2.1. Main Driver Genes
The main druggable genetic alterations in non-small cell lung cancer involved EGFR (epidermal growth factor receptor), KRAS (Kirsten rat sarcoma viral oncogene homolog), ALK (anaplastic lymphoma kinase), BRAF (V-raf murine sarcoma oncogene homolog B1) and ROS1 (c-ros oncogene 1) genes.
EGFR is a transmembrane protein with intrinsic tyrosine kinase activity, crucial for regulating cell proliferation, survival, and differentiation. Activating mutations in the EGFR gene were first identified in non-small cell lung cancers (NSCLC) in 2004, marking one of the most significant advancements in understanding the molecular biology of these tumors [
54]. EGFR gene mutations in lung tumors are primarily found in exon 19 (deletions) and exon 21 (L858R substitution) [
55]. Specifically, these mutations lead to constitutive activation of the receptor's tyrosine kinase, independent of ligand presence, promoting growth, survival, and cell proliferation through downstream signaling pathways such as PI3K/AKT and RAS/RAF/MEK/ERK. EGFR mutations are more common in women, non-smokers, patients with adenocarcinoma histology, and Asian patients [
56,
67]. In these populations, EGFR mutation prevalence can reach up to 50%, whereas in Caucasian patients it ranges around 10-15% [
55,
56]. The discovery of activating mutations of the epidermal growth factor receptor (EGFR) in patients with lung adenocarcinoma led to the development of a new family of biological agents, called tyrosine kinase inhibitors (TKIs), that have revolutionized the clinical management of LC patients.
Mutations in the KRAS gene are among the most common genetic alterations in lung cancers, particularly in NSCLC [
65,
66]. KRAS is a proto-oncogene that encodes a GTPase protein, which is involved in signal transduction that influences cell proliferation, differentiation, and survival. Under normal conditions, the KRAS protein alternates between an active (GTP-bound) state and an inactive (GDP-bound) state [
64,
66]. Mutations, however, result in a constitutively active protein that continuously stimulates downstream signaling pathways, such as the MAPK and PI3K-AKT pathways, promoting uncontrolled growth of cancer cells [
65,
66]. KRAS mutations are present in approximately 20-25% of NSCLC cases, with a higher prevalence in patients with adenocarcinoma compared to those with other histological subtypes, suggesting a strong correlation with tobacco exposure [
67]. The most common types of mutations in lung cancers are primarily concentrated in codons 12, 13, and 61, with G12C, G12D, and G12V being the most frequent among these [
68]. Each specific mutation can differently influence tumor biology and treatment response, generally leading to an unfavorable prognosis for the patient [
69].
The ALK gene encodes a receptor tyrosine kinase that is involved in the regulation of cell growth. Originally identified in anaplastic large cell lymphomas [
57], ALK alterations are now also recognized in various solid tumors, including lung cancers [
58]. ALK mutations in lung cancers are often the result of genetic rearrangements, leading to the formation of fusion proteins with oncogenic activity [
58]. In lung cancers, the most common rearrangement involves the fusion of the ALK gene with EML4 (echinoderm microtubule-associated protein-like 4). This rearrangement, known as EML4-ALK, was first identified in 2007 and results in the production of a fusion protein with constitutive tyrosine kinase activity, promoting cell proliferation and tumor survival [
58,
59]. This translocation accounts for about 5% of lung carcinomas, and among the multiple genetic alterations involved in the development of these tumors, it has emerged as an important biomarker [
63]. In addition to the known rearrangement with EML4, other ALK fusion partners have been identified, including KIF5B, TFG, and KLC1 [
60,
61,
62]. Although these rearrangements are less common, they still contribute to the pathogenesis of lung cancer through similar mechanisms of aberrant activation of the ALK pathway. ALK-positive NSCLC exhibits highly aggressive behavior and is often diagnosed at advanced stages compared to wild-type patients.
