Carrillo and Hebert13 and may become unregulated by oncogenic mechanisms such as somatic gene mutation, increased gene copy number and overexpression of EGFR. Reference da Cunha Santos, Shepherd and Tsao14 When the epidermal growth factor or transforming growth factor-alpha binds to the extracellular domain, it results in a dimerisation of proteins that trigger a series of intracellular phosphorylation pathway cascades and leads to the activation of internal domain tyrosine kinase. Reference da Cunha Santos, Shepherd and Tsao14,Reference Yarden15 This activation leads to downstream signalling in the rat sarcoma (RAS)-Rapidly accelerated fibrosarcoma (RAF)–mitogen-activated protein kinase (MAPK) pathway or phosphoinositide 3-kinase (PI3K) and promotes cell proliferation, protein synthesis and inhibition of p53 Reference Oda, Matsuoka, Funahashi and Kitano16 ; moreover, mutations in the EGFR gene or overexpression of EGFR causes continuous and uncontrolled cell proliferation and inhibition of apoptosis. Reference Khalil and Altiok12 EGFR is reported to be overexpressed in 40–80% of NSCLC cases and its mutations in NSCLC are commonly found in people with East Asian ethnicity, adenocarcinoma histological subtype, females and nonsmokers. Reference Villalobos and Wistuba11,Reference Syrigos, Georgoulias, Zarogoulidis, Makrantonakis, Charpidou and Christodoulou17 Studies Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18,Reference Martínez-Carretero, Pascual, Rus and Bernardo19 have reported that most EGFR mutations occur in exons 18–22 of the tyrosine kinase domains, with the most common mutation being exon 19 deletions and a point mutation in exon 21 (L858R). Furthermore, there are other rare EGFR mutations including substitutions such as glycine 719 with serine, cysteine or alanine in exon 18, which confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs), or mutations associated with resistance to first-generation TKIs such as the T790M mutation in exon 20 or insertions in exon 20. Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18
Patients with exon 19 or 21 deletions or L858R point mutations are reported to be more sensitive to TKIs such as erlotinib and gefitinib, Reference Mok, Wu and Thongprasert20–Reference Douillard, Ostoros and Cobo22 and several studies Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18,Reference Mok, Wu and Thongprasert20–Reference Douillard, Ostoros and Cobo22 have reported that targeted TKIs such as erlotinib, gefitinib, osimertinib and afatinib inhibit EGFR mutations. These inhibitors work by binding to the adenosine triphosphate site on the EGFR tyrosine kinase domain, thereby selectively blocking phosphorylation of the intracellular tyrosine kinase domain of EGFR. Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18 A Pan-Asia Phase III randomised study compared gefitinib and carboplatin-paclitaxel chemotherapy in 1,217 patients with stage IIIB or IV NSCLC and reported that the 12-month progression-free survival for patients using gefitinib as a first-line treatment was 24·9% compared to 6·7% for patients using standard carboplatin-paclitaxel chemotherapy. Reference Mok, Wu and Thongprasert20,Reference Wu, Chu and Han23,Reference Fukuoka, Wu and Thongprasert24 Furthermore, a subgroup of EGFR mutation-positive patients showed a longer progression-free survival with gefitinib compared to mutation-positive patients using carboplatin-paclitaxel chemotherapy, and they concluded that EGFR mutations are strong predictive biomarker for progression-free survival and tumour response to first-line gefitinib versus carboplatin–paclitaxel. Reference Mok, Wu and Thongprasert20,Reference Fukuoka, Wu and Thongprasert24 A similar study by Maemondo et al. Reference Maemondo, Inoue and Kobayashi25 also showed significantly longer progression-free survival for EGFR mutation-positive patients undergoing gefitinib treatment compared to those undergoing carboplatin–paclitaxel chemotherapy, however, progression-free survival of patients undergoing gefitinib treatment did not differ significantly between EGFR exons 19 deletions and L858R point mutation. Reference Maemondo, Inoue and Kobayashi25 Ahmadzada et al. Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18 reported that patients treated with EGFR-TKIs can develop drug resistance over time through a variety of mechanisms such as secondary acquired mutations that render the TKIs ineffective and, according to Demuth et al., Reference Demuth, Madsen, Weber, Wu, Meldgaard and Sorensen26 will eventually occur in all patients undergoing TKI treatment of NSCLC. The most common resistance mutation of first-generation TKIs is the T790M mutation in exon 20, whereby threonine is replaced by methionine at position 790 in the tyrosine kinase domain of EGFR and reduces the effectiveness of early generation EGFR-TKIs. Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18 According to Ahmadzada et al., Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18 osimertinib is currently used for advanced NSCLC patients with T790M mutation, with disease progression on or after EGFR-TKI therapy and is recommended as a second-line therapy for patients with metastatic EGFR T790M-positive NSCLC Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18 and recommended biomarker testing for EGFR T790M mutation for EGFR mutant patients that have progressed following EGFR-TKI treatment. Reference Ahmadzada, Kao, Reid, Boyer, Mahar and Cooper18
Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA)
The PIK3CA gene is located on the long arm of chromosome 3 at position 26·3 (3q26·3) and encodes the p110 α catalytic subunit of the mitogenic-signalling protein PI3K. Reference Villalobos and Wistuba11 The PI3Ks are heterodimeric lipid kinases composed of catalytic and regulatory subunits and are involved in cell signalling, proliferation, cell growth, transformation, adhesion, apoptosis, survival and motility. Reference Villalobos and Wistuba11,Reference Jean and Kiger27,Reference Chaft, Arcila and Paik28 Mutations at exon 9 and exon 20 in PIK3CA results in constitutive activation of PI3K and increased kinase activity Reference Scheffler, Bos and Gardizi29 and have been associated with tumorigenesis and treatment resistance in various malignancies. Reference Chaft, Arcila and Paik28 PIK3CA amplifications, deletions and somatic missense mutations have been reported in lung cancers, and the mutations have been found in about 2–4% of NSCLC patients, usually affecting exons 9 and 20, Reference Villalobos and Wistuba11 with higher incidences being found in squamous cell, adenocarcinoma histology and nonsmokers. Reference Chaft, Arcila and Paik28,Reference Scheffler, Bos and Gardizi29
A study by Chaft et al. Reference Chaft, Arcila and Paik28 analysed tissue samples from 1,125 patients with lung adenocarcinoma and reported that PIK3CA mutations occur in approximately 2% of lung adenocarcinoma patients and that most tumours with PIK3CA mutations coexisted with other driver mutations involved in regulatory pathways of cells such as mutations in kirsten rat sarcoma viral oncogene (KRAS), anaplastic lymphoma kinase (ALK) and EGFR genes. Reference Chaft, Arcila and Paik28 Scheffler et al. Reference Scheffler, Bos and Gardizi29 investigated tumour samples of 1,144 NSCLC patients for PIK3CA mutations and compared the clinical, pathological and genetic characteristics of PIK3CA-mutated patients with a control group of PIK3CA-wild-type patients. They identified about 3·7% patients with PIK3CA mutations in exon 9 and exon 20, with a higher frequency in squamous cell carcinoma (8·9%) compared to adenocarcinoma (2·9%). Spoerke et al. Reference Spoerke, O’Brien and Huw30 investigated the effect of PI3K pathway inhibitors on a collection of NSCLC tumour samples and observed PIK3CA amplification in 37% of squamous tumours and 5% of adenocarcinomas, whereas PIK3CA mutations were found in 9% of squamous and none in adenocarcinomas. Furthermore, they observed that the cell lines harbouring PI3K pathway alterations [such as receptor tyrosine kinase (RTK) activation, PI3K mutation or amplification] were sensitive to pictilisib (a PI3K inhibitor) across the cell line panel and in tumour xenografts. They further indicated that a combination of pictilisib with paclitaxel or erlotinib had greater effects on cell viability than PI3K inhibition alone and demonstrated the prognostic and predictive value of PI3K inhibitors in preclinical models. Chaft et al. Reference Chaft, Arcila and Paik28 also reported that cell lines harbouring PIK3CA mutations are sensitive to downstream inhibitors such as everolimus [an inhibitor of mammalian target of rapamycin (mTOR)], though this sensitivity can be annulled by coincident KRAS mutations. Massacesi et al. Reference Massacesi, Di Tomaso and Urban31 reported that early clinical studies with PI3K inhibitors such as buparlisib and alpelisib demonstrated antitumour activities and acceptable safety profiles in preclinical models of solid tumours and that currently several PI3K inhibitors such as pictilisib, copanlisib, taselisib and alpelisib are under clinical investigation. Reference Massacesi, Di Tomaso and Urban31,Reference Vansteenkiste, Canon and De Braud32 Vansteenkiste et al. Reference Vansteenkiste, Canon and De Braud32 conducted an open-label, two-stage phase II study BASALT-1 to investigate the safety and efficacy of buparlisib in patients with relapsed NSCLC harbouring documented aberrations in the PI3K pathway. They reported that although preclinical evidence supported the rationale for PI3K inhibition in advanced NSCLC, other pathways may play an oncogenic role; however, given the PI3K pathway activation involvement in treatment resistance, a combination of PI3K inhibitors with chemotherapy, immunotherapy or other targeted agents may improve efficacy compared to monotherapy. According to some studies, Reference Chaft, Arcila and Paik28,Reference Vansteenkiste, Canon and De Braud32 however, a number of questions that still remained unanswered regarding PI3K inhibition and its prognostic and predictive potential in cancer treatment and thus warrants further investigation.
