Fig. 12.1
Targetable oncogenes in NSCLC: (a) preferentially or exclusively expressed by lung adenocarcinoma; (b) preferentially or exclusively expressed by lung squamous cell carcinoma
In the present chapter we will analyze the molecular alterations and the potential role of targeted drugs in advanced lung ADC and in SqCC, focusing particularly on human epidermal growth factor receptor 2 (HER2), mesenchymal epithelial transition (MET), BRAF, and gene fusions in the former, and on fibroblast growth factor receptor 1 (FGFR1), discoidin domain recptor 2 (DDR2), and PI3KCA/AKT alterations in the latter.
Adenocarcinoma of the Lung
An impressive number of studies evaluating gene expression, mutations and other genomic alterations as well as the proteomic profile of lung ADC have been published in the past two decades. These studies led to the identification of a great number of alterations, many of them with incidence less than 5 % of NSCLC. Their low frequency makes difficult to evaluate associations to clinical characteristics and outcome and to set up clinical trials with target agents in the subcategories of patients harboring these specific alterations. In order to get larger study cohorts, academic and non-academic research groups, either united in leagues or consortiums, have started to collect data in large databases. The American National Cancer Institute (NCI) sponsored the formation of the Lung Cancer Mutation Consortium under the leading supervision of the University of Colorado. The consortium prospectively evaluated 10 driver mutations in 1,000 lung ADCs using the SnapShot platform and the Food and Drug Administration (FDA) approved fluorescence in situ hybridization (FISH) assays for ALK translocation and MET amplification. An actionable driver alteration was detected in 622 (62 %) of 1,007 samples with any genotyping, and in 465 (63 %) of the 733 fully genotyped cases [39]. KRAS and sensitizing EGFR mutations were found in 25 and 15 % of cases respectively, and ALK rearrangements in 8 %. Other aberrations included mutations in BRAF (2 %), HER2 (2 %), PIK3CA (1 %), NRAS (1 %), MEK1 (<1 %) and amplification of MET (1 %). Importantly, patients with an actionable alteration treated with a targeted agent lived longer than patients with a targetable driver mutation that didn’t receive a tailored treatment (3.5 vs 2.4 years, p < 0.0001). Recently, results from data collection through a common database by the French oncologic centers (Biomarkers France study) were presented during the American Society of Clinical Oncology (ASCO) meeting of 2013 [2]. Among the 10,000 NSCLC specimens analyzed (76 % were lung ADC and 5 % SqCC), a known target was found in 46 % of cases. In particular the group found mutations of EGFR in 10.2 % of samples, KRAS in 26.8 %, BRAF in 1.8 %, HER2 in 0.9 %, PIK3CA in 2.4 % and ALK rearrangement in 3.9 %. The frequency of such aberrations is low and they could be considered as niche conditions. However, taking into account the high incidence and mortality of lung cancer, the absolute number of patients bearing such aberrations is not insignificant and the value of their recognition for therapeutic purposes is considerable.
HER2 Aberrations
HER2 is a membrane receptor also known as human EGFR2 or ERBB2/neu and belongs to the ERBB family together with EGFR (HER1), HER3 and HER4. Unlike other family members, HER2 has no known ligand but it acts as preferred dimerization partner for other family members, including EGFR. Given the central role in tumor genesis, sustenance and progression [33] and the potential regulatory activity on EGFR [46], HER2 represents an appealing target for treatment of lung cancer.
HER2 may be deregulated through alterations of gene copy number, gene sequence and protein expression. HER2 overexpression has been described in up to 20 % of cases [34], while HER2 amplification and mutation occur in up to 10 and 3 % of lung adenocarcinoma, respectively [1, 7, 80]. The most common mutation is an inframe duplication/insertion at codon 776 of exon 20 [1].
