Biologic Basis of Lung Cancer | 2 |
LUNG CARCINOGENESIS
Exposure and Susceptibility
Tobacco smoking is the major cause of lung cancer (1). Individuals with predisposition to nicotine addiction are more prone to continue smoking, and thus, be exposed to high doses of tobacco-associated carcinogens, including the polycyclic aromatic hydrocarbon (PAH) benzo-[a]-pyrene and the key nicotine metabolite nicotine-derived nitrosamine ketone (NNK) (Figure 2.1). These carcinogens can be activated or eliminated through endogenous enzyme systems whose activity is determined by specific genetic polymorphisms. For example, the glutathione-S-transferase (GST) system detoxifies carcinogens by adding a glucuronide metabolite to PAHs. Individuals with a GST-Mu 1 (GSTM1) genotype are deficient in this process, so GSTM1 smokers will have an increased risk of lung cancer. Conversely, the cytochrome P450 system metabolically activates carcinogens, such as PAHs, by adding an epoxide metabolite. Individuals with a P450 CYP1A1 genotype have increased activity of this enzyme, so CYP1A1 smokers have an increased risk for lung cancer. Thus, individual genetic differences can confer susceptibility to lung cancer upon specific carcinogen exposure.
Activated PAHs can covalently bind to DNA to form specific DNA adducts, an early sign of lung carcinogenesis. These adducts can be repaired by endogenous DNA repair enzymes, which have differential activity conferred by genetic variation. If not repaired, damaged DNA can induce cell death. However, if these alterations persist, they can lead to oncogenic, somatic alterations of tumor suppressor genes (e.g., p53, Rb) or oncogenes (e.g., KRAS, BRAF), or to DNA instability as indicated by loss of heterozygosity. These alterations are commonly found in the bronchial epithelium of cigarette smokers leading to field carcinogenesis. In some individuals, this process gives rise to a cell that undergoes clonal transformation and develops into a tumor. The full series of events that facilitate cellular transformation remain unclear, but likely includes a stochastic process of mutation accumulation, transformation of key pluripotent cells or stem cells, and cooperation between mutations in epithelial cells and a permissive immune environment that allows the proliferation of cells bearing foreign neoantigens (2).
Figure 2.1 Mechanistic Framework for Understanding How Cigarette Smoking Causes Lung Cancer. All Events Can Occur Chronically Since a Smoker Typically Uses Multiple Cigarettes per Day for Many Years
Source: From Ref. (1). Hecht SS. Lung carcinogenesis by tobacco smoke. Int J Cancer. 2012;131:2724-2732. Reprinted by permission of John Wiley & Sons, Inc.
Field Carcinogenesis
Recent studies of bronchial field carcinogenesis provide insight into the molecular pathogenesis of lung cancer, and may directly impact the care of those at risk for lung cancer by providing novel strategies for early detection and targeted chemoprevention. For example, two multicenter trials (Airway Epithelial Gene Expression in the Diagnosis of Lung Cancer [AEGIS] 1 and 2) demonstrated that a gene expression classifier from normal-appearing bronchial epithelial cells was a sensitive biomarker for diagnosing lung cancer among smokers undergoing bronchoscopy for suspected disease (3). Other studies have identified spatial and temporal variations in cellular alterations within field of cancerization in smokers, including transcriptional dysregulation of AKT and ERK1/2, epigenetic alterations of micro-RNAs (miRNAs) such as miR4423, and somatic chromosomal alterations. These aberrations can have a significant impact on the entire bronchial epithelial field, including basal cells that may serve as progenitors for lung carcinoma (4).