ALK
The ALK gene is located on the short arm of chromosome 2 at position 23 (2p23) and encodes a ALK receptor tyrosine kinase (RTK) protein, which is a member of the insulin receptor superfamily. Reference Villalobos and Wistuba11,Reference Chiarle, Voena, Ambrogio, Piva and Inghirami33 In NSCLC, ALK fuses with echinoderm microtubule-associated protein-like 4 (EML4) genes that encodes for a cytoplasmic chimeric protein with constitutive kinase activity, which promotes malignant growth and proliferation. Reference Villalobos and Wistuba11,Reference Kwak, Bang and Camidge34 The EML4 replaces the extracellular and intramembranous parts of ALK to create a new molecular target. Reference Du, Shao, Qin, Tai and Gao35 According to Du et al., Reference Du, Shao, Qin, Tai and Gao35 ALK rearrangement creates an ALK tyrosine kinase that activates downstream-signalling pathways, leading to increased cell proliferation and survival. The EML4-ALK fusion has been reported to occur in 3·7–7% of NSCLC cases, are more common in younger patients who have never smoked, usually in adenocarcinomas with signet-ring cells or cribriform histology features, and can form secondary mutations such as L1196M, C1156Y and F1174L. Reference Villalobos and Wistuba11
The prognostic or predictive value of EML4-ALK fusion for patients with metastatic NSCLC has been investigated by several authors. Reference Villalobos and Wistuba11,Reference Zhang, Shiratsuchi, Palanisamy, Nagrath and Ramnath36,Reference McCoach, Blakely and Banks37 According to Villalobos and Wistuba, Reference Villalobos and Wistuba11 patients with ALK mutations often respond well to ALK inhibitors such as crizotinib; and as a result, testing for ALK rearrangements in patients with advanced lung adenocarcinoma was recommended in current clinical practice guidelines. Zhang et al. Reference Zhang, Shiratsuchi, Palanisamy, Nagrath and Ramnath36 have reported that treating patients harbouring EML4-ALK gene rearrangement with crizotinib have resulted in rapid (within 6 weeks), greater response rates (65%) and longer progression-free survival (7·7 months) compared with standard chemotherapy. They also indicated that most patients with ALK-positive lung cancer who respond to crizotinib are to have a relapse due to the development of resistance mechanisms such as mutations in the ALK gene (L1196M). Villalobos and Wistuba Reference Villalobos and Wistuba11 have also reported that although the initial response of patients to ALK inhibitor treatment was very favourable, some patients developed resistance to the inhibitor due to secondary mutations within the kinase domain of EML4-ALK Reference Villalobos and Wistuba11 ; however a second-line inhibitor, ceritinib, has been reported to overcome the crizotinib-resistant mutation. Therefore, several second-generation ALK inhibitors that target ALK-positive NSCLC, such as alectinib, ceritinib and brigatinib, have been developed and are currently under evaluation in clinical trials. Reference Villalobos and Wistuba11 Furthermore, third-generation ALK inhibitors such as lorlatinib in clinical trials target wild-type EML4-ALK as well as resistant mutations. It has been demonstrated in a case study of an advanced stage ALK-positive NSCLC lung adenocarcinoma patient harbouring EML4-ALK gene rearrangement in the tumour that it is feasible to detect serial genetic alterations (ALK rearrangement and secondary resistance mutation L1196M) in expanded circulating tumour cells from a patient’s blood. Reference Zhang, Shiratsuchi, Palanisamy, Nagrath and Ramnath36 McCoach et al. Reference McCoach, Blakely and Banks37 also demonstrated a noninvasive approach to detect ALK fusions and actionable resistance mechanisms by analysing cell-free circulating tumour DNA without an invasive biopsy, which was observed to be more specific and sensitive towards molecular mutation. The current standard to diagnose a patient with ALK mutations in most studies is through tumour samples analysed by fluorescence in situ hybridisation (FISH) using an ALK break-apart probe, immunohistochemistry (IHC), and reverse-transcription polymerase chain reaction (RT-PCR).
Mitogen-Activated Extracellular Signal-Regulated Kinase Kinases (MEK1/2)
The MEK is a family of seven (i.e., MEK1–MEK7) human MAPK kinases (MAP2K), with dual specificity for tyrosine and serine–threonine residues. Reference Arcila, Drilon and Sylvester38 The MEK1 protein kinase is encoded by the MAP2K1 gene located on the long arm of chromosome 15 at position 22·31 (15q22·31), whereas the MEK2 protein kinase is encoded by the MAP2K2 gene located on the short arm of chromosome 19 at position 13·3 (19p13·3). Reference Marks, Gong and Chitale39 It is reported that MEK1 and MEK2 are the only known activators of the MAPK pathway that comprises the RAS-RAF-MEK-extracellular signal-regulated kinase (ERK)-signalling pathway and is essential for normal cellular functions including growth, proliferation, differentiation, survival, apoptosis, motility, transcription, metabolism and migration of cells. Reference Marks, Gong and Chitale39,Reference Kim and Giaccone40 The MAPK pathway is implicated in the tumorigenesis of a broad array of cancers including NSCLC, and the majority of patients with MEK1 mutations are usually current or former smokers. Reference Kim and Giaccone40 Mutations in MEK1 are uncommon, making up less than 1% of the NSCLC cases, reported to be recurrent, found in lung adenocarcinoma patients and define a distinct subset of lung cancer that is strongly associated with smoking but with potential sensitivity to MEK inhibitors. Reference Arcila, Drilon and Sylvester38,Reference Kim and Giaccone40
Studies Reference Mas, Boda and Caulfuty41–Reference Troiani, Vecchione and Martinelli44 have assessed the preclinical evidence of the potential role of MEK inhibitors (such as selumetinib, erlotinib, trametinib, dabrafenib, cobimetinib, binimetinib, pimasertib, refametinib) in the management of NSCLC, especially those harbouring KRAS mutations or as an add-on therapy (EGFR-MEK inhibition) for overcoming cells resistant to EGFR-TKIs. Mas et al. Reference Mas, Boda and Caulfuty41 used a human in vitro three-dimensional lung adenocarcinoma model (OncoCilAir™) harbouring KRAS mutation to assess the antitumour effectiveness of selumetinib and trametinib MEK inhibitors. They reported a reduced tumour growth in response to the MEK inhibitors; however, halting the selumetinib dosing resulted in tumour relapse. Chen et al. Reference Chen, Cheng and Walton42 used genetically engineered mouse model to conduct an animal trial that mimic a human clinical trial in patients with KRAS-mutant lung cancers to determine whether the MEK inhibitor selumetinib increases the efficacy of docetaxel. They observed that the addition of selumetinib provided substantial benefit for mice with lung cancer caused by KRAS. Huang et al. Reference Huang, Lee and Chang43 used PC-9 cells harbouring EGFR exon 19 deletion to investigate effective strategies against cells resistant to EGFR-TKIs. They reported that persistent activation of the ERK (also known as MAPK) pathway contributes to the acquired gefitinib resistance and that combined treatment of gefitinib and MEK inhibitors may be therapeutically useful for acquired gefitinib-resistant lung adenocarcinoma cells harbouring EGFR mutations. Although the MEK inhibitors have potential to manage NSCLC, Troiani et al. Reference Troiani, Vecchione and Martinelli44 reported the development of resistance to MEK inhibitor therapy; however, they further indicated that several mechanisms have been used to describe the development of resistance and that potential techniques are being developed to overcome it.
Mesenchymal–Epithelial Transition (MET) Proto-Oncogene
The MET gene, which was discovered in 1984 is located on the long arm of chromosome 7 at position 31 (7q31) and it is identified as an oncogene. Reference Villalobos and Wistuba11,Reference Furlan, Kherrouche, Montagne, Copin and Tulasne45 This oncogene can be found on the surface of epithelial cells, fibroblasts, endothelial cells, pericytes and smooth muscle cells Reference Schmitz, Koeppen and Binot46 and encodes for the hepatocyte growth factor (HGF) receptor, which is a RTK and is a ligand with high affinity for MET-RTK. The HGF receptor activates multiple-signalling pathways that play fundamental roles in cell proliferation, survival, motility, embryogenesis, tissue reparation and invasion. Reference Villalobos and Wistuba11,Reference Zhang, Du and Zhang47 Aberrant activation of MET include gene amplification, mutation and protein overexpression Reference Villalobos and Wistuba11,Reference Zhang and Babic48 and can result in tumour formation or metastasis. MET activation can occur in different ways, including paracrine or autocrine activation by HGF, overexpression of the receptor through gene activation mutations or gene amplifications, Reference Schmitz, Koeppen and Binot46 and the overexpression of HGF is reported to be the most common cause of MET overactivation in lung cancer. Reference Furlan, Kherrouche, Montagne, Copin and Tulasne45 MET overexpression occurs in about 2·4–22% of NSCLC patients depending on the method used for the detection, and it is reported that about 3·9% are found using FISH, whereas about 18% are found using quantitative PCR (qPCR). Reference Zhang, Du and Zhang47 MET mutations are reported to occur in about 3% of squamous cell lung cancers and 8% of lung adenocarcinomas, whereas MET amplifications are reported in 4% of lung adenocarcinomas and 1% of squamous cell lung cancers. Reference Villalobos and Wistuba11
According to Salgia, Reference Salgia49 MET mutation or deletion is currently being used as a predictive biomarker to guide patient selection for MET-targeted therapies and to predict patient response but can also be used as a prognostic biomarker. Salgia Reference Salgia49 reported that MET-targeted agents in clinical development include monoclonal antibodies such as emibetuzumab, ficlatuzumab, and rilotuzumab and TKIs such as crizotinib, tepotinib, cabozantinib and capmatinib. Although MET mutations rarely occur in lung cancer, its presence is often correlated with increased tumour development and growth, often indicating an adenocarcinoma or a pleiotropic tumour, Reference Smolen, Sordella and Muir50 and increased MET copy number or gene amplification has been reported to correlate with poor patient survival rates. Reference Zhang, Du and Zhang47 Various techniques including IHC, RT-PCR, Western blots and enzyme-linked immunosorbent assays have been used to measure varying levels of MET and HGF amplification in different types of tumours, Reference Zhang, Du and Zhang47 and higher serum or plasma HGF or soluble MET (sMET) levels is reported to be associated with disease progression, development of tumour resistance to treatment, metastasis and poorer survival. Reference Matsumoto, Umitsu, De Silva, Roy and Bottaro51
KIT Proto-Oncogene RTK