Despite BRAF gene mutations in lung carcinoma being identified before EML4-ALK translocations, there have been few clinical studies completed on this type of lung carcinoma with BRAF mutation. To date, BRAF gene mutations are recognized as one of the genetic factors contributing to the development of various types of cancer, including lung cancer [
71]. BRAF is a gene that encodes a serine/threonine kinase protein, which is part of the MAPK/ERK signaling pathway involved in the regulation of cell growth, differentiation, and survival [
72]. Mutations in this gene can lead to abnormal cell signaling and, consequently, uncontrolled proliferation of cancer cells. Specifically, conformational changes occur in the BRAF protein, causing constitutive activation of the MAPK/ERK signaling pathway [
73]. This persistent activation stimulates cell proliferation, inhibits apoptosis, and promotes the survival of cancer cells. Several BRAF mutations have been identified, the most common being the substitution from valine to glutamate at codon 600 (V600E), which accounts for over 90% of BRAF mutations in melanoma [
74]. This mutation, in particular, creates a form of BRAF that is independent of upstream regulatory signals, leading to incessant kinase activity. BRAF gene mutations are present in a minority of lung cancer cases, with an estimated prevalence of about 1-3% in NSCLC. These mutations have been identified mainly in adenocarcinoma subtypes [
75]. BRAF gene mutations can be classified into three main classes. Class I BRAF mutations are commonly identified in human tumors and represent about 50% of BRAF mutations in lung tumors [
75,
76]. They affect the V600 amino acid, including V600D/E/K/R, and act as active monomers independent of RAS, resulting in marked activation of BRAF kinase activity and constant activation of the MAPK pathway [
77]. Class II and Class III mutations, on the other hand, are non-V600 mutations and represent 50-80% of BRAF mutations in lung tumors [
75]. Class II mutations are mainly found in the activation segment (such as K601, L597) or in the P-loop (such as G464, G469) [
78,
79], and have intermediate to high kinase activity. Class III mutations have been found in the P-loop (G466), the catalytic loop (N581), and the DFG motif (D594, G596) [
78,
79], and are characterized by absent or low kinase activity. In terms of prognosis, class I BRAF mutations generally show a slightly better prognosis compared to class II and III mutations. The latter are linked to more aggressive behavior, a less favorable clinical course, and earlier disease progression following initial chemotherapy. BRAF mutations globally exhibit a significant predominance among males (61%) and individuals who have a history of smoking (81%), with varying frequencies across different mutational classes.
The ROS1 gene encodes a receptor tyrosine kinase involved in the regulation of cell growth and intracellular signaling. Initially, it was found to be implicated in the development of human glioblastoma [
80]. and was later recognized in other malignant neoplasms, including lung, ovarian, and gastric tumors [
81,
82,
83]. ROS1 gene rearrangements are described in 1-2% of patients with NSCLC and were first identified in 2007 [
80,
81]. The majority of recorded patients are young, light smokers, or non-smokers. These rearrangements are more frequent in adenocarcinoma but have also been reported in large-cell carcinoma cases [
84,
85]. ROS1 mutations are primarily represented by gene translocations, where a part of the ROS1 gene fuses with another gene, creating a fusion oncogene. Among the most common translocations are fusions between ROS1 and the genes CD74, SLC34A2, TPM3, and SDC4 [
81,
84,
86,
87]. These fusions result in the production of potent oncogenic drivers that promote cell proliferation, activation, and cell cycle progression by activating downstream signaling pathways, accelerating the development and progression of lung carcinoma due to the upregulation of JAK/STAT, PI3K/AKT, and MAPK/ERK signaling pathways [
88]. Despite the low frequency of ROS1 gene mutations, their identification is clinically significant as patients harboring this mutation tend to respond well to specific tyrosine kinase inhibitors [
89]. This ensures that the therapeutic choice for the patient is targeted and precise.
2.2. Emerging Biomarkers
Lung cancer is a heterogeneous disease that requires precise patient stratification to optimize treatment options. In recent years, specific biomarkers such as PD-L1 (Programmed death-ligand 1), MET (Mesenchymal Epithelial Transition), RET, NTRK, PIK3CA, HER2 (human epidermal growth factor receptor) and STK11 have gained attention as crucial tools for the diagnosis, prognosis, and therapeutic management of lung cancer [
70].