Lung Development and Carcinogenesis
Many of the interactions that occur between epithelial cells and stromal cells during lung carcinogenesis are similar to those that are essential to normal lung development. The pathways involved in the development and differentiation of the normal lung are also important for tumor initiation, progression, and histologic differentiation. Current paradigms suggest that lung carcinomas arise from pluripotent stem cells and progenitor cells that are capable of differentiation into several histologic cell types. The hypothesis that lung cancer is caused by the aberrant expression of genes involved in lung development is supported by gene expression studies demonstrating similarities between genetic signatures obtained from human lung tumors and those that are characteristically noted during normal lung development (5, 6). These studies show that the molecular profiles of high-grade tumors (e.g., large-cell carcinoma) demonstrate derangements in genes involved in the regulation of both the cell cycle and transcription, resulting in developmental arrest at a stage close to the undifferentiated progenitor cell. However, the molecular signatures of low-grade tumors (e.g., adenocarcinoma) are comprised of genes involved in terminal differentiation pathways. This linkage between poorly differentiated tumors and the molecular parameters of early development suggests that these genetic signatures are important for lung cancer progression and are potential biomarkers of clinical outcome.
MOLECULAR DETERMINANTS OF HISTOLOGIC SUBTYPES
The clinical significance of the developmental regulation of histologic differentiation is highlighted by recent clinical trials showing the differential efficacy of selected lung cancer therapies within specific histological and molecular subtypes. These findings have helped transform the clinical management of non–small-cell lung cancer (NSCLC) from a “one-size-fits-all” approach to a strategy based on personalized medical care (7).
In the most recent World Health Organization (WHO) lung cancer classification scheme, the major subtypes of lung cancer have been reorganized and renamed based upon seminal clinical and biological studies (8). The major changes include grouping all neuroendocrine tumors together, restricting the designation of large-cell carcinoma to tumors that lack differentiation by both morphologic and immunohistochemical criteria, and the adoption of the IASLC/ATS/ERS Lung Adenocarcinoma Classification scheme (9). These modifications are supported by biological studies that demonstrated that specific molecular signatures are associated with clinically relevant histological subtypes (10). This refined histologic classification and routine molecular testing protocols now facilitate the assignment of lung cancer treatment by specific histological and molecular subtypes (Figure 2.2). For example, the chemotherapeutic agent pemetrexed has activity in non-squamous NSCLC, but not squamous cell carcinoma, while necitumumab, an anti-epidermal growth factor receptor (EGFR) monoclonal antibody, may augment the activity of standard chemotherapy in some patients with squamous cell carcinoma, but not non-squamous NSCLC.
For early stage lung adenocarcinoma, the revised classification scheme captures distinct histological and biological subtypes with prognostic significance. For example, tumors with a preinvasive histology, such as adenocarcinoma in situ (AIS) or microinvasive adenocarcinoma (MIA), have 5-year survival rates of 95% to 100% after complete surgical resection. The histologic classification is also supported by the biological data demonstrating unique molecular properties of specific subtypes. Gene expression profiling studies from North America, Asia, and Europe have reproducibly demonstrated that early stage lung adenocarcinomas cluster into three major genomic subgroups that correlate with histology (Figure 2.3) (11). These studies have led to incorporation of these subgroups into the new WHO classification scheme and the revised lung cancer TNM staging system, with annotations for AIS and MIA now included for T1 tumors (12). It will be important to prospectively validate the prognostic accuracy of the revised staging system for preinvasive cancers and to evaluate the clinical significance of specific subtypes of invasive adenocarcinoma. For example, recent studies indicate that the micropapillary and solid variants are associated with a poorer prognosis after resection than other subtypes, such as lepidic-predominant adenocarcinoma.
Figure 2.2 Classification of Lung Carcinoma
The major lung cancer histologies are neuroendocrine, squamous cell, and adenocarcinoma. Within adenocarcinoma, the major histological subtypes include the preinvasive adenocarcinoma in situ (AIS), microinvasive adenocarcinoma; lepidic predominant adenocarcinoma (LPA), and solid adenocarcinoma. For patients with advanced adenocarcinoma with actionable molecular alterations in EGFR, ALK, or ROS1, treatment with targeted therapy is recommended.