The KIT gene is located on the long arm of chromosome 4 at position 12 (4q12) and is identified as an oncogene. The KIT proto-oncogene (CD117) encodes a transmembrane protein belonging to the RTK type III, which transmit signals from cell surfaces into the cells through transduction and binds its stem cell factor (SCF) ligand. Reference Roudi, Kalantar, Keshtkar and Madjd52,Reference Patel, Ersek and Kim53 It is involved in intracellular signalling and plays an important role in cell proliferation, differentiation, apoptosis and survival. Reference Roudi, Kalantar, Keshtkar and Madjd52 KIT expression allows for the development of germ cells, melanocytes, mast cells and erythrocytes and eventually result in autophosphorylation, dimerisation and signal transduction. Reference Patel, Ersek and Kim53 However, mutations in KIT that cause autophosphorylation without the presence of the ligand lead to uncontrolled cell proliferation, which eventually induces tumour development. Reference Xu, Wang and Zhang54 Donnenberg et al. Reference Donnenberg, Zimmerlin, Landreneau, Luketich and Donnenberg55 reported that KIT mutations provide mechanisms for upregulated cellular proliferation and resistance to apoptosis and may also render tumours entirely dependent on KIT receptor-signalling activity and hence susceptible to its inhibition. According to Donnenberg et al., Reference Donnenberg, Zimmerlin, Landreneau, Luketich and Donnenberg55 cells that overexpress KIT are present in NSCLC tumours and account for 22% of all NSCLC tumour types. Perumal et al. Reference Perumal, Pillai, Nguyen, Schaal, Coppola and Chellappan56 reported that SCF (which is the ligand for KIT receptor) is elevated in adenocarcinomas and metastatic carcinomas of the lung, but not in squamous cell carcinomas or normal lung tissue, and this elevated level is correlated with a poor prognosis and often results in increased mutation to KIT receptor. Furthermore, SCF-KIT ligand can potentially differentiate smokers from nonsmokers, suggesting a role of this gene in lung carcinogenesis induced by smoking. The progression of precancerous stem cells to cancer is associated with an upregulation of KIT, suggesting that KIT-SCF signalling may be involved in the formation and survival of cancer stem cells. Reference Perumal, Pillai, Nguyen, Schaal, Coppola and Chellappan56
Donnenberg et al. Reference Patel, Ersek and Kim53 studied tumours from 58 patients, which were KIT positive and KIT negative and further investigated the effect of imatinib on tumours expressing high levels of KIT. They observed that overexpression of KIT was about 3·1-fold when compared to normal lung, and tumours expressing high KIT levels were sensitive to imatinib. They concluded that NSCLC tumours can be categorised into low-level KIT and high-level KIT. Hence, overexpression of KIT in KIT-positive NSCLC supports exploring KIT as a therapeutic target in a subset of patients. Xu et al. Reference Xu, Wang and Zhang54 investigated mutations and prognosis of NSCLC harbouring KIT mutations in 402 patients and the status of KIT mutation was detected by next-generation sequencing (NGS). They observed a KIT gene mutation rate of 3·48% in NSCLC, and the median overall survival for patients was 23·0 months. They further reported that HER2-accompanied mutations might play a worse prognosis in KIT gene mutated NSCLC. Yoo et al. Reference Yoo, Kim and Song57 analysed tissue samples obtained from the primary tumours of 147 NSCLC patients to determine the frequency of KIT expression and any potential relationships between KIT expression, pathological factors and clinical outcomes. They reported that KIT is expressed relatively frequently in NSCLC and may become a therapeutic target for patients, particularly those with inoperable or recurrent KIT-positive tumours.
Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2)
The DDR2 gene is located on the long arm of chromosome 1 at position 23·3 (1q23·3) and is a type I transmembrane RTK, which is activated by fibrillar collagen and has been reported to play roles in cell migration, cell adhesion, proliferation and survival when activated by ligand binding and phosphorylation. Reference Xu, Buczkowski and Zhang58,Reference Sasaki, Shitara and Yokota59 According to Xu et al., Reference Xu, Buczkowski and Zhang58 the DDR2 plays a major role in cancer progression by regulating the interactions of tumour cells with their surrounding collagen matrix and DDR2 mutant cells are more likely to become oncogenic. DDR2 mutations have been reported to occur in several cancers including NSCLCs, with somatic mutations in the gene reported at a frequency of 3·8% in a sample set of 290 squamous cell lung cancer samples. Reference Xu, Buczkowski and Zhang58 Villalobos and Wistuba Reference Villalobos and Wistuba11 have also reported DDR2 mutation prevalence rate of 3–4% for lung squamous cell carcinomas and 0·5% for adenocarcinomas; however, these mutations were only observed in smokers. Terai et al. Reference Terai, Tan and Beauchamp60 reported DDR2 mutation in approximately 4% of patients with lung squamous cell carcinoma. Most DDR2 mutations are found in the kinase domain, can induce an increase in cell proliferation rate Reference Lee, Jung and An61 and are of higher prevalence in later stages of the disease, so the possibilities are that mutation may be correlated with advanced disease. Reference Terai, Tan and Beauchamp60
Kobayashi-Watanabe et al. Reference Kobayashi-Watanabe, Sato and Watanabe62 investigated DDR2 expression and mutation status in 44 human clinical samples and 7 cell lines and reported that DDR2 overexpression might contribute to tumour progression in lung squamous cell carcinoma and could be a potential molecular target of the disease. Teria et al. Reference Terai, Tan and Beauchamp60 reported the identification of mutations in the DDR2 can be the potential therapeutic targets in NSCLCs, and it is effectively targeted by US Food and Drug Administration (FDA)-approved multitargeted kinase inhibitors such as dasatinib, imatinib, nilotinib and ponatinib. These have been identified as inhibitors of DDR2 and potentially suppress the proliferation of DDR2 mutated cancer cell lines. Reference Xu, Buczkowski and Zhang58,Reference Terai, Tan and Beauchamp60,Reference Kobayashi-Watanabe, Sato and Watanabe62 In preclinical studies, dasatinib was demonstrated to inhibit proliferation in lung cancer cell lines with DDR2 mutations in both in vitro and in vivo models. However, Xu et al. Reference Xu, Buczkowski and Zhang58 reported that DDR2 inhibition with dasatinib leads to both adaptive and acquired resistance, with resistance mechanisms such as DDR2 gatekeeper mutation, neurofibromatosis type 1 loss and activation of parallel RTK pathways such as EGFR, insulin-like growth factor 1 receptor and MET. Reference Xu, Buczkowski and Zhang58,Reference Beauchamp, Woods and Dulak63 According to Xu et al., Reference Xu, Buczkowski and Zhang58 tumour-derived cell lines with DDR2 mutations are not solely reliant on the mutation for cell proliferation, but they often display upregulation and partial dependence on neuroblastoma-derived avian myelocytomatosis viral oncogene homolog (MYCN). DDR2 has an extremely long juxtamembrane domain that is thought to play a key role in the receptor’s function, and investigations are ongoing to discover whether preventing the overexpression of this portion of the receptor will have a potential therapeutic effect. Reference Kim, Ko, You and Rhee64 Terai et al. Reference Terai, Tan and Beauchamp60 investigated the development of effective and selective DDR2 inhibitors that could be used to pharmacologically address the impact of inhibiting the kinase activity of DDR2. They demonstrated that dual targeting of SRC proto-oncogene and DDR2 kinases may be more effective than selective inhibition of either kinase alone in DDR2 mutated lung cancer models and indicated that a combination of these two activities may be sufficient to target DDR2 mutated tumours.
AKT Serine–Threonine Kinase 1 (AKT1)
The AKT serine–threonine kinase family includes three members, namely, AKT1, AKT2 and AKT3, which are encoded by three distinct genes. Although AKT1 and AKT2 are expressed in most tissues, AKT3 is expressed only in a few organs. Reference Rao, Pierobon and Kim65 The AKT1 gene is located on the long arm of chromosome 14 at position 32·33 (14q32·33) and belongs to a class of genes known as oncogenes. It encodes protein kinase B, a serine–threonine protein kinase that is rapidly and specifically activated by platelet-derived growth factor through PI3K and helps to regulate the growth and division of many cell types throughout the body by degradation of the nuclear factor of activated T cells. Reference Rao, Pierobon and Kim65 AKT1 also plays a key role in multiple cell processes including apoptosis, cell motility, invasion, cell growth, proliferation, survival, angiogenesis and metastasis Reference Malanga, Scrima and De Marco66 ; however, its activation phosphorylates and inactivates components of the apoptotic mechanism. AKT1 mutations occur very rarely in lung cancer, with a prevalence rate of about 1·9%, but its oncogenic properties may contribute to the development of approximately 5·5% squamous cell lung cancers. Reference Malanga, Scrima and De Marco66,Reference Do, Solomon, Michell, Fox and Dobrovic67 It is reported that AKT1 mutations can be found in both adenocarcinoma and squamous cell carcinoma lung cancers, and about 1 in 100 NSCLCs have a mutation in the AKT1 gene that changes the AKT1 protein. 68
Liu et al. Reference Liu, Zhou, Qian, Shi, Fang and Jiang69 demonstrated that AKT1 overexpression and gene amplification regulate human lung cancer cell resistance to cisplatin. They reported that the AKT1-forced expression in the cells was enough to make the cells cisplatin resistant. However, AKT1 inhibition by its dominant negative mutant reversed the cisplatin-resistant cells to become cisplatin sensitive. They indicated that AKT amplification and the mTOR pathway play important roles in human lung cancer cells acquiring cisplatin-resistance, which may represent a new mechanism for acquiring cisplatin resistance and a potential novel therapeutic target for overcoming cisplatin resistance in human cancer in the future. Reference Liu, Zhou, Qian, Shi, Fang and Jiang69 Hollander et al. Reference Hollander, Maier, Hobbs, Ashmore, Linnoila and Dennis70 used genetically engineered mice deficient in AKT1, AKT2 and AKT3 isoforms to investigate the isoform(s) required for mutant KRAS-mediated lung tumorigenesis. They observed that AKT1 is the primary AKT isoform activated by mutant KRAS in lung tumours and that AKT3 may oppose AKT1 in lung tumorigenesis and lung tumour progression. They reported that deletion of AKT1 prevented lung tumorigenesis in both tobacco-induced cancer and genetic mutant KRAS models. The AKT1 deletion also prevented both tumour initiation and progression in vivo and prevented transformation by mutant KRAS in vitro. They concluded that since AKT1 inhibitors considered as cancer therapeutics in clinical development are not isoform selective, their results support specific targeting of AKT1 to mitigate the effects of mutant KRAS in lung cancer. Reference Hollander, Maier, Hobbs, Ashmore, Linnoila and Dennis70 According to My Cancer Genome, 68 AKT1 mutation is currently an inclusion criterion in clinical trials for NSCLCs and selumetinib, and AKT1 inhibitor MK-2206 and vistusertib are the therapies used in these trials for NSCLCs that contain AKT1 mutation. 68 The Chemietek 71 has also reported other AKT1 inhibitors in clinical trials such as afuresertib (GSK2110183), AZD5363, ipatasertib (GDC-0068), GSK690693, M2698 (MSC2363318A), MK-2206 monohydrochloride, MK-2206 dihydrochloride and uprosertib (GSK2141795). 71 Kim et al. Reference Kim, Kang and Lee72 investigated various single-nucleotide polymorphisms (SNPs) of the AKT1 gene, including rs3803300, rs1130214, rs3730358, rs1130233 and rs2492732, to determine the impact on survival in early stage NSCLC patients. They observed that the SNPs had a significant effect on the expression and activity of the AKT1 gene and were associated with poorer overall survival of patients as well as poorer disease-free survival. Furthermore, they reported that the SNPs impacted the capacity for AKT1-mediated apoptosis to prevent micro-metastatic tumour cells and could be used as a potential prognostic marker for patients with early stage NSCLC. Zhang et al. Reference Zhang, Fan and Li73 studied the impact of the rs1130214 SNP of the AKT1 gene and found that patients with either one or two mutant copies of the gene were significantly associated with poorer patient outcome and disease control. Lee et al. Reference Lee, Choi and Jeon74 investigated the rs3803300 and found results similar to Zhang et al.’s Reference Zhang, Fan and Li73 in that the SNP presence was associated with poorer overall survival as well as disease-free survival.
