Frequency of oncogenic drivers in lung adenocarcinoma as part of the Lung Cancer Mutation Consortium
The opportunities for potential therapeutic targets in SQCLC were supported by the observation that 96 % of tumors contained one or more mutations in tyrosine kinases, serine/threonine kinases, phosphatidylinositol-3-OH kinase (PI3K) catalytic and regulatory subunits, nuclear hormone receptors, G-protein-coupled receptors, proteases, and tyrosine phosphatases (Fig. 8.2). Targetable genetic alterations were considered present in 64 % of TCGA samples based on the availability of an FDA-approved targeted therapy or one under study in clinical trials, the confirmation of the altered allele in RNA sequencing, and the mutation assessor score. Of these, mutations or amplifications were reported in three families of tyrosine kinases: the erythroblastic leukemia viral oncogene homologues (ERBBs), fibroblast growth factor receptors (FGFRs), and Janus kinases (JAKs). An alteration in at least one of the core cellular pathways known to represent potential therapeutic opportunities (PI3K-AKT, receptor tyrosine kinase (RTK), and RAS) was identified in 69 % of samples. Specifically, one of the components of the PI3K-AKT pathway was altered in 47 % of tumors, and RTK signaling was probably affected by events such as EGFR amplification, BRAF mutation, or FGFR amplification or mutation in 26 % of tumors. While this analysis suggests new areas for potential therapeutic development, the dependence of SQCLC on many of these alterations needs to be defined clinically.
Alterations in targetable oncogenic pathways in SQCLCs. Sixty-nine percent of 178 SQCLCs revealed alteration in at least one of the three pathways: PI3K-AKT, RTK, and RAS. Alterations are defined by somatic mutations, homozygous deletions, and high-level, focal amplifications and, in some cases, by significant up- or downregulation of gene expression (AKT3, FGFR1, PTEN)
FGFR is a transmembrane receptor tyrosine kinase that participates in the regulation of embryonal development, cell proliferation, differentiation, and angiogenesis. The FGFR family has four members, including FGFR1, FGFR2, FGFR3, and FGFR4, and binds up to 22 FGF ligands. After binding with their FGF ligands, FGFRs undergo dimerization followed by activation of downstream signaling via PI3K-AKT and RAS-RAF-MAPK pathways central to survival in tumorigenesis.
Alterations in FGFR1–4 were seen in 27 % of tumor samples reported by TCGA, with FGFR1 mutations or amplification in 18 % of samples . In a set of 153 SQCLC tumors, Weiss and colleagues found FGFR1 amplification in 22 % of the samples by FISH . A separate study by Dutt and colleagues identified an amplification frequency of 21 % by SNP array analysis of 57 SQCLC samples . In an Asian population with surgically resected SQCLC, FGFR1 amplification was found in 13 % of 262 specimens and correlated with cigarette smoking and a shorter overall survival (51.2 vs. 115 months) . A study conducted by Heist and colleagues found that FGFR1 amplification status did not correlate with smoking history or survival in a Caucasian population .
In Weiss and colleagues’ study, treatment with an FGFR inhibitor PD173074 resulted in tumor regression in a mouse xenograft model . Malchers and colleagues found that FGFR1-amplified tumor cells that co-expressed MYC were more sensitive to FGFR inhibition. The authors reported two SQCLC patients with amplified FGFR1 and high MYC expression who responded to FGFR inhibition: one responded to BGJ398, a highly specific FGFR inhibitor, and the other responded to pazopanib, a multikinase inhibitor with weak activity against FGFR . FGFR inhibitors have also been found to have activity in other solid tumors such as breast cancer and gastric cancer. Several trials evaluating selective FGFR1 inhibitors are currently in early phase clinical testing .
The PI3K pathway is a signal transduction pathway central to cell survival, metabolism, motility, and angiogenesis. Abnormalities in this pathway including in PI3K/PTEN/AKT/mTOR are more common in SQCLC than in lung adenocarcinoma, suggesting an increased dependence on this pathway [92–94]. The TCGA analysis found 47 % of tumors to have an alteration in one of the components of the PI3K-AKT pathway . The mutation rate for PIK3CA in SQCLC ranges from 3.6 to 6.5 % [93, 95], and PIK3CA amplification occurs in 42–43 % in an Asian population by FISH  and PCR , respectively. Soria and colleagues reported a loss of PTEN expression by IHC and PTEN methylation in 24 % and 35 % of NSCLC, respectively . Jin and colleagues showed a PTEN mutation rate of 10 in SQCLC compared to 1.7 % in lung adenocarcinoma samples .