HER2 prognostic role has been investigated in several studies with conflicting results. The protein expression did not affect survival in our study published in 2002 [34]. Conversely, a recent meta-analysis shows a weak poor prognostic role of HER2 overexpression in lung adenocarcinoma [47]. HER2 amplification does not seem to affect prognosis in NSCLC patients, as recently published by Cappuzzo and colleagues [10]. The prognostic impact of HER2 mutations hasn’t been so far clarified [1, 54].
Anti-HER2 strategies are current treatment options for HER2 overexpressing breast and gastric cancer. Several trials evaluated trastuzumab (Genentech Inc. San Francisco CA, USA) and pertuzumab (Genentech Inc. San Francisco CA, USA), two monoclonal antibodies targeting HER2, in lung cancer patients selected for the expression or amplification of HER2. None of these studies demonstrated sufficient activity to plan further investigation in HER2 amplified and overexpressing lung tumors [23, 32, 43–45, 93]. On the other hand, preclinical studies revealed that HER2 mutation is tumorigenic [63] and that NSCLC cell lines harboring HER2 mutation are sensitive to trastuzumab [85]. Several case reports supported these findings [8, 54, 82] and more recently Mazières and colleagues published a retrospective series of 16 HER2 mutation positive patients receiving an anti-HER2 treatment (trastuzumab, lapatinib, afatinib or masatinib). Authors found an impressive overall response rate (ORR) of 50 % and a disease control rate (DCR) of 82 %, with the greatest benefit obtained with trastuzumab (15 patients, DCR 96 %) and afatinib (4 patients, 100 %). Another class of drugs under investigation in HER2 mutant lung cancer patients is the irreversible pan-HER inhibitors family, including lapatinib, afatinib (Boehringer Ingelheim Pharm. Ingelheim, Germany), dacomitinib (Pfizer, New York City NY, USA) and neratinib (Puma Biotechnology, Los Angeles CA, USA) among the others. Lapatinib, a reversible pan-ErbB inhibitor, registered for HER2-positive breast cancer, has shown minimal activity in NSCLC [76]. A Belgian exploratory phase II trial showed a promising activity of afatinib in HER2 mutated NSCLC: all the five patients bearing HER2 mutation experienced response to treatment [16]. Dacomitinib was evaluated in NSCLC patients with HER2 amplification or mutation. Among the 22 HER2 positive patients the agent provided only few responses (ORR 17 %) [42]. Notably, no patient with gene amplification responded to the inhibitor. On the other hand, neratinib has shown some activity in HER2 mutant lung cancer in a phase I trial [22], and a randomized phase II trial (NCT01827267) is already evaluating the compound alone or in combination with a mTOR inhibitor in 84 HER2 mutated NSCLC subjects.
In conclusion HER2 overexpression and gene amplification seem not to affect the response to treatment with specific anti-HER2 agents. On the other hand, the presence of HER2 mutation may select a sub-group of NSCLC patients suitable for anti-HER2 therapies, either with monoclonal antibodies or small molecule inhibitors. Further investigation and the setup of larger phase II and III trials are urgently warranted.
MET Amplification
The MET proto-oncogene is located on chromosome 7q21 and encodes for the tyrosine kinase receptor of the hepatocyte growth factor (HGF). MET deregulation may be due to protein overexpression, gene amplification, and mutation. MET protein is widely expressed in lung cancer, with a frequency ranging from 25 up to 75 % of cases [35]. It is known that MET over-expression is a negative prognostic factor for outcome in lung cancer [35]. MET gene copy number gain was described in about 4 % of lung cancers and has been associated with poor patient outcome in several studies [9, 56], but not in all of them [20]. Mutation of MET gene can occur in approximately 5 % of lung cancer specimens. The prognostic value of MET mutation and its role in oncogenesis have not been sufficiently elucidated [17, 49], although more recent findings suggest a role in disease progression [24].