Rearranged During Transfection (RET) Proto-Oncogene
The RET proto-oncogene encodes a receptor-type tyrosine kinase for members of the glial cell line-derived neurotrophic factor family of extracellular-signalling molecules. Reference Villalobos and Wistuba11 It is located on the long arm of chromosome 10 at position 11·2 (10q11·2) and it is involved in cell proliferation, migration and differentiation as well as neuronal navigation. Reference Villalobos and Wistuba11,Reference Knowles, Murray-Rust and Kjær75 RET chromosomal rearrangements are reported to occur in approximately 1–2% of NSCLC cases, and they tend to be mutually exclusive with other major lung-cancer drivers such as EGFR, KRAS mutations and ALK or ROS proto-oncogene 1 (ROS1) rearrangements. Reference Villalobos and Wistuba11,Reference Wang, Xu and Wang76,Reference Mendoza77 RET rearrangements usually occur in adenocarcinomas with more poorly differentiated solid features, are found in young never smokers and are observed in equal proportions in males and females. Reference Villalobos and Wistuba11,Reference Wang, Xu and Wang76,Reference Mendoza77 In addition, the activation or gain in function of RET through fusion with genes such as KIF5B, CCDC6, and NCOA4 has been linked to lung adenocarcinoma. Reference Villalobos and Wistuba11,Reference Wang, Xu and Wang76–Reference Gozgit, Chen and Song78 According to Mendoza, Reference Mendoza77 activation of RET can occur by somatic or germ line alterations, which then will lead to autophosphorylation of intracellular tyrosine residues and initiation of PI3K-AKT, RAS-MAPK and phospholipase C pathways that signal cell proliferation and survival. Furthermore, somatic rearrangements of RET induces the formation of RET fusion protein kinases that localise in the cytosol and have transforming and oncogenic properties. Reference Mendoza77 Currently the standard diagnostic assay for the detection of RET chromosomal rearrangements include FISH, RT-PCR, IHC and NGS methodologies.
Several studies Reference Villalobos and Wistuba11,Reference Lee, Choi and Jeon74,Reference Wang, Xu and Wang76 have shown that RET rearrangement or fusion potentially causes oncogenic alterations but can be inhibited by multitargeted kinase inhibitors such as sunitinib, vandetanib, sorafenib and cabozantinib. Wang et al. Reference Wang, Xu and Wang76 reported a case study of a 50-year-old male patient with stage IV NSCLC lung adenocarcinoma. After four cycles of chemotherapy with gemcitabine and cisplatin, the disease stabilised and chemotherapy was stopped due to the adverse effects but 6 months later the cancer recurred. Gene testing shows the tumour to be KIF5B-RET fusion positive and the patient was treated with cabozantinib, an RTK inhibitor of RET, which considerably improved the patient’s condition. Reference Wang, Xu and Wang76 Ju et al. Reference Ju, Lee, Shin, Lee, Bleazard and Won79 performed a combined analysis of parallel whole-genome and transcriptome sequencing for cancer and paired normal tissue of a 33-year-old lung adenocarcinoma patient who was a never smoker and had no familial cancer history. They reported KIF5B-RET fusion as a driver mutation of lung adenocarcinoma and may be a good therapeutic molecular target for treatments, and the development of specific agents targeting KIF5B-RET will provide more advanced therapeutic strategies for lung adenocarcinoma. Reference Ju, Lee, Shin, Lee, Bleazard and Won79 Gozgit et al. Reference Gozgit, Chen and Song78 investigated the therapeutic sensitivity of ponatinib as a potent RET inhibitor in RET fusion-positive patient-derived xenograft (PDX) models. They compared ponatinib activity with sunitinib, sorafenib, lenvatinib and cabozantinib activities in RET fusion-positive and RET fusion-negative PDX models alongside a standard-of-care chemotherapeutic agent and demonstrated that RET fusions in NSCLC are therapeutically responsive to RET inhibitors and that ponatinib is a more potent inhibitor. Reference Gozgit, Chen and Song78
BRAF Proto-Oncogene
The BRAF proto-oncogene is a serine–threonine protein kinase that belongs to the RAS-RAF-MEK-ERK-MAPK pathway involved in the transduction of mitogenic signals from the membrane to the nucleus and affects cell division, differentiation and secretion. Reference Pao and Girard80,Reference Garnett and Marais81 The BRAF gene is located on the long arm of chromosome 7 at position 34 (7q34); and when activated by oncogenic mutations, it phosphorylates MEK and promotes cell proliferation, cell growth and survival. Reference Villalobos and Wistuba11 It is reported that over 97% of BRAF mutations are located in codon 600 of the BRAF gene and the BRAF-V600E point mutations, which represent about 70–90% of all mutations in BRAF gene, are the most frequently identified cancer-causing mutations in melanoma, colorectal cancer, non-Hodgkin lymphoma, thyroid carcinoma and NSCLC and account for about 50% of all BRAF mutations in NSCLC. Reference Alvarez and Otterson82 Other less frequent BRAF gene mutations include V600K (8–20%), V600D (0·1%), V600M (0·3%) and V600R (1%). The prevalence of BRAF-mutated lung cancer is between 1·5 and 3·5% without any ethnic predilection, and mutations are observed in 3–5% of nonsquamous NSCLC. Reference Pao and Girard80,Reference Alvarez and Otterson82 BRAF mutations are reported to be more frequent in female patients, though non-V600E mutations occur at a higher frequency in males. Most patients harbouring BRAF mutations are either current or former smokers. Reference Alvarez and Otterson82 Alvarez et al. Reference Alvarez and Otterson82 reported that most BRAF V600E-mutated tumours have an aggressive micropapillary pattern that is associated with shorter progression-free survival and overall survival.
Marchetti et al. Reference Marchetti, Felicioni and Malatesta83 retrospectively investigated the prevalence and prognostic role of BRAF mutations in tumour specimens from 1,046 NSCLC patients using high-resolution melting analysis. They reported 37 cases of BRAF mutations in the samples and that most (57%) were V600E mutations that were also significantly more prevalent in females than males. They observed that all the non-V600E mutations were found in smokers and that the V600E-mutated tumours exhibited an aggressive histotype characterised in 80% of patients and were also significantly related to shorter disease-free and overall survival rates. Reference Marchetti, Felicioni and Malatesta83 They demonstrated a prognostic role for BRAF-V600E mutations in patients with NSCLC, though they also suggested that additional large multicentre studies will be required to extend and confirm their data. Paik et al. Reference Paik, Arcila and Fara84 reviewed data from 697 lung adenocarcinoma patients who underwent molecular testing for BRAF, EGFR and KRAS mutation and FISH for ALK rearrangements. They reported that 3% of the patients harboured the BRAF mutations of which 50% were V600E, 39% G469A and 11% D594A mutations and that all the patients with BRAF mutations were current or former smokers. The BRAF-V600E mutations have been determined to be an activating mutation, and cells that harbour it are sensitive to the TKIs: dabrafenib, trametinib, sorafenib and vemurafenib. Reference Alvarez and Otterson82 Alvarez et al. Reference Alvarez and Otterson82 investigated agents capable of treating BRAF-mutant lung cancer by analysing several clinical trials that used different BRAF and MEK inhibitors as a treatment option for BRAF-V600E mutation-positive patients. They concluded that targeting BRAF-V600E mutations with a combination of BRAF and MEK inhibitors provides the potential to be the best front-line option for patients with this oncogenic driver. Planchard et al. Reference Planchard, Kim and Mazieres85 conducted a multicentre, nonrandomised, open-label, phase 2 trial to study the efficacy of dabrafenib (a BRAF inhibitor) in patients with BRAF V600E-positive advanced NSCLC. In all 78 patients received dabrafenib after one or more prior chemotherapy regimens for metastatic disease and 6 patients received dabrafenib as the first-line treatment. They reported that the dabrafenib showed improved clinical activity in BRAF V600E-positive NSCLC and that dabrafenib could represent a treatment option for patients with limited therapeutic options. Reference Planchard, Kim and Mazieres85
Fibroblast Growth Factor Receptors (FGFRs)
The FGFRs constitute a family of four transmembrane RTKs, namely, FGFR1, FGFR2, FGFR3 and FGFR4, which belong to the immunoglobulin superfamily Reference Ahmad and Iwata86 and mediate cellular signalling after binding to their fibroblast growth factors’ high-affinity ligands. Reference Desai and Adjei87 FGFR1, FGFR2, FGFR3 and FGFR4 genes encode the FGFR1, FGFR2, FGFR3 and FGFR4 proteins and are located on the short arm of chromosome 8 at location 11·23 (8p11·23), on the long arm of chromosome 10 at location 26·13 (10q26·13), on the short arm of chromosome 4 at location 16·3 (4p16·3) and on the long arm of chromosome 5 at location 35·2 (5q35·2), respectively. The fibroblast growth factors activate FGFRs to regulate cell proliferation, growth, differentiation and migration during embryogenesis and homeostasis and act both in mesenchymal and in epithelial cells. Reference Desai and Adjei87–Reference Ornitz and Itoh89 According to Desai and Adjei, Reference Desai and Adjei87 the FGFR pathway controls cellular processes such as cell cycle progression, migration, metabolism, survival, proliferation and differentiation and plays a key role in signal transduction in lung cancer. Furthermore, it also activates multiple signal transduction pathways, such as RAT sarcoma (RAS) kinase and MAPK, thus making the FGFR central in angiogenesis, embryogenesis, inflammation and malignant tumour cell proliferation. Reference Desai and Adjei87 Weiss et al. Reference Weiss, Sos and Seidel90 demonstrated that FGFR1 amplification is common in squamous cell lung cancer and it is associated with tumour growth and survival, suggesting that FGFR inhibitors may be a viable therapeutic option for this group of patients.