Programmed death-ligand 1 (PD-L1), also known as CD274, is considered an immune checkpoint that facilitates the suppression of the antitumor immune response. PD-L1 can be present on the surface of various cells, including macrophages, antigen-presenting cells, B and T lymphocytes, epithelial cells, muscle cells, and endothelial cells [
90]. The factor inducing its expression is interferon-gamma (IFN-γ), released by CD8 T cells [
91]. The receptor of PD-L1 (PD-1) is primarily expressed by activated cytotoxic T cells [
90]. When PD-L1 binds to the PD-1 receptor on activated T cells, immune system suppression occurs [
90,
92]. This interaction prevents autoimmune responses in peripheral tissues during inflammations, contributing to maintaining immunological tolerance [
93]. The ligand-receptor complex triggers two reactions that inhibit the immune response: the first is the inhibition of interleukin 2 (IL-2) synthesis [
94], and the second is related to the inhibition of the T cell receptor, known as the "stop signal," which can modify the duration of contact between T cells and target cells or antigen-presenting cells [
95]. The PD-L1-PD-1 interaction is exploited in carcinogenesis to evade the immune system [
96]. Elevated levels of PD-L1 have been detected on the surface of various tumor cell types, including NSCLC. This mechanism allows tumor cells to escape the immune response; PD-L1 acts as a pro-tumorigenic factor by activating proliferative and survival signaling pathways, also promoting tumor progression [
97]. In lung tumors, PD-L1 expression can be regulated by various mechanisms, including genetic mutations, gene amplifications, and transcriptional regulation induced by inflammation or hypoxia. PD-L1 overexpression has been associated with a worse prognosis and greater tumor aggressiveness [
97]. Mutations in the PD-L1 gene in lung tumors are relatively rare compared to other mechanisms of overexpression. However, some mutations can significantly impact PD-L1 function and the immune response. Specifically, point mutations in PD-L1 have been found that can influence its ability to interact with PD-1, altering the antitumor immune response. Some studies have identified that these mutations lead to a loss of function or stabilization of the protein [
98]. Additionally, amplifications of the 9p24.1 locus, where PD-L1 resides, have been observed in a fraction of lung tumors. These amplifications are often correlated with high PD-L1 expression and have been associated with an unfavorable response to PD-1/PD-L1 inhibitor therapies [
99,
100]. Finally, although rare, gene translocations involving PD-L1 can lead to the fusion of PD-L1 with other genes, influencing its expression and function. These translocations can generate chimeric isoforms with new functional properties for tumor cells [
101]. Due to the clinical relevance in the context of immunotherapy observed in lung tumors, the molecular bases of PD-L1 regulation and its role in the tumor microenvironment are still being explored. The aim is to understand hidden molecular mechanisms to identify new therapeutic targets and treatment combinations that can improve patient outcomes [
102].
The MET gene encodes a tyrosine kinase receptor known as hepatocyte growth factor receptor (HGFR). This receptor plays a crucial role in regulating cell proliferation, motility, morphogenesis, and survival [
103]. Upon binding to its ligand HGF, MET undergoes dimerization and auto-phosphorylation, activating important intracellular signaling pathways such as PI3K/AKT and MAPK/ERK [
104]. Alterations in the MET gene, including mutations, amplifications, and fusions, are implicated in the pathogenesis of various cancers, including lung cancers [
103,
104,
105]. Point mutations in the tyrosine kinase domain of MET can lead to constitutive activation of the receptor, promoting the growth and survival of tumor cells [
106]. Among these, the most studied is the MET exon 14 skipping mutation (METex14), which prevents receptor degradation and results in prolonged MET signaling [
107]. MET exon 14 skipping mutations occur in approximately 3-4% of NSCLC cases and are generally not associated with other driver mutations [
108,
109,
110]. Amplification of the MET gene, detected in 1-6% of NSCLC patients, leads to an increased number of gene copies and overexpression of the MET receptor with aberrant signaling. This amplification is often observed in cases of acquired resistance to treatments with epidermal growth factor receptor inhibitors [
110,
111,
112,
113,
114]. Recently, rearrangements of the MET gene, including KIF5B-MET fusion, have been identified and although rare, have shown significance in NSCLC. These fusions are present in approximately 0.2% - 0.3% of NSCLC cases and are considered potential driver mutations targetable by specific therapies [
115,
116,
117]. In conclusion, MET gene alterations are associated with poor prognosis and resistance to standard treatments in lung cancers. Therefore, detecting these alterations is critically important for the therapeutic management of patients [
118].