MET amplification has been investigated as mechanism of resistance to anti-EGFR treatment, occurring in about 20 % of EGFR mutation positive lung cancer patients who progressed on an EGFR TKI. Therefore, much effort has been put in investigating anti-MET agents in combination with EGFR TKIs with the aim of delaying or overcoming the appearance of resistance. Onartuzumab (METMab; Genetech Inc., San Francisco CA, USA) is a fully human monoclonal antibody against MET. Although the promising prolongation of progression free survival (PFS) registered in one phase II study in MET immunohistochemistry (IHC) positive patients [78], the phase III trial (METLung) evaluating this agent in combination with the EGFR TKI erlotinib in previously treated and MET IHC positive NSCLC was recently halted for lack of clinically meaningful activity (http://www.gene.com/media/press-releases/14562/2014-03-02/genentech-provides-update-on-phase-iii-s). Detailed data from the study have not been yet released and data from other two phase III trials (NCT01519804 and NCT01496742) evaluating onartuzumab in combination to chemotherapy in SqCC and adenocarcinoma, respectively, are still missing. Similar results occurred in the clinical investigation of tivantinib (ARQ197, Arqule; Daiichi Sankyo, Tokyo, Japan), a non-ATP competitive small molecule MET inhibitor that showed a significant activity in combination to erlotinib in non-squamous lung cancer in one phase II trial [72]. However, the following phase III trial comparing the combination of tivantinib and erlotinib to placebo and erlotinib in biologically unselected NSCLC did not meet its primary end-point of improving survival and was prematurely stopped in October 2012 (http://investors.arqule.com/releasedetail.cfm?ReleaseID=710618). A subset analysis showed longer PFS and OS for patients with at least 2+ immunostaining in more than 50 % of tumor cells [70], but further investigation is needed to prospectively confirm these findings.
Crizotinib (Pfizer, New York City NY, USA) is a MET inhibitor, primarily developed as anti-ALK agent for ALK rearranged lung cancer. In a case report, a patient with NSCLC with a de novo MET amplification and no ALK rearrangement achieved rapid response to crizotinib [59], suggesting a potential role of anti-MET strategies in patients with de novo amplification of the gene.
The negative results of the above mentioned trials evaluating anti-MET agents should not be generally regarded as failure of anti-MET therapy approach. At the present time, no study has evaluated these agents in patients with de novo MET amplification or mutations and investigation in these subcategories of lung adenocarcinoma is warranted.
BRAF Mutations
BRAF is a member of the RAS family of serine/threonine kinases and is the first downstream effector of KRAS. Activating mutations of BRAF lead to increased kinase activity and are transforming in vitro [14]. BRAF mutations are well characterized tumor drivers in metastatic melanoma and were also described in 1–5 % of NSCLC, almost exclusively in adenocarcinoma [52, 62]. Whereas BRAF mutation positive melanoma harbors a V600E mutation in 80 % of cases [75], lung cancer displays a non-V600E mutation in about 50 % [52, 62]. Non-V600E mutations are more commonly associated to cigarette smoking, while V600E mutations are more common in females and never smokers. BRAF mutations are mutually exclusive with EGFR and KRAS mutations and ALK rearrangement. The presence of mutations in the BRAF gene seems to significantly worsen patients’ prognosis. In the study by Marchetti and collaborators, V600E mutations were associated to a more aggressive tumor histotype (papillary features) and shorter disease free survival and overall survival (OS). No prognostic impact was found for non-V600E mutations [52].
Clinical data on the activity of BRAF inhibitors in lung cancer harboring BRAF mutations are poor, although several compounds are currently under investigation. Vemurafenib (Zelboraf, Daiichi Sankio, Japan) is a first generation BRAF inhibitor initially developed in melanoma. It has been tailored to have specific activity against the protein with the V600E amino acidic change and its inhibitory activity against non-V600E mutations is unknown. Preclinical data and one case report suggest that non-V600E mutations are resistant to vemurafenib [26, 91]. Conversely, some case reports describe considerable responses to treatment with vemurafenib in NSCLC patients with V600E BRAF mutations [25, 64]. Another V600E BRAF inhibitor with promising activity is dabrafenib (GlaxoSmithKline, Brentoford, United Kingdom). During the ASCO annual meeting of 2013, preliminary results of a single arm phase II trial were presented [65]. Among 13 evaluable patients treated with dabrafenib, 5 achieved a partial response (ORR 54 %) and the study could continue to the second stage.