Helsten et al. Reference Helsten, Elkin, Arthur, Tomson, Carter and Kurzrock91 studied the frequencies of FGFR mutations in 4,853 solid tumours and found FGFR aberrations in 345 (7·1%) of all tumour types, with 227 (66%) of the aberrations being gene amplifications, 90 (26%) being mutations and 28 (8%) being rearrangements. The aberration proportions were further analysed for the different FGFR subfamilies, and the most frequent alterations affected FGFR1 (49%) followed by FGFR3 (23%), FGFR2 (19%) and FGFR4 (7%), therefore suggesting that FGFR inhibition could be an important therapeutic option for multiple tumour types. Reference Helsten, Elkin, Arthur, Tomson, Carter and Kurzrock91 Quintanal-Villalonga et al. Reference Quintanal-Villalonga, Molina-Pinelo and Cirauqui92 investigated tumour samples from 87 patients with advanced (stage IIIC–IV) NSCLC and who had been given erlotinib or gefitinib as the first or a further line of treatment. They demonstrated that high FGFR1 expression levels predict higher resistance to erlotinib or gefitinib in patients with TKI-treated EGFR-mutated and EGFR wild-type lung adenocarcinoma and a poorer prognosis to patients treated with these inhibitors. The potential of FGFR as a predictive biomarker has been reported by Porta et al. Reference Porta, Borea and Coelho88 who reviewed several clinical trials involving multitarget TKI inhibitors with FGFR status-based patient selection and reported that FGFR TKIs are promising targeted therapy drugs for various cancers, including NSCLC. They further reported that lucitanib (a strong inhibitor of FGFR1 and FGFR2), nintedanib (an FGFR TKI that targets FGFR1-3) and ponatinib are all being used in clinical trials with tumours harbouring FGFR aberrations. Reference Porta, Borea and Coelho88 Desai and Adjei Reference Desai and Adjei87 reported that genomic alterations in squamous cell lung cancer have shown that FGFR2 mutations are present in 3% of cases; however, in a comprehensive analysis that sequenced 623 genes from 188 cases of primary lung adenocarcinoma, the FGFR family of receptors was among the highly dysregulated genes, with mutations found in 19% of cases. Reference Desai and Adjei87 Weiss et al. Reference Weiss, Sos and Seidel90 conducted a systematic search for alterations that are therapeutically amenable and performed a high-resolution gene-copy number analyses in a set of 232 squamous cell lung cancer specimens and found 155 (67%) FGFR1 amplification and confirmed the presence of FGFR1 amplifications in an independent group of squamous cell lung cancer samples using FISH (22% of cases). They demonstrated that the FGFR inhibitor PD173074 inhibited growth and induced apoptosis in lung cancer cells harbouring amplified FGFR1. Reference Weiss, Sos and Seidel90
KRAS Homologue
KRAS is a member of the RAS family of membrane-associated G proteins and encodes a protein that belongs to the intrinsic guanosine triphosphatase superfamily which is involved in cell proliferation, differentiation, cytoskeletal reorganisation and survival. Reference Villalobos and Wistuba11,Reference Acquaviva, Smith and Sang93 The KRAS gene is located on the short arm of chromosome 12 at position 12·1 (12p12·1) Reference Villalobos and Wistuba11 and it is associated with the activation of several signalling pathways including RAS-RAF-MAP kinase kinase (MEK)-ERK and RAS-MAPK and acts downstream of a number of RTKs, including EGFR. Reference Villalobos and Wistuba11,Reference Barbie, Tamayo and Boehm94 KRAS is essential for normal development; however, its activating mutation can be oncogenic due to the consistent activation of its downstream-signalling pathways that are involved in deregulated cell proliferation, growth, invasion and metabolism. Reference Zhang, Park, Shin and Deng95 According to Jancík et al., Reference Jancík, Drábek, Radzioch and Hajdúch96 activating mutations in the KRAS gene impair the ability of the KRAS protein to switch between active and inactive states, thereby leading to cell transformation and increased resistance to chemotherapy and biological therapies that targets the EGFRs. Reference Villalobos and Wistuba11,Reference Jancík, Drábek, Radzioch and Hajdúch96 KRAS-mutant tumours make up about 20–35% of NSCLCs patients with adenocarcinoma and are typically found in tumours from patients who are current or former smokers, and it constitutes the most frequent potentially targetable molecular subtype of NSCLC. Reference Ferrer, Zugazagoitia, Herbertz, John, Paz-Ares and Schmid-Bindert97–Reference Riely, Marks and Pao99 Ferrer et al. Reference Ferrer, Zugazagoitia, Herbertz, John, Paz-Ares and Schmid-Bindert97 reported that KRAS-mutant advanced-stage lung cancers have generally been associated with poorer overall survival than KRAS wild-type tumour, though other studies in early stage or advanced stage cohorts have not been consistent in validating this poorer survival and hence have been reported that the prognostic significance of KRAS mutational status in lung cancer remains uncertain. Reference Ferrer, Zugazagoitia, Herbertz, John, Paz-Ares and Schmid-Bindert97–Reference Riely, Marks and Pao99
According to Roman et al. Reference Román, Baraibar and López100 and Villalobos and Wistuba, Reference Villalobos and Wistuba11 although the predictive and prognostic value of KRAS-mutational status remains unclear and there are no targeted therapies approved for patients with lung cancer and KRAS mutation, several clinical trials are underway to investigate KRAS mutations and its clinical impact. Villalobos and Wistuba Reference Villalobos and Wistuba11 in a review of biomarkers reported that the presence of KRAS mutations may be associated with unfavourable outcome, be a negative predictor of responsiveness to chemotherapy, associated with an increased likelihood of having a second primary tumour and is a predictor of resistance to EGFR-TKIs (e.g., gefitinib or erlotinib) targeted therapy in patients with NSCLC. Furthermore different trials have reported improvements in both progression-free survival and response rate with the combination of selumetinib (MEK1/MEK2 inhibitor) and docetaxel compared to docetaxel alone, and there are promising results with sorafenib (RAS-RAF pathway inhibitor) with a disease control rate of approximately 50%. Reference Villalobos and Wistuba11 Roberts et al. Reference Roberts, Stinchcombe, Der and Socinski98 reported a trial that compared carboplatin–paclitaxel with gefitinib in patients with adenocarcinoma and light smoking or never-smoking history and demonstrated the superiority of gefitinib to carboplatin–paclitaxel in the overall response rate, progression-free survival, quality of life and a lower rate of toxicity in the intent-to-treat patient population. Reference Roberts, Stinchcombe, Der and Socinski98 Acquaviva et al. Reference Acquaviva, Smith and Sang93 examined the activity of ganetespib, a small-molecule inhibitor of Hsp90 for NSCLCs in a panel of lung cancer cell lines harbouring a diverse spectrum of KRAS mutations and reported the potential of ganetespib as a single agent or as a combination treatment of KRAS-driven lung tumours. Reference Acquaviva, Smith and Sang93 Janes et al. Reference Janes, Zhang and Li101 demonstrated that mutant KRAS can be selectively targeted and that ARS-1620 is a new generation of KRAS-G12C-specific inhibitors with promising therapeutic potential. Dompe et al. Reference Dompe, Klijn and Watson102 in a study to identify agents to sensitise KRAS-mutant NSCLC cells to MEK inhibitor treatment reported that MEK inhibitors are a potential treatment option for tumours harbouring KRAS mutations and demonstrated that FGFR inhibitors combined with trametinib are effective in KRAS-mutant NSCLC xenografts as well as genetically engineered mouse models. Roman et al. Reference Román, Baraibar and López100 reported that KRAS mutations is a negative prognostic factor in lung adenocarcinoma histology tumours; and worse progression-free survival, disease-free survival or overall survival is reported in patients harbouring KRAS-mutated genotypes. Furthermore, KRAS mutation has the potential as a negative predictor of response to chemotherapy, an increased likelihood of having a second primary tumour, and is a predictor of resistance to targeted therapy with EGFR-TKIs, such as gefitinib or erlotinib in patients with NSCLC. Reference Román, Baraibar and López100
Phosphate and Tensin Homologue Deleted on Chromosome 10 (PTEN)