LUNG CANCER GENETICS
Large-scale efforts to comprehensively evaluate the biology of lung cancer by high-throughput transcriptional analysis, sequencing, and copy number analysis has provided a catalog of genomic alterations that are associated with specific histological subtypes of lung cancer (Table 2.1) (8,13,14). For adenocarcinoma, the most common somatic mutations occur in KRAS, EGFR, and P53, with gene rearrangements of ALK, RET, and ROS1. For squamous cell carcinoma, mutations in P53 and PIK3CA are common, as is amplification of FGFR1. Small-cell carcinoma typically harbors mutations in RB and P53, with frequent amplification of MYC.
Figure 2.3 Adenocarcinoma Molecular Profiles Correlate With Histological Subtype Class
Independent studies (A–C) with different genomics analysis platforms all show that early-stage lung adenocarcinomas cluster into three major genomic subgroups that correlate with three major adenocarcinoma pathological subtypes (preinvasive adenocarcinoma in-situ and minimally invasive; lepidic predominant adenocarcinoma; solid adenocarcinoma).
Panel A, from Ref. (33), reprinted with permission of the American Thoracic Society, copyright © 2016 American Thoracic Society. Panel B, from Ref. (34), reprinted by permission from Macmillan Publishers Ltd. Panel C, from Ref. (35), reprinted with permission.
KRAS Mutation
KRAS mutations are the most common signal transduction pathway driver mutations in lung adenocarcinomas in Whites. RAS proteins mediate cellular proliferation in many growth factor signaling pathways, including the EGFR pathway. Oncogenic missense mutations in KRAS result in the loss of intrinsic GTPase activity that is required to return the KRAS protein to its inactive form, resulting in a loss of intrinsic negative-feedback of RAS activity and constitutive activation of KRAS (15). KRAS mutations localize to codons 12, 13, and 61, and are detectable in approximately 30% of lung adenocarcinomas. KRAS-mutant adenocarcinomas typically occur in smokers and in the central region of the lung. In smokers, tobacco carcinogens might specifically induce KRAS mutations with subsequent activation of proliferative signaling pathways, while in never smokers as yet unidentified carcinogens might selectively induce EGFR mutations, thus activating the EGFR signaling pathway (15).
KRAS codon 12 mutations have been associated with inferior disease-free survival and increased lung cancer mortality (16). Furthermore, KRAS mutations are associated with a lack of response to EGFR-targeted agents in both lung and colorectal cancer. Thus far, no targeted treatments have demonstrated consistent clinical efficacy against KRAS-mutant NSCLC.
EGFR Mutation
Unlike KRAS, other genetic alterations are actionable in that they predict response to molecularly targeted drugs (8). Advances in this field have provided proof-of-principle for the concept of oncogene addiction, that despite the diverse array of genetic lesions typical of cancer, some tumors rely on a single dominant oncogene for growth and survival such that inhibition of this specific oncogene is sufficient to kill the tumor (17). In lung cancer, the first successful example of this approach was the use of anti-EGFR tyrosine kinase inhibitors (TKIs) in patients whose tumors harbored mutations in the EGFR gene. EGFR mutations typically occur in the receptor tyrosine kinase domain, leading to constitutive activation of downstream signaling and hypersensitivity to EGFR-TKIs, such as gefitinib or erlotinib. The two most common EGFR sensitizing mutations are the L858R point mutation in exon 21 and in-frame deletions in exon 19, which account for greater than 90% of EGFR mutations found in lung cancer. These EGFR mutations are highly specific for lung adenocarcinoma and are mutually exclusive with other major lung cancer driver mutations, including KRAS, ALK, ROS1, RET, BRAF, and ERBB2/HER2. For reasons that have not been determined, the prevalence of EGFR mutations in lung cancer show significant ethnic variations, ranging from 10% to 15% in whites to 30% to 50% in East Asians. In patients with advanced EGFR-mutated NSCLC, response rates to EGFR-TKIs are 60% to 70%, significantly higher than the 30% response rate achieved with conventional chemotherapy. EGFR-TKIs also yield a significant improvement in progression-free survival in patients with EGFR-mutated tumors, and EGFR-TKIs are now the standard treatment for this molecularly defined patient subgroup.