Taken together, data on BRAF mutations suggest this molecular aberration can be considered as a targetable driver in lung adenocarcinoma. Clinical data are still limited, but the only available phase II trial with a BRAF inhibitor shows a promising activity of this class of compounds. The future challenge will be to develop new BRAF inhibitors that can inhibit both V600E and non-V600E mutations.
Gene Fusions
After the discovery of ALK fusions, other hybrid genes have been found in lung cancer. Gene fusions may occur as consequence of chromosomal translocation, inversion or interstitial deletion. Oncogenic rearrangements may cause the expression of new proteins or of a protein with different activity than the native one. Alternatively, the fusion can bring a gene under the control of a strong promoter, causing aberrant expression of a protein in cells where usually the protein has no or low expression.
ROS1 is a proto-oncogene located on chromosome 6q22. Rearrangements of ROS1 gene were firstly described in lung tumors by Rikova and colleagues in 2007 [66]. The physiological role of the protein is still not completely clarified, although it’s known to be a transmembrane receptor with tyrosine kinase activity. A large number of partner genes have been discovered, including SLC34A2, SDC4, EZR, FIG, TPM3, CD74, KDELR2, and LRIG3 among the others [67, 81]. In all these fusions, the chromosomal translocation fuses the 3′-end region of ROS1, containing the kinase domain, to the 5′-end region of the partner gene. Both in vitro and in vivo experiments proved the tumorigenic properties of ROS-1 fusion protein [12, 13]. ROS1 rearrangements may be found in approximately 2 % of lung adenocarcinoma and are more frequent in never smokers [4]. On the basis of the high homology between ROS1 and ALK amino acidic sequences [92], it was postulated that ALK inhibitors, such as crizotinib (PF-02341066; Xalkori, Pfizer, New York, USA), could have inhibitory activity also on ROS1. As matter of fact, the recently presented preliminary data of the PROFILE 1001 study evaluating crizotinib in ROS1 positive patients showed a striking antitumor activity with an ORR of 56 % [60]. The fusion protein is also client of the heat shock protein (HSP) pathway and, therefore, similarly to ALK, ROS-1 positive tumors are supposed to be sensitive to treatment with HSP90 inhibitors [69]. At present time, no clinical data on HSP90 targeting agent is available in ROS1 positive patients.
RET gene is located on chromosome 10 and codifies a transmembrane receptor with tyrosine kinase activity. It is involved in cell proliferation, migration, differentiation, and in neuronal navigation. Germline and somatic mutations of RET are known to cause the multiple endocrine neoplasia type 2 (MEN2) syndrome and are involved in the tumorigenesis of sporadic medullary thyroid cancer [88]. Furthermore, RET rearrangements are involved in sporadic and radiation induced papillary thyroid carcinoma. RET rearrangements have been identified in lung ADC as well, with an incidence of 1–2 %. Several genes, such as KIF5B, CCDC6, NCO4 and TRIMM33, can act as fusion partners [81, 86]. RET positive lung carcinomas are more common in poorly differentiated tumors and in never smokers [86]. Therapeutically, several multiple kinases inhibitors, such as vandetanib (Astrazeneca, London, United Kingdom), cabozantinib (Exelis Inc. San Fransisco CA, USA), ponatinib (Ariad, Cambridge MA, USA), axitinib (Pfizer, New York City NY, USA), sunitinib (Pfizer, New York City NY, USA) and sorafenib (Bayer Healthcare Pharm, Leverkusen, Germany), are able to block RET function. Phase III trials data in biologically unselected NSCLC are available for some of these agents both as monotherapy and in combination. However, all these studies results are negative and none of these drugs was approved for lung cancer treatment probably for the absence of genotypic selection. On the other hand, some case reports describe anecdotal responses to treatment with vandetanib and cabozantinib in RET positive lung cancer patients [18, 27]. In particular, some authors observed two partial responses and one long-term stable disease among the first 3 patients treated in the context of a phase II trial evaluating cabozantinib in RET positive lung adenocarcinoma (NCT01639508). Phase II trials are currently evaluating also vandetanib (NCT01823068), sunitinib (NCT01829217) and ponatinib (NCT01813734) monotherapy in RET rearrangement positive lung cancer. Their results are earnestly expected and hopefully in the next few years new treatment options will be available for this subtype of lung ADC.