PTEN is a tumour-suppressor gene located on the long arm of chromosome 10 at position 23·31 (10q23·31) and encodes a phosphatidylinositol-3,4,5-trisphosphate 3-phosphatase protein with lipid phosphatase activity. Reference Gkountakos, Sartori and Falcone103 PTEN plays an essential role in tumorigenesis and both its mutation and inactivation can influence proliferation, apoptosis and cell cycle progression in tumour cells. Reference Lu, Cao, Chen, Xiao, Zou and Chen104 In normal physiological conditions, PTEN facilitates functions related to smooth muscle differentiation, angiogenesis and T-reg cell stability; and it commonly loses its function through mutation, deletion, transcriptional silencing or protein instability, which has consequent alterations in important pathways implicated in cell proliferation, survival, migration and genomic stability. Reference Gkountakos, Sartori and Falcone103,Reference Xiao, Hu and He105 It is one of the most frequently inactivated tumour suppressor genes, with its protein expression significantly reduced in a number of cancer types, including NSCLC. Reference Gkountakos, Sartori and Falcone103,Reference Xiao, Hu and He105 The tumour-suppression function of PTEN involves negative regulation of PI3K-AKT-mTOR (PI3K/mTOR/AKT) oncogenic-signalling pathway (an important signalling pathway involved in a large number of cellular events). Furthermore, PTEN also has functions related to the maintenance of chromosomal stability and competence of DNA repair. Reference Gkountakos, Sartori and Falcone103 The inactivation, deletion or mutation of PTEN allows the PI3K/mTOR/AKT pathway to be activated even in the absence of exogenous stimulus, leading to the possible initiation of tumorigenesis. Reference Cully, You, Levine and Mak106 According to Gkountakos et al., Reference Gkountakos, Sartori and Falcone103 smoking can induce downregulation of PTEN expression and therefore increase mTOR/AKT-signalling activation in the airway epithelium of healthy and chronic obstructive pulmonary disease in smokers compared to nonsmokers. Reference Gkountakos, Sartori and Falcone103 The standard current diagnostic assays for the detection of PTEN are IHC, PCR, NGS and FISH methodologies. Reference Gkountakos, Sartori and Falcone103
Xiao et al. Reference Xiao, Hu and He105 performed a systematic review and meta-analysis of 23 studies evaluating the survival data of 2,505 patients, regarding the prognostic value of PTEN downregulation in NSCLC patients. They reported that decreased PTEN expression is associated with poor overall survival, unfavourable disease-free survival and unfavourable prognostic value for progression-free survival of patients with NSCLC and recommended using PTEN protein expression detected by IHC as a prognostic factor for treatment of NSCLC. Reference Xiao, Hu and He105 Lu et al. Reference Lu, Cao, Chen, Xiao, Zou and Chen104 conducted a study on human lung adenocarcinoma cell line A549 overexpressing wild-type or mutant PTEN and expressing an small interfering ribonucleic acid directed towards endogenous PTEN. They demonstrated that overexpression of wild-type PTEN inhibited cell proliferation, promoted cell apoptosis, caused cell cycle arrest at G1, downregulated p-AKT and decreased the expression of the telomerase protein human telomerase reverse transcriptase (hTERT). Furthermore, they confirmed that PTEN downregulates the PI3K/AKT/hTERT pathway, thereby suppressing the growth of lung adenocarcinoma cells. Lim et al. Reference Lim, Zhang and Miller107 reported that PTEN loss can potentially predict an aggressive subset of lung tumours that have a poor prognosis, and hence PTEN is an important determinant of prognosis in patients with advanced NSCLC, and by identifying this subgroup using IHC will allow identification of a poor prognosis patient subgroup that can be targeted with novel treatments that restore PTEN function. Reference Lim, Zhang and Miller107 Endoh et al. Reference Endoh, Yatabe, Kosaka, Kuwano and Mitsudomi108 investigated 78 lung cancer patients who had recurrent disease after surgical resection and were treated with gefitinib and observed that high expressions of PTEN were associated with prolonged survival.
Cytotoxic T-lymphocyte-Associated Gene 4 (CTLA4)
The CTLA4 gene is located on the long arm of chromosome 2 at position 33·2 (2q33·2) and encodes the cytotoxic T-lymphocyte associated protein 4 (CTLA4) which is a member of the immunoglobulin superfamily and plays an important role in normal immune function as an immune checkpoint receptor by inhibiting proliferation of activated T cells upon binding to CTLA4 ligands. Reference Villalobos and Wistuba11,Reference Kucuk, Charbonnier, McMasters, Chatila and Bleesing109,Reference Salama and Hodi110 The regulatory checkpoint’s role of CTLA4 in regulating the immune system has made CTLA4 an attractive therapeutic target for cancer, with the development of human monoclonal antibodies that have successfully targeted CTLA4 in clinical trials. Reference Salama and Hodi110 Kucuk et al. Reference Kucuk, Charbonnier, McMasters, Chatila and Bleesing109 reported that CTLA4 haploinsufficiency can lead to an immune dysregulation syndrome which is characterised by lymphoproliferation, characteristic lymphocytic infiltration in nonlymphoid organs, autoimmune cytopenias, hypogammaglobulinaemia and recurrent infections. Reference Kucuk, Charbonnier, McMasters, Chatila and Bleesing109 Furthermore, integration of positive and negative co-stimulatory signals in the B7:CD28-CTLA4 pathway can modulate the generation and maintenance of immune responses, and inhibition of negative regulation through binding of CTLA4 has been shown to promote stimulation of adaptive immunity and potentiation of T-cell activation. Reference Grosso and Jure-Kunkel111 Kucuk et al. Reference Kucuk, Charbonnier, McMasters, Chatila and Bleesing109 described murine studies that showed that deletion of CTLA4 results in increased numbers of follicular B helper T cells and follicular regulatory T cells and loss of CTLA4 in follicular B helper T cells resulted in increased antigen-specific B-cell responses, whereas the loss of CTLA4 in follicular regulatory T cells resulted in defective suppression of antigen-specific and auto antigen-specific antibody responses. Reference Kucuk, Charbonnier, McMasters, Chatila and Bleesing109
Formenti et al. Reference Formenti, Rudqvist and Golden112 conducted a single-institution pilot-feasibility two-stage phase II trial of local radiotherapy and ipilimumab in chemotherapy-refractory metastatic NSCLC patients and demonstrated that a combination of radiation therapy and CTLA4 blockade (ipilimumab) induces systemic tumour responses in NSCLC patients. Grosso and Jure-Kunkel Reference Grosso and Jure-Kunkel111 conducted a detailed review of both preclinical advances and clinical application of anti-CTLA4 therapy in cancer treatment and reported complete regression or delayed tumour growth observed in several types of murine transplantable tumour models after anti-CTLA4 immunotherapy in preclinical studies. Furthermore, human antibodies such as ipilimumab and tremelimumab (both of which bind CTLA4 and block its interaction with B7 ligands to increase T-cell activation and proliferation) have been reported to be used in phase II and III clinical trials for various cancers including NSCLC. Reference Salama and Hodi110,Reference Grosso and Jure-Kunkel111 The role of CTLA4-directed treatment in NSCLC in a phase II clinical trial compared ipilimumab to carboplatin–paclitaxel chemotherapy alone in patients with stage IV NSCLC. Patients who received ipilimumab had an improvement in immune-related progression-free survival and improved the overall survival. Modulation of the immune system via blockade of CTLA4 represents a significant advance in the field of cancer treatment, and the future holds promise for developing additional T-cell-modulating agents. Reference Salama and Hodi110 Paulsen et al. Reference Paulsen, Kilvaer and Rakaee113 investigated tissue microarrays from tumour tissue samples and evaluated the immunohistochemical expression of CTLA4 in 536 patients with primary resected stage I–IIIA NSCLC. They analysed CTLA4 expression in tumour and stromal primary tumour tissue and in locoregional metastatic lymph nodes. They observed that CTLA4 expression in tumour epithelial cells or stromal cells of primary tumours was not significantly associated with disease-specific survival in the patients. However, expression of CTLA4 in high stromal cells independently predicted significantly improved disease-specific survival in squamous cell carcinoma subgroup, and there was an independent negative prognostic impact of tumour epithelial cells on CTLA4 expression in metastatic lymph nodes. Reference Paulsen, Kilvaer and Rakaee113 Expression of CTLA4 has a potential for both prognostic and predictive impacts on NSCLC patients; however, CTLA4 is an emerging biomarker and still requires more research and a deeper understanding of its mechanism.