ALK Rearrangement
The discovery of chromosomal rearrangements of the ALK gene locus in adenocarcinoma of the lung has led to similar advances in therapy. Although ALK aberrations are common in anaplastic large cell lymphoma, the specific EML4–ALK rearrangement is almost exclusively found in lung adenocarcinoma with an acinar or solid growth pattern. ALK rearrangements are found in approximately 4% of NSCLCs, without any ethnic variations in prevalence. ALK-rearranged NSCLCs are highly sensitive to treatment with ALK inhibitors, such as crizotinib. In randomized clinical trials, crizotinib has demonstrated significant improvements in response rate and progression-free survival in patients with ALK-rearranged NSCLC when compared to standard chemotherapy. Similarly, high response rates have been seen with crizotinib in patients with ROS1-rearranged NSCLC.
Resistance to Targeted Therapy
The widespread implementation of molecularly targeted therapy has resulted in significant clinical benefits for selected subgroups of patients with advanced adenocarcinoma of the lung. However, there are limitations to targeted therapy. Acquired resistance inevitably develops to first-line TKIs directed against EGFR and ALK. For EGFR-mutated tumors, the most common form of acquired resistance is the development of a secondary, EGFR T790M point mutation in 50% of patients who have disease progression on an EGFR-TKI. Other mechanisms contributing to resistance to EGFR-TKIs include overexpression of other oncogene products (e.g., MET), bypass signaling pathways mediated by heat shock protein or TGFβ signaling, and induction of cellular differentiation pathways leading to histologic transformation to small-cell carcinoma.
Second- and third-generation EGFR-TKIs have been developed in response to the need for agents to override acquired resistance. For example, osimertinib is a mutation-selective EGFR-TKI that specifically targets T790M-positive tumors. Similarly, ceritinib and alectinib have been developed to target secondary ALK mutations in patients whose cancer has progressed on crizotinib (Table 2.2) (18).
Rebiopsy of driver mutation-positive tumors upon disease progression can guide the use of second- and third-line therapy by identifying specific mechanisms of resistance. Secondary mutations can be identified from biopsies of actual tumor deposits (primary or metastatic) or from a “liquid biopsy” that evaluates circulating tumor DNA in the blood. Ongoing research is being directed toward the development of treatment approaches targeting other driver mutations found in lung cancer and the identification of strategies to overcome other mechanisms of acquired resistance.
EPIGENETICS
Epigenetic changes within the human genome, including chromatin remodeling, histone modification, and DNA methylation, can alter the expression of critical oncogenes and tumor suppressor genes and can have a profound impact on malignant transformation (19). Both global and gene-specific epigenetic changes are being evaluated as biomarkers for the early detection and prognosis of lung cancer.
Methylation
Hyper- and hypo-methylation of CpG islands within gene promoters is an epigenetic modification of DNA that can culminate in loss of gene transcription (20). Aberrant methylation of the promoter region of tumor suppressor genes, with resultant gene silencing, plays an important role in the pathogenesis of most types of human cancer, with individual tumor types having their own acquired pattern of methylation (21). In lung cancer, hypermethylation of CpG islands has been found in 15% to 80% of tumors, affecting many genes, including APC, p16, and RASSF1A. Techniques such as methylation-specific PCR and restriction landmark genomic scanning can be used to visualize different patterns of methylation that can distinguish tumor from normal tissue, and categorize clinically relevant subgroups. In sputum samples, the methylation pattern for a specific panel of genes has been reported to be predictive for the development of lung cancer among high-risk smokers (22,23).