Recently, Doebele and his research group described a novel fusion involving the gene NTRK1, located on chromosome 1 and codifying the TRKA receptor tyrosine kinase [83]. In the initial report, authors found the fusion in 3 out of 91 (3.3 %) lung adenocarcinoma samples with no other common oncogenic alterations. Preclinical data showed antitumor activity of crizotinib, leustartinib (CEP-701, Cephalon, Frazer PA, USA) and ARRY-470. Crizotinib inhibitory activity was inferior to the other agents, but given its availability in clinic, the research group sought to determine if treatment of NTRK1 positive patients with crizotinib could lead to clinical benefit. One NTRK1 rearranged patient received crizotinib obtaining a minor response that lasted almost 3 months. Clinical trials with existing or novel inhibitors are highly warranted.
Squamous Cell Carcinoma of the Lung
Despite the recent discoveries of many recurrent genomic alterations that could act as therapeutic targets in lung ADC, the biological characterization of SqCC is still at a relative earlier stage. This disparity has led to significant differences in the way patients with advanced NSCLC are currently treated. However, in the past few years the scientific community has shown growing interest in understanding SqCC biology and identifying new treatment targets. Initial studies on single-neuclotide polymorphisms (SNP) array identified FGFR1 gene as potential actionable marker of SqCC [3, 19]. Sequencing studies aiming at identifying mutated kinases [29] and phosphotyrosine signaling studies [66] identified mutations in DDR2 that could be therapeutically targetable. More recently, the Cancer Genome Atlas Research Network described a comprehensive landscape of genomic and epigenomic alterations of 178 SqCC of the lung, using SNP array profiling, whole-exome sequencing, RNA and miRNA sequencing, and methylation profiling [6]. The research group observed mutations in tyrosine kinases, serine/threonine kinases, PI3K catalytic and regulatory subunits, nuclear hormone receptors, G protein-coupled receptors, proteases and tyrosine phosphatases in 96 % of the analyzed tumors. In particular, they identified a somatic alteration of a potentially targetable gene in 64 % of cases. These data are exciting and could open the way to personalized treatment also for lung SqCC. Moreover, in these days a significant private-public partnership initiative is going to be launched in the U.S.A., the “Master Protocol” for advanced SqCC, which is an initiative from the US National Cancer Institute and FDA with the goal of rapid identification of active drugs in a randomized phase II study which will lead to a well randomized phase III registration trial if endpoints in phase II are met.
FGFR Alterations
FGFR family includes 4 receptors with tyrosine kinase activity, namely FGFR-1, -2, -3 and -4. The activation of the receptor leads to downstream signaling through the PI3K/AKT and RAS/RAF/MEK pathways [53]. Interest in this family came from initial studies showing amplification of the chromosome 8p12 containing the FGFR1 gene in 10–20 % of lung SqCC and <5 % of ADC [19, 71, 87]. In some of these studies, authors showed that lung cancer cell lines with FGFR1 amplification were sensitive to small-molecule FGFR kinase blockers [19, 87]. Studies on the prognostic association of this molecular event are not univocal. A Korean study demonstrated a negative independent prognostic significance of FGFR1 amplification, although Heist and colleagues found no association of the genetic alteration to survival [31, 87]. FGFR mutations have been described in all the four members of the family by whole-exon and RNA sequencing [6]. Mutations of FGFR2 and FGFR3 cover up to 5 % of SqCC, are oncogenic and sensitize cancer cells to FGFR kinase inhibitors [38].