Spoerke and colleagues found that cell lines harboring alterations in this pathway, including PI3K mutation or amplification, PTEN loss, or receptor tyrosine kinase activation, predict for sensitivity to PI3K inhibitors . There are a number of ongoing evaluations of PI3K and mTOR inhibitors in various solid tumors including in lung cancer, although no efficacy data are available to date.
The discoidin domain receptor (DDR) is a plasma membrane receptor tyrosine kinase that regulates cell adhesion, proliferation, and extracellular remodeling upon binding to its endogenous ligand, type 1 collagen [99, 100]. Upregulation of DDR1 in NSCLC, particularly squamous tumors, has been associated with improved disease-free and overall survival . Hammerman and colleagues sequenced 290 SQCLC tumors and cell lines and found 3.8 % tumors to harbor a DDR2 mutation. These tumors showed a gain-of-function phenotype that was abrogated by treatment with dasatinib, a multikinase inhibitor with activity against both DDR1 and DDR2 . Two phase II studies showed no advantage of dasatinib in unselected patients with advanced NSCLC [103, 104]. A recent case report describes a patient with chronic myelogenous leukemia (CML) and SQCLC harboring a DDR2 mutation who was treated with dasatinib, resulting in normalization of blood counts and a near complete reduction in size of the primary lung mass . A phase II trial is currently underway to investigate the efficacy of dasatinib selected in SQCLC with DDR2 mutations.
Insulin-like growth factor-1 receptor (IGF1R) is a cell surface receptor with tyrosine kinase activity that binds to the ligands IGF-1 or IGF-2 and activates the PI3K and RAS signaling pathways [106–108]. IGF1R protein expression is seen more commonly in SQCLC compared to other subtypes [109, 110], but thus far has not been found to be of prognostic value . A phase II study with figitumumab, a monoclonal antibody against IGF1R, with paclitaxel and carboplatin showed a response rate of 78 % in patients with advanced SQCLC . However, a subsequent randomized phase III trial of figitumumab with paclitaxel/carboplatin in unselected NSCLC patients was discontinued due to futility and increased toxicity . Early clinical trials evaluating small molecular inhibitors of IGF1R are ongoing , and the utility of free IGF-1 or IGF1R protein expression as a predictive biomarker remains to be seen.
Platelet-derived growth factor receptor (PDGFR) tyrosine kinase, classified as PDGFRA and PDGFRB, plays a crucial role in cell proliferation and angiogenesis . PDGFRA amplification occurs in 8.7 % SQCLC compared to 3.8 % of lung adenocarcinomas . A randomized phase III study with the addition of sorafenib, a multi-targeted TKI that targets PDGFRA, to platinum-based chemotherapy failed to show an improved survival in patients with advanced NSCLC. A subset of SQCLC in the study had increased mortality with the addition of sorafenib . Further clinical trials are now evaluating selective anti-PDGFRA inhibitors as well as PDGFRA-targeting monoclonal antibodies .
EGFR vIII is a variant of EGFR with deletion of exons 2–7, which was first described in human glioma . A study in Japan revealed an EGFR vIII mutation in 8 of 252 patients, among which 7 had SQCLC . Ji and colleagues examined 179 NSCLC samples and found an EGFR vIII mutation in 5 % SQCLC but none of the lung adenocarcinomas. The investigators also noted tumor regression in EGFR vIII mutant murine models treated with an irreversible EGFR inhibitor, HKI-272. Similarly cell lines that were resistant to gefitinib and erlotinib in vitro proved sensitive to HKI-272 . HKI-272 is currently in the early phase of clinical development .
MET is a proto-oncogene located on 7q21-31 and encodes a tyrosine kinase receptor for hepatocyte growth factor. MET activation either by increased gene copy number or amplification leads to enhanced proliferation, motility, apoptosis resistance, and angiogenesis. MET amplification has been recognized as a mechanism of resistance to EGFR TKI therapy in lung adenocarcinoma . MET protein expression occurs in approximately 40 % of SQCLC and associated with a poorer prognosis . A phase II trial of onartuzumab, a monoclonal antibody against MET, in combination with erlotinib showed improvements in OS and PFS compared with erlotinib alone in a MET-overexpressed subgroup of advanced NSCLC, of which 30 % had SQCLC . A randomized phase III study is currently evaluating onartuzumab in combination with a platinum doublet in SQCLC.
Small Cell Lung Cancer
Small cell lung cancer (SCLC) is characterized by frequent inactivating mutations in the critical tumor suppressor genes TP53 (75–90 %)  and RB1 (60–90 %) [127, 128]. A mouse model with conditional inactivation of these two tumor suppressors in the lung generates lung tumors histologically and biologically similar to human SCLC .