The proto-oncogene RET, first identified in 1985 [
119], encodes a receptor tyrosine kinase that activates downstream signaling pathways (RAS/MAPK/ERK, PI3K/AKT, and phospholipase C-γ), thereby promoting cellular proliferation, migration, and differentiation [
120]. Genetic alterations, including chromosomal rearrangements and point mutations, lead to aberrant RET activation, contributing to tumorigenesis. Specifically, chromosomal rearrangements of RET have been found in approximately 1%-2% of NSCLC patients [
87,
121]. These alterations result in the formation of chimeric proteins with constitutive oncogenic activity, stimulating downstream signaling pathways that promote tumor cell proliferation and survival [
122]. The most commonly identified rearrangement in NSCLC is the KIF5B-RET fusion [
87,
123,
124], although other fusion partners such as CCDC6, NCOA4, TRIM33, and CUX1 have also been identified [
125]. Patients with RET fusion-positive NSCLC represent a distinct molecular subgroup with specific clinical and pathological characteristics. RET gene fusions appear to be mutually exclusive with other mutations, including those in EGFR, KRAS, ALK, HER2, and BRAF genes, suggesting independent oncogenic driver roles. Furthermore, initial reports indicate that RET rearrangements are more frequent among younger (< 60 years old), female, non-smoking patients with adenocarcinoma histology [
87,
123,
124,
126,
127]. In addition to rearrangements, point mutations in the RET gene have also been identified in lung tumors, albeit less commonly than fusions. Similar to rearrangements, these mutations can lead to RET receptor activation, contributing to oncogenesis [
22]. Therefore, RET gene mutations represent a significant class of oncogenic alterations in lung tumors, and understanding their molecular mechanisms is facilitating the development of new targeted inhibitors, advancing therapies for RET-positive patients.
Mutations in NTRK genes are emerging as significant molecular markers in lung cancers, influencing diagnosis, prognosis, and therapeutic options [
128]. The NTRK1, NTRK2, and NTRK3 genes encode the TRKA, TRKB, and TRKC receptors, respectively. These transmembrane proteins belong to the TRK receptor family and play a crucial role in cellular signaling and neural growth by activating pathways such as PIK3/PLCγ/MAPK [
129,
130]. NTRK gene fusions are rare but frequent oncogenic alterations, present in up to 1% of all solid tumors [
131]. In NSCLC, the frequency of these fusions is approximately 0.1-0.2%, occurring when the 3' sequence of the NTRK gene fuses with the 5' sequence of a fusion partner gene [
132]. The resulting fusion protein is aberrantly expressed, constitutively activating the receptor's kinase domain, which leads to persistent activation of cellular signaling pathways necessary for oncogenesis [
131,
133]. NTRK gene fusions typically exclude other canonical oncogenic mutations, suggesting they act as sole oncogenic drivers in the development and maintenance of their host tumors [
134]. Most patients with lung cancers harboring NTRK gene fusions exhibit clinical characteristics similar to those with ALK, RET, or ROS1 fusions [
132], and are often found in a younger population with minimal or no smoking history. However, studies have also identified NTRK gene fusions in patients of various ages and with a previous smoking history [
132,
135]. Additionally, many patients with TRK fusion-positive lung carcinoma have developed metastases in the central nervous system [
136]. Generally, NTRK gene mutations in lung cancers can be associated with variable prognoses. Some studies suggest that patients with these mutations may benefit from targeted therapies with NTRK inhibitors, as these inhibitors block aberrant tyrosine kinase activity, thereby reducing tumor growth and improving clinical response [
137]. Despite current knowledge, further studies are necessary to evaluate the exact incidence of NTRK gene mutations in different populations and to better understand the clinical implications of these mutations. These mutations represent a promising field for the personalization of therapy in NSCLC patients.
The PIK3CA gene encodes a catalytic subunit of phosphatidylinositol-3-kinase (PI3K), a class of enzymes involved in numerous cellular processes, including cell growth, proliferation, survival, and metabolism. Mutations in the PIK3CA gene have been implicated in various types of cancer [
138], including lung cancer, with a frequency of 2-4% in NSCLC cases [
138,
139]. PIK3CA is often mutated or amplified due to a missense variant that mainly affects the helical binding domain (exon 9, E545K or E542K) or the catalytic subunit (exon 20, H1047R or H1047L) [
138,
140,
141]. These alterations lead to constitutive and PI3K/AKT/mTOR pathway-independent PI3K enzymatic activation, resulting in uncontrolled proliferation and survival of cancer cells with consequent drug resistance [
142]. PIK3CA gene mutations in lung adenocarcinomas have not been reported as mutually exclusive. On the contrary, co-occurrence with alterations in EGFR, BRAF, ALK, and more frequently, KRAS genes has been observed. This observation raises the question of whether PIK3CA mutation alone can be a sufficient oncogenic driver for NSCLC tumor formation [
143,
144]. PIK3CA mutations have been reported to be associated with smoking exposure. Specifically, patients with PIK3CA mutation exhibit greater smoking exposure than patients with EML4-ALK translocation or EGFR mutation, and less smoking exposure than patients with smoking-associated aberrations such as KRAS [
145,
146]. Additionally, PIK3CA mutation has been observed to occur more frequently in patients with various prior malignancies compared to NSCLC [
146]. The presence of PIK3CA mutations in lung cancer has several clinical implications, including worse prognosis due to increased tumor aggressiveness and metastatic potential [
142,
145]. Furthermore, PIK3CA/EGFR co-mutation has been associated with reduced efficacy of EGFR inhibitors, thus necessitating alternative or combinatorial therapeutic strategies [
147]. This underscores the need for further studies to ensure a deeper understanding of molecular mechanisms to improve NSCLC treatment.