Micro-RNA
miRNAs are small, noncoding RNA strands containing about 22 nucleotides that function in the transcriptional and posttranscriptional regulation of gene expression. It is estimated that miRNAs may regulate up to two-thirds of the human genome (19). More than 50% of miRNAs are located at cancer-related chromosomal regions, supporting the concept that miRNAs may act as oncogenes or tumor suppressor genes. The genome-wide expression profile of miRNAs is significantly different in primary lung cancers than in corresponding noncancerous lung tissues, and human lung cancers have extensive alterations of miRNA expression that may deregulate cancer-related genes. Thus, patterns of miRNA expression may be linked to relevant clinical parameters, such as prognosis and therapeutic response (24). For example, one study determined that expression of miRNA-21 consistently correlated with poor outcome in patients with early stage lung cancer (25), while another study suggested that patterns of plasma miRNA may be used for risk stratification in lung cancer screening programs (26). Overall, large-scale prospective trials will be required to establish the potential role of miRNA biomarkers in the clinical setting.
TUMOR MICROENVIRONMENT
The importance of cooperation between tumor cells and their surrounding microenvironment has been thoroughly reviewed by Hanahan and Weinberg (27,28). Tumor cells cooperate with surrounding cells to promote inflammation and to activate pathways involved in invasion, metastasis, and angiogenesis. Many of these processes represent potential targets for therapeutic interventions directed against tumor cells and/or the microenvironment. The tumor microenvironment is a complex system composed of stromal fibroblasts, macrophages, lymphocytes, other bone marrow-derived cells, and extracellular matrix that, in a reciprocal fashion, can contribute to tumor regression or progression.
TGFβ Signaling
The TGFβ signaling pathway mediates many of the interactions required for tumor cell progression (29). TGFβ, the ligand for the TGFβ-type II receptor (TGFβ-RII), is a family of pleiotropic cytokines (TGFβ 1, 2, 3) that regulate tissue homeostasis and prevent tumor initiation by inhibiting cellular proliferation, differentiation, and survival. TGFβ is secreted as a latent molecule and is activated by protease cleavage. TGFβ signaling primarily occurs through SMAD protein-dependent pathways in which binding to TGFβ-RII induces phosphorylation and activation of TGFβ-RI. TGFβ signaling may also proceed via less well defined SMAD-independent pathways. Depending upon context, TGFβ signaling may alternatively suppress tumor growth in early stage cancers or promote tumor cell invasion and metastasis in late-stage cancers. Both in vitro and in vivo models have demonstrated the importance of TGFβ signaling in the progression of lung adenocarcinoma from preinvasive lesions to invasive, metastatic tumors by mediating changes in both tumor cells and the stromal compartment.
Epithelial–Mesenchymal Transition
In order for epithelial tumor cells to metastasize, they must acquire mesenchymal cell properties, such as the loss of cell–cell adhesion, invasiveness, vascular intravasation and extravasation, and angiogenesis (30). This process of EMT involves numerous signaling pathways relevant to cancer, including those mediated by TGFβ, AKT, MEK-ERK, ZEB, SNAIL, FAS, and EZH2 (31). Studies in in vitro and in vivo model systems have demonstrated that both EMT within primary tumors and mesenchymal-to-epithelial transition (MET) within metastatic foci are required for lung cancer progression and metastasis.
Immune Checkpoints
The clinical significance of the immune cell component of the tumor microenvironment has been revealed by recent studies showing that the immune checkpoint mediator PD-L1, expressed by tumor cells, can bind to the PD-1 receptor on tumor infiltrating T-lymphocytes, resulting in T-cell inactivation (32). Through this mechanism, tumors can successfully evade immune surveillance. In 2015, two anti-PD-1 monoclonal antibodies, nivolumab, and pembrolizumab, were approved by the U.S. Food and Drug Administration (FDA) as second-line treatment for NSCLC after progression on standard chemotherapy (Table 2.2) (18). The response rate to these immune checkpoint inhibitors is approximately 20%, but many responses are durable and both agents have demonstrated an improvement in overall survival over standard second-line chemotherapy. Current research is focused on the identification of biomarkers to predict which patients will benefit the most from PD-1/PD-L1 targeted therapy. Thus far, PD-L1 expression by immunohistochemistry has yielded conflicting data, but the response to immune checkpoint inhibitors does appear to be directly correlated with the mutational load of the tumor, likely as a surrogate for increased neoantigen expression.