Several clinical studies are currently evaluating the impact of anti-FGFR strategies. Ponatinib (AP26113; Ariad Pharm Inc, Cambridge MA, USA) is a multi-inhibitor of all the four members of the FGFR family and other kinases, which showed preclinical activity on amplified or mutated lung cancer cell lines [28]. One phase II trial is currently evaluating ponatinib in molecularly selected patients at the University of Colorado (NCT01935336). The study includes an important analysis of FGFR alterations (gene copy number and mRNA expression) aiming at prospectively identifying the prevalence of each biomarker and the extent of their overlap. Indeed, preclinical data suggest that FGFR1 amplification alone may be insufficient to determine the category of patients that more likely will respond to treatment [89]. Other agents are under investigation in FGFR1 amplified SqCC of the lung, such as dovitinib (TKI258; Novartis, Basel, Switzerland. NCT01861197), AZD4547 (Astrazeneca, London, United Kingdom), and NVP-BGJ398 (Novartis, Cambridge MA, USA). Data from these studies will elucidate whether FGFR inhibition can be a successful strategy in patients with lung cancer harboring alterations of the receptor or of its gene.
DDR2 Mutation
The discoidin domain receptors (DDRs) are membrane receptors with tyrosine kinase activity that bind collagen and, when activated, interact with Src and Shc [36]. DDR2 gene is located on chromosome 1q23.3 and mutations in its sequence have been detected in 3.8 % of lung SqCC [29]. No data are currently available on the potential prognostic significance of DDR2 mutations. Therapeutically, some authors described high affinity and inhibitory activity of dasatinib (BMS-354825; Brystol-Myers Squibb, New York City NY, USA), imatinib (Novartis, Basel, Switzerland) and nilotinib (Novartis, Basel, Switzerland) on the DDRs [15]. Preclinical experiences on lung cancer cell lines showed that DDR2 mutations are oncogenic. Moreover, treatment with interfering RNA or dasatinib was able to selectively inhibit DDR2 mutated cells [29].
This strong preclinical data supported the development of dasatinib in the clinical setting. Two clinical trials evaluated dasatinib as monotherapy or in combination with erlotinib in biologically unselected NSCLC [30, 40]. Both trials showed a lower activity of the experimental treatment compared to cytotoxic chemotherapy, but one of the patients who responded to the combination of erlotinib and dasatinib harbored mutation of DDR2. A phase II study (NCT01514864) is currently evaluating dasatinib monotherapy in patients with DDR2 mutation positive SqCC and its results are eagerly anticipated.
PI3KCA/AKT/mTOR Pathway Aberrations
The PI3K/AKT/mTOR pathway is an important signal transduction pathway involved in regulation of cell proliferation, differentiation, motility and angiogenesis [21]. Several membrane tyrosine kinases, including EGFR, HER2, c-MET, IGF1R can activate PI3K, leading to phosphorylation of AKT and subsequent activation of mTOR, Bcl-2, tuberous sclerosis 2 and other target effectors. The PI3K/AKT pathway can also interact with other signaling pathways; for example, RAS can directly activate PI3K [84]. Deregulation of the pathway at several levels has been described in a number of malignancies, including lung cancer. The Cancer Genome Atlas Network project found significant alterations of the pathway in 47 % of samples analyzed, most commonly point mutations and amplification of PI3KCA, deletions and deactivating mutation of PTEN and overexpression of AKT [6]. The overall frequency of these molecular events makes the blockade of this pathway particularly appealing for therapeutic purposes. On the other hand, alterations of the PI3K pathway often occur simultaneously with other molecular aberrations in contrast with the mutual exclusiveness of driver mutations, suggesting that PI3K alterations may be a secondary event [6]. This could have huge impact on the development of inhibitors of this pathway.
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