Programmed Cell Death-1 (PD-1)
The PD-1 gene is located on the long arm of chromosome 2 at position 37·3 (2q37·3) and encodes the programmed cell death protein 1 (PD-1) which belongs to the immunoglobulin superfamily. PD-1 is a transmembrane immune checkpoint protein that expresses on T cells and binds the two programmed cell death ligands 1 and 2 (PD-L1 and PD-L2). Reference Zhang, Dutta and Liu114 According to Yu et al., Reference Yu, Boyle, Zhou, Rimm and Hirsch115 PD-L1 is expressed broadly in hematopoietic cells, including dendritic cells, macrophages, mast cells, T cells and B cells, and in nonhematopoietic cells, including endothelial, epithelial and tumour cells. The PD-1-PD-L1 pathway has a number of immunosuppressive functions, including inhibiting T-cell production, limiting T-cell proliferation and inducing apoptosis in activated T cells and plays an important role in inhibiting anti-tumour immunity in tumour cells. Reference Jia, Zhang and Zhang116,Reference Meyers, Bryan, Banerji and Morris117 The PD-L1 expression in patients with NSCLC has been reported Reference Yu, Boyle, Zhou, Rimm and Hirsch115 to range from 24 to 60%, and the current methodology for the detection of PD-L1 protein expression is IHC analysis. According to Zhang et al., Reference Zhang, Dutta and Liu114 the cell-intrinsic PD-1 engages with its ligand (PD-L1) to promote tumorigenesis and modulate downstream mTOR signalling in the absence of adaptive immunity, and knowledge of the PD-1-PD-L1 pathway has contributed to the production of immunotherapeutic agents targeting the PD-1 and PD-L1 and also has the potential to block the negative regulatory signal. Reference Zhang, Dutta and Liu114 Several of these inhibitors including pembrolizumab, nivolumab and imfinzi durvalumab (PD-1 antibodies) and avelumab and atezolizumab (PD-L1 antibodies) have been approved for clinical trials or patients treatments. Reference Yu, Boyle, Zhou, Rimm and Hirsch115,Reference Ernani and Ganti118–Reference Gettinger, Horn and Jackman120
A general agreement is that PD-L1 expression on tumour cells predicts responsiveness to PD-1 inhibitors in several tumour types, and therefore, PD-L1 expression in tumour tissue, evaluated by IHC, is in use currently as the core strategy to select patients for PD-L1 immune checkpoint inhibitor antibody treatment. Reference Ernani and Ganti118–Reference Gettinger, Horn and Jackman120 A number of clinical trials investigated the clinical efficacy of PD-L1 as a predictive biomarker for PD-L1 inhibitors or its prognostic role in NSCLC. Most studies Reference Brahmer, Reckamp and Baas119–Reference Pabani and Butts121 observed that PD-L1 overexpression is associated with significantly higher objective response rates, though others have not identified a similar relationship. Brahmer et al. Reference Brahmer, Reckamp and Baas119 conducted a randomised, open-label, international, phase 3 study to evaluate the efficacy and safety of nivolumab, as compared with docetaxel in 272 patients and observed that nivolumab disrupts PD-1-mediated signalling and restores antitumour immunity and also reported that the overall survival, response rate and progression-free survival were significantly better with nivolumab than with docetaxel. Gettinger et al. Reference Gettinger, Horn and Jackman120 reported that nivolumab significantly prolonged patients’ overall survival, had a favourable safety profile, and was associated with lower symptom burden and better quality of life compared with docetaxel in clinical studies that evaluated nivolumab, and also a 5-year follow-up results indicated that nivolumab treatment resulted in long-term overall survival. Pabani and Butts Reference Pabani and Butts121 reviewed published clinical trials that showed significant change in the treatment of advanced NSCLC and in the ongoing clinical trials that offer hope to further improve outcomes for patients with advanced NSCLC. They reported that the introduction of atezolizumab, nivolumab and pembrolizumab proved to be superior to chemotherapy in the second-line setting; and for patients with tumours strongly expressing PD-L1, pembrolizumab has been associated with improved outcomes in the first-line setting. The 5-year overall survival was 16% compared to the expected 5-year survival of less than 5%; and in patients with tumours expressing 50% or more PD-L1, the 5-year overall survival was 43%.
Human EGFR 2 (HER2)
The HER2 (or ERBB2) is a member of the HER family of RTKs, which include HER1 (or ERBB1), HER3 (or ERBB3) and HER4 (ERBB4); and these receptors consist of a ligand-binding extracellular domain and an intracellular tyrosine kinase domain. Reference Pillai, Behera and Berry122 The HER family of receptors plays a central role in the pathogenesis of several human cancers, is involved in the regulation of cell growth, survival and differentiation via multiple signal transduction pathways and participates in cellular proliferation and differentiation. Reference Villalobos and Wistuba11,Reference Pillai, Behera and Berry122 The HER2 gene is a proto-oncogene located on the long arm of chromosome 17 at position 12 (17q12) and encodes for a RTK member of the ERBB receptor family. Reference Villalobos and Wistuba11 Homodimerisation or heterodimerisation of the HER2 receptor allows for autophosphorylation of tyrosine residues within the cytoplasmic domain of the receptors and initiates important signal transduction pathways, including the MAPK, PI3K and protein kinase C, involved in cell proliferation, survival, differentiation, angiogenesis and invasion. Reference Villalobos and Wistuba11,Reference Pillai, Behera and Berry122,Reference Iqbal and Iqbal123 Overexpression of HER2 is associated with oncogenic transformation of cells and has been reported in 7–34·9% of NSCLCs and its associated with poor prognosis in patients. Furthermore, activating HER2 mutations have been found in 1.6–4% of lung cancers, and these mutations are found more often in adenocarcinomas in female, Asians, never or light smokers. Reference Villalobos and Wistuba11,Reference Pillai, Behera and Berry122,Reference Iqbal and Iqbal123
HER2 has been studied in patients with lung cancer for its potential role as a target for therapy, and different studies reinforce the importance of screening lung adenocarcinomas for HER2 mutation as a method to select patients who could benefit from HER2-targeted therapies. Reference Pillai, Behera and Berry122 Pillai et al. Reference Pillai, Behera and Berry122 reported on HER2 mutations in patients who were enrolled in the Lung Cancer Mutation Consortium and observed a prevalence of HER2 mutations of 2·6% in a population of 920 patients. The median overall survival of patients with HER2 mutations who received HER2-targeted therapies was 2·1 years compared to 1·4 years for those who did not and the survival of patients with wild-type HER2 was reported at 2·6 years. Reference Pillai, Behera and Berry122 Takenaka et al. Reference Takenaka, Hanagiri and Shinohara124 investigated the clinical significance of HER2 expression in NSCLC specimens from 159 adenocarcinomas and 77 squamous cell carcinoma patients who had been treated by complete resection of NSCLC. They reported identifying HER2 overexpression in 3·1 and 1·2% of patients with adenocarcinoma and squamous cell lung cancer, respectively. Furthermore, they observed HER2 overexpression in adenocarcinoma was a significantly unfavourable prognostic factor. However, since the number of NSCLC patients with HER2 overexpression was small, further investigations will be necessary to clarify the efficacy of molecular-targeted therapy for this subgroup. Reference Takenaka, Hanagiri and Shinohara124 In a report published by the Cancer Genome Project, sequencing of HER2 in 120 lung tumours revealed somatic mutations in the kinase domain of the protein, and the prevalence of HER2 mutations was 4%, all of which occurred in adenocarcinomas. Reference Pillai, Behera and Berry122,Reference Chandrashekhar125 Several ongoing clinical trials are investigating HER2-targeted therapies in patients with HER2-mutated and amplified NSCLC. Targeted agents such as trastuzumab, afatinib, neratinib, pyrotinib, dacomitinib, poziotinib, neratinib, lapatinib and bevacizumab are being used for treatment of patients with HER2 mutation. Some of these agents have been shown to offer promising therapies for patients with HER2-mutated NSCLC, Reference Villalobos and Wistuba11,Reference Pillai, Behera and Berry122 for example, Villalobos and Wistuba Reference Villalobos and Wistuba11 reported that afatinib and trastuzumab showed response rates of approximately 50%. More research is needed into these new targeted therapies targeting HER2 in lung cancer and has the potential to improve outcomes in this molecular subgroup of patients.
ROS Proto-Oncogene 1 (ROS1) RTK
The ROS proto-oncogene 1 (ROS1) is located on the long arm of chromosome 6 at position 22·1 (6q22·1) and encodes for an ‘orphan’ RTK with no known ligand and biologic function in humans. Reference Sehgal, Patell, Rangachari and Costa126,Reference Lin and Shaw127 Phylogenetic sequence analysis revealed that ROS1 is related to the ALK-leukocyte tyrosine kinase and insulin receptor RTK families and is involved in downstream-signalling processes of cell growth and differentiation, Reference Lin and Shaw127,Reference Kerr128 and its expression has been observed in epithelial cells of the kidneys, male reproductive organs, small intestines, the heart and the lungs in animal models. Reference Lin and Shaw127 ROS1 oncogenic fusion was first reported in NSCLC in 2007, and it describes a distinct molecular subgroup in approximately 1–2% of patients with NSCLC. It has been reported that ROS1 fusion and ALK rearrangements are mutually exclusive; however, they share similar clinicopathological features and are generally associated with younger age, history of light or never smoking and adenocarcinoma histologic type. Reference Sehgal, Patell, Rangachari and Costa126,Reference Lin and Shaw127 Although the exact mechanism of ROS1 kinase activation in the fusion proteins is unknown, when activated, ROS1 signals through the MAPK-ERK, P13K-AKT, Janus kinase-signal transducer and activator of transcription 3 and protein tyrosine phosphatase, nonreceptor-type 6-protein tyrosine phosphatase, nonreceptor-type 11 pathways to promote cell growth and survival. Reference Lin and Shaw127 The ROS1 and ALK tyrosine kinase domains are found to share significant homology, including bindings sites for adenosine triphosphate and crizotinib and this homology has principal relevance to the development of ROS1-targeted treatments. Reference Sehgal, Patell, Rangachari and Costa126,Reference Lin and Shaw127 Currently, the commonly used approach for ROS1 fusion detection in clinical samples has included FISH, IHC, RT-PCR and NGS. Reference Sehgal, Patell, Rangachari and Costa126,Reference Lin and Shaw127
A multitargeted MET-ALK-ROS1 inhibitor, Crizotinib is reported to have considerable clinical efficacy in ROS1-positive patients, and it is approved by the FDA for the treatment of patients with advanced ROS1-positive NSCLC. Reference Sehgal, Patell, Rangachari and Costa126,Reference Joshi, Pande and Noronha129 Furthermore, according to Lin et al., Reference Lin and Shaw127 several other ROS1 inhibitors including ceritinib, brigatinib, lorlatinib, entrectinib, cabozantinib, DS-6051b and TPX-0005 are also being developed. Joshi et al. Reference Joshi, Pande and Noronha129 retrospectively analysed data from 22 ROS1-positive lung cancer patients to determine the effect of crizotinib in patients with ROS1 rearrangement. They observed that the ROS1-positive cohort was relatively younger (mean age of 54 years compared to 60 years in a ROS1-negative cohort), mostly (86%) nonsmokers and 95% had adenocarcinoma histology. They demonstrated crizotinib as an effective treatment option potentially predicting significantly better progression-free survival and improvement in the overall survival. Shaw et al. Reference Shaw, Ou, Bang, Camidge, Solomon and Salgia130 reported the safety, pharmacokinetics and efficacy of response to therapy of 50 patients with advanced NSCLC who tested positive for ROS1 rearrangement in a phase 1 study of crizotinib. They reported a median age of 53 years. The majority (78%) of the patients had never smoked, 22% were former smokers and 98% had histologic features of adenocarcinoma. Furthermore, 6% had a complete response, 66% had a partial response and 18% had stable disease as their best response had an overall response rate of 72%. They found that crizotinib has marked antitumour activity in patients with advanced ROS1-rearranged NSCLC, and therefore ROS1 was validated as a therapeutic target in ROS1-rearranged lung cancers. Wu et al. Reference Wu, Yang and Kim131 also conducted a phase II, open-label, single-arm trial of 127 patients with ROS1-positive advanced NSCLC who had received three or fewer lines of prior systemic therapies. They reported a median age of 52 years, 72% were never smokers and 98% had adenocarcinoma histologic classification, 13% had complete response, 58% had partial response, 17% had stable disease and an objective response rate of 72%. They found from the study that crizotinib provides significant clinical benefit in patients with ROS1-positive advanced NSCLC with durable clinical responses and improvements in quality of life and symptom burden.