Mutations in the HER2 gene in lung tumors are a topic of growing interest in oncological research. HER2, also known as ERBB2, is an oncogene that encodes a tyrosine kinase receptor involved in the regulation of cell growth, differentiation, migration, and apoptosis [
148]. Although HER2 alterations are well documented in various types of cancers, such as breast carcinoma, their role in lung tumors has only recently started to emerge [
149]. HER2 mutations are relatively rare, found in approximately 1-3% of NSCLC, and are observed more frequently in adenocarcinomas compared to other NSCLC subtypes [
22,
148,
149,
150]. HER2 alterations in lung tumors can primarily manifest through two mechanisms: gene amplification and point mutations. Amplification of the HER2 gene leads to overexpression of the HER2 protein on the cell surface, promoting uncontrolled cell proliferation and tumor cell survival [
151,
152]. The point mutations described to date are insertions within a small stretch of exon 20 with A775_G776insYVMA insertion/duplication at the COOH-terminal end of the αC-helix; these can increase the receptor's enzymatic activity independently of ligand presence, leading to constant proliferative signaling [
151,
152,
153,
154]. HER2 mutations in lung tumors are predominantly observed in female, non-smoking patients and are often associated with an unfavorable prognosis [
150,
153,
155]. Patients with these mutations tend to present with more aggressive tumors and a less favorable response to standard therapies [
154,
155]. For this reason, it is necessary to further investigate HER2 mutations which, although rare, are emerging as mutations of particular interest, especially for the development and progression of adenocarcinoma. Further studies might indeed highlight HER2 as a promising and relevant therapeutic target.
Mutations in the STK11 gene, also known as LKB1, represent a tumor suppressor gene implicated in the autosomal dominant disorder that predisposes individuals to cancer known as Peutz-Jeghers syndrome (PJS) [
156]. Located on chromosome 19p13.3, the STK11 gene encodes a protein that functions as a kinase involved in the regulation of energy metabolism, cell growth, apoptosis, and cell polarity. Disruption of these processes is implicated in carcinogenesis. STK11 mutations have been identified as significant genetic events in various cancer types, notably within the context of NSCLC [
157,
158]. STK11 mutations are recorded in approximately 20-30% of NSCLC cases, with a higher prevalence in adenocarcinoma subtypes compared to squamous cell carcinomas, and they deactivate the LBK1 protein [
159]. LKB1 is a key regulator of the AMPK (AMP-activated protein kinase) pathway, a crucial pathway for cellular responses to energy metabolism and stress. It is one of the few known serine/threonine kinases to be inactivated, which implicates dysfunction of the AMPK pathway, leading to dysregulated cell growth and survival under energy-stress conditions [
160]. This contributes to the uncontrolled proliferation of cancer cells. STK11 mutations are often associated with a more aggressive tumor phenotype and a poorer prognosis [
161]. Additionally, STK11 mutations can influence therapeutic responses. For instance, tumors harboring STK11 mutations tend to exhibit intrinsic resistance to immune checkpoint inhibitors, such as anti-PD-1/PD-L1 antibodies [
161,
162,
163]. This resistance may be due to the decreased presence of tumor-infiltrating lymphocytes (TILs) and a less inflammatory tumor microenvironment, defined as a "cold" immunosuppressive microenvironment [
165,
166]. Despite the complexity and critical nature of STK11 mutations, ongoing research continues to explore the interactions between STK11 and other molecular pathways to identify additional therapeutic targets and optimize treatment combinations.