Recent reports including exome, transcriptome, and limited whole-genome sequencing have provided insights into the fuller landscape of genetic alterations in SCLC [130, 131]. In addition to confirming TP53 and RB1 inactivation, these studies define other alterations of interest in SCLC, with potential therapeutic implications. One consistent finding from both reports was an exceptionally high degree of genomic alteration in this tumor type, including mutations, insertions, deletions, large-scale copy number alterations, and gross inter- and intra-chromosomal rearrangements. MYC family member alterations, including gene amplification of MYC, MYCN, and MYCL1, as well as a recurrent gene fusion involving MYCL1, are frequent in SCLC and may represent important drivers of SCLC oncogenesis. The tumor suppressor PTEN appears to be inactivated in approximately 10 % of SCLC, and mutations of other factors in the same signaling pathway were also identified. Other alterations implicated as potential drivers in subsets of SCLC include amplification of the tyrosine kinase FGFR1 (in a reported 6 % of cases) and of the developmental regulator and transcription factor SOX2 (in up to 27 % of cases). The therapeutic implications of the large majority of the genetic alterations documented to date in SCLC have not been defined.
Importantly, pure SCLC lacks EGFR mutations and ALK rearrangements, even in those patients with this malignancy who are never smokers [132, 133]. However, in the rare cases of SCLC transformation as a mechanism of acquired resistance to EGFR TKIs, there is persistence of the original EGFR mutation in the tumors confirmed on biopsy. In all cases where SCLC has been documented as a mechanism of acquired resistance to EGFR TKIs, the original tumor was a pure adenocarcinoma prior to EGFR TKI treatment, and the transformation was validated by histologic examination and confirmed by expression of neuroendocrine markers.
The Role of Small Biopsies in the Age of Genotype-Driven Therapy
The majority of patients with lung cancers present with metastatic disease and are diagnosed, as a result, from small biopsy or cytology specimens alone. Bronchial biopsy samples obtained from patients with lung cancer frequently contain only limited amounts of primary carcinoma, and often one or more of the biopsy fragments will not contain tumor at all . Despite these challenges, cytology can be used to distinguish histology in NSCLC with accuracy up to 96 %, and in a majority of cases, cytology is suitable for molecular analysis as well .
As evident in the preceding text, oncologists are increasingly faced with the challenge of obtaining sufficient tumor material to perform standard of care or exploratory molecular analyses prior to determining a patient’s treatment plan, all while minimizing the need for further invasive procedures. In tertiary cancer centers, most patients present with a pathologic diagnosis, with variable amounts of diagnostic material leftover. In addition to surgical pathology, immunohistochemistry and fluorescence in situ hybridization typically require at least 4–6 five micrometer recuts, PCR-based genotyping requires approximately 10, and next-generation sequencing requires over 10. Stepwise algorithms that prioritize specific molecular studies are clearly needed.
Recognizing the need to reassess and formally address the optimal utility of small biopsy and cytology specimens to meet the modern day clinical needs, a new lung adenocarcinoma classification has recently been published under the joint sponsorship of the International Association for the Study of Lung Cancer (IASLC), the American Thoracic Society (ATS), and the European Respiratory Society (ERS) . The major changes introduced include the greater use of special stains to classify difficult cases further into adenocarcinoma or SQCLC, the diagnosis using small samples, and the need to manage tissue strategically for molecular studies. In particular, for those cases where a small biopsy specimen shows NSCLC lacking either definite squamous or adenocarcinoma morphology, the immunohistochemistry workup should be as limited as possible to preserve tissue for molecular testing. A multidisciplinary and institutional approach should be implemented to incorporate clinical information in selecting the appropriate methods for obtaining tissue samples and prioritizing molecular studies including DNA sequence analysis, fluorescence in situ hybridization, and RNA-based studies. While the list of targets will continue to evolve, recommendations for testing by the National Comprehensive Cancer Network working group are driven by the availability of rationally targeted therapies which can, in practice, be given to patients (Table 8.1).
Targeted agents for patients with genetic alterationsa
Genetic alteration (i.e., driver event)
Available targeted agents with activity against driver event in lung cancer
While next-generation sequencing is ideal for patients with adequate tissue samples, as it may discover potential targets for therapy and/or generate clinical trial opportunities for patients, symptomatic patients may not have the luxury of waiting, untreated, the 4–8-week turnaround time for results. In such cases, other assays that can be completed in a shorter time period, such as IHC for mutant forms of EGFR or ALK, can be prioritized to help determine treatment plans. Indeed, immunostaining to detect mutant EGFR correlates well with sequencing-based assays and is particularly useful for small biopsies when the material is scant or bone biopsies in which the decalcification processes often results in DNA degradation .
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