Neurotrophic Receptor Tyrosine Kinase (NTRK)
The NTRK genes NTRK1, NTRK2 and NTRK3 belong to a family of nerve growth factor receptors that play an essential role in several aspects of neuronal function and development, including promoting proliferation and survival. Reference Amatu, Sartore-Bianchi and Siena132,Reference Terry, De Luca and Leung133 However, the NTRK gene expression is limited within the human nervous system after embryogenesis. Reference Marchiò, Scaltriti and Ladanyi134 The NTRK1, NTRK2 and NTRK3 genes are located on the long arm of chromosome 1 at position 23·1 (1q23·1), on the long arm of chromosome 9 at position 21·33 (9q21·33) and on the long arm of chromosome 15 at position 25·3 (15q25·3) and encodes the tropomyosin receptor kinase (TRK) protein family of receptors, TRKA, TRKB and TRKC, respectively. Reference Amatu, Sartore-Bianchi and Siena132,Reference Farago, Taylor and Doebele135 According to Farago et al., Reference Farago, Taylor and Doebele135 NTRK gene fusions have been described in different adult and paediatric solid tumours and are considered to drive tumour growth and survival through expression of a constitutive activation of TRK fusion proteins which are caused by ligand-independent dimerisation. Reference Farago, Taylor and Doebele135 The prevalence of NTRK chromosomal rearrangement is low in common cancer types, including NSCLC, and it is reported to occur at a frequency of approximately 0·1–1% in NSCLC, Reference Farago, Taylor and Doebele135–Reference Hirsch, Suda, Wiens and Bunn137 though others Reference Hirsch, Suda, Wiens and Bunn137 have also reported up to 3%. Aberrations in NTRK genes in lung adenocarcinomas include gene fusions (0·2%), mutations (13%) and genomic amplifications (6%); and Ma reported that protein overexpression and fusion is the most validated mechanism of oncogenic transformation. Reference Marchiò, Scaltriti and Ladanyi134
Cocco et al. Reference Cocco, Scaltriti and Drilon138 reported that patients with NTRK fusion-positive cancers treated with first-generation TRK inhibitors, such as larotrectinib or entrectinib demonstrated over 75% response rates irrespective of the tumour histology and that the TRK inhibitors were well tolerated by most patients. Farago et al. Reference Farago, Le and Zheng136 investigated the frequency of NTRK1 rearrangements in 1378 NSCLC tumour specimens and further assessed the clinical activity of entrectinib in a patient who was enrolled into a phase 1 dose-escalation study of entrectinib in adult patients with locally advanced or metastatic disease. The patient had a history of smoking and developed progressive disease despite prior lines of therapy, which included carboplatin and pemetrexed, pembrolizumab, docetaxel and vinorelbine. They reported the prevalence of NTRK1 gene rearrangements at a frequency of 0·1% in the tumour specimens and also observed a significant anti-tumour activity with entrectinib treatment in the patient with NSCLC harbouring an sequestosome 1 (SQSTM1)-NTRK1 gene rearrangement. Although the antitumour activity occurred in one patient, it still suggests that entrectinib is a potentially effective therapy for tumours with NTRK gene rearrangements. They reported that the potent clinical response of the patient with NTRK1-rearranged NSCLC to entrectinib is a strong indication to screen patients with NSCLC for NTRK gene rearrangements. In another study, Farago et al. Reference Farago, Taylor and Doebele135 analysed a database of 4,872 consecutively screened patients with NSCLCs harbouring NTRK fusions and characterised the clinical, molecular and histologic features and reported a prevalence rate of 0·25% (11 of 4,872) NTRK fusions, which consisted of 64% NTRK1 (7 of 11) and 36% NTRK3 (4 of 11). Furthermore, they observed that NTRK fusions in NSCLCs occur across sexes, ages, smoking history and histology. Terry et al. Reference Terry, De Luca and Leung133 investigated 686 lung cancer cases (adenocarcinoma, large-cell carcinoma, squamous cell carcinoma, small-cell carcinoma and carcinoid tumour) with clinical outcome data in tissue microarray format that were immunohistochemically stained for NTRK1 and NTRK2. They reported that both NTRK1 and NTRK2 expressions strongly correlate with squamous carcinoma histology and are also highly specific markers of the disease when compared with the other carcinoma subtypes, including adenocarcinoma. Furthermore, NTRK2-positive staining in squamous carcinoma is associated with improved disease-specific survival and overall survival, whereas NTRK1-positive staining in squamous carcinoma and adenocarcinoma had no prognostic significance with respect to disease-specific survival or overall survival. They determined that NTRK1 and NTRK2 are potentially useful biomarkers in separating squamous cell carcinoma from adenocarcinoma and that NTRK2 is a useful independent positive prognostic indicator for disease-specific survival or overall survival. Reference Terry, De Luca and Leung133
Epithelial Cell Adhesion Molecule (EpCAM)
EpCAM was discovered in the late 1970s as one of the first identified tumour-associated antigens; however, its actual contribution to carcinogenesis was unexplored until recently. Reference Baeuerle and Gires139 It is a type I transmembrane glycoprotein that is expressed on the basolateral surface of most normal epithelial tissues such as the colon, gastric, prostate and lung epithelia. Reference Kim, Kim and Cui140 The EpCAM gene is located on the short arm of chromosome 2 at position 21 (2p21) and is involved in intercellular adhesions and interacts with E-cadherin to induce cell adhesion. Overexpression of EpCAM is reported to be linked to stimulation of the cell cycle and proliferation by upregulating c-myc and cyclin A/E, though the relationship between EpCAM overexpression and the invasiveness or metastatic ability of cancer cells and the underlying molecular mechanisms still remains unclear. Reference Kim, Kim and Cui140 Although Kim et al. Reference Kim, Kim and Cui140 reported on overexpression of EpCAM in NSCLC, the clinical implication has not been fully investigated.
Several studies Reference Baeuerle and Gires139,Reference Kim, Kim and Cui140 are currently investigating the role of EpCAM as an oncogenic-signalling molecule for cancer cells, the frequency and level of EpCAM expression on various cancers and its prognostic potential, EpCAM-directed immunotherapeutic approaches and the interaction of EpCAM with other proteins to provide a basis for a potential therapeutic window and repression of its growth-promoting signalling in carcinomas. Reference Baeuerle and Gires139 Baeuerle and Gires Reference Baeuerle and Gires139 recently reported on the potential prognostic significance of EpCAM overexpression in cancers and EpCAM-targeted immunotherapeutic approaches with edrecolomab in clinical development. Kim et al. Reference Kim, Kim and Cui140 investigated the frequency of EpCAM expression in NSCLC cells and human tissues and also evaluated the clinicopathological significance of EpCAM expression in lung adenocarcinoma. They reported the detection of 51·3% (120/234) EpCAM overexpression in surgically resected adenocarcinoma tissues, and the overall survival was not significantly different between EpCAM overexpression and EpCAM-negative patients. They concluded that EpCAM may play a role in the carcinogenesis of adenocarcinoma of the lung and may provide a promising molecule for targeted therapy in NSCLC. Reference Kim, Kim and Cui140 Thompson et al. Reference Thompson, Fan and Black141 conducted a single-centre, prospective, observational study of 101 NSCLC patients who underwent sampling of pleural effusion to demonstrate the potential of enumeration and characterisation of pleural EpCAM-positive cells (PECs) from pleural effusions for the management of NSCLC patients. They reported that the median number of PECs was significantly greater in the malignant (n = 84) than in the non-malignant group (n = 17) and also demonstrated that higher numbers of PECs were independently associated with inferior overall survival. Went et al. Reference Went, Vasei and Bubendorf142 analysed 3,360 samples by immunohistochemical staining tissue microarrays of primary human carcinoma from colon, stomach, prostate and lung cancers for both frequency and intensity of EpCAM expression. They reported high-level EpCAM expression in 64% (823/1,287) of lung cancers. They also observed a trend towards a longer survival in patients with adenocarcinomas, large-cell and bronchoalveolar carcinomas and high EpCAM expression which supports EpCAM as a potential prime target for immunotherapies in major human malignancies.
Conclusion
Lung cancer is now recognised as a heterogeneous disease that develops from genetic mutations and gene expression patterns, which initiate unregulated cellular growth, proliferation and progression. Recent breakthroughs in molecular technologies have created avenues in search of genes that are overexpressed in lung cancers and which may serve as novel targets for treatment. The early detection of lung cancer has the potential to greatly impact the disease burden through timely identification and treatment of patients at a manageable stage of the cancer. Moreover, the identification of lung cancer biomarkers that can potentially be used in screening for early detection and diagnosis, to monitor patient response to specific treatment options, to provide information about a likely cancer outcome independent of treatment received or to provide clinicians the possibility of prospectively identifying the group of patients who will benefit from a particular treatment can potentially lead to patient-specific targeted treatment and increased survival. Therefore, identification of accurate biomarkers to determine the mutational status of driver mutations, which can be targeted by specific TKIs, is essential for treatment decision-making in advanced stage NSCLC and has the potential to shift the treatment paradigm of NSCLC to more personalised and targeted therapy.
Acknowledgements
The authors would like to acknowledge with much gratitude the initial contributions from Patricia Bui, Hillary Ho and students in the PHYS 383 class at the University of Waterloo.
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
