Non-small cell lung cancer exhibits substantial histological and molecular heterogeneity. Alterations involving EGFR, ALK, ROS1, KRAS, BRAF, RET, MET, HER2, and other oncogenic drivers can continuously activate tumor-cell proliferation and survival signaling and generate molecular subtypes with distinct biological characteristics and drug sensitivities.

Keywords: non-small cell lung cancer; molecular classification; EGFR; ALK; ROS1; KRAS; BRAF; RET; MET; HER2; PIK3CA; immune checkpoint; targeted therapy; resistance mechanism

1 Pathological Types and Molecular Characteristics of Non-Small Cell Lung Cancer

1.1 Major Pathological Types

Non-small cell lung cancer (NSCLC) accounts for approximately 85% of lung cancers and mainly includes lung adenocarcinoma, lung squamous cell carcinoma, and large-cell carcinoma. Receptor tyrosine kinase mutations, gene fusions, and abnormalities in the RAS-MAPK pathway are relatively common in lung adenocarcinoma, whereas TP53 alterations, cell-cycle dysregulation, and chromosomal copy-number changes are more frequent in lung squamous cell carcinoma.

1.2 Oncogenic Driver Alterations

Oncogenic driver alterations allow tumor cells to maintain proliferation and survival signaling in the absence of normal extracellular stimulation. Representative driver alterations in NSCLC include EGFR mutations, ALK and ROS1 fusions, KRAS and BRAF mutations, RET fusions, MET exon 14 skipping, and HER2 mutations. Different driver alterations generally define relatively independent molecular subtypes, although co-mutations and resistant subclones may emerge during tumor evolution or under the selective pressure of targeted therapy.

1.3 Major Forms of Molecular Alteration

Molecular abnormalities in NSCLC include point mutations, short insertions or deletions, exon skipping, gene fusions, and gene amplification. Point mutations and insertions or deletions can alter protein conformation or kinase activity. Exon skipping can affect receptor stability. Gene fusions can induce constitutive kinase activation through abnormal dimerization, whereas gene amplification increases target-protein expression and signaling output. Different forms of alteration correspond to different detection methods and drug sensitivities, and their research significance cannot be determined solely from the gene name.

Table 1 Major Molecular Alterations in NSCLC and Their Functional Characteristics

2 EGFR Mutations and Targeted Intervention

2.1 EGFR Signaling Activation

Epidermal growth factor receptor (EGFR) belongs to the ErbB receptor tyrosine kinase family. After ligand binding to the extracellular domain, EGFR forms homodimers or heterodimers, activates its intracellular tyrosine kinase domain, and undergoes autophosphorylation. This initiates RAS-RAF-MEK-ERK, PI3K-AKT-mTOR, JAK-STAT, and related pathways that regulate cell proliferation, survival, and differentiation.

2.2 Common EGFR Mutations

EGFR exon 19 deletions and exon 21 L858R are the most common classic sensitizing mutations in NSCLC. These alterations enhance kinase activity and create dependence of tumor cells on EGFR signaling. EGFR exon 20 insertions, G719X, L861Q, S768I, and other abnormalities have different structural characteristics and drug sensitivities. The specific mutation site should therefore be defined rather than treating all EGFR mutations as a single category.

2.3 EGFR Tyrosine Kinase Inhibitors

(1) First-Generation EGFR Inhibitors

Gefitinib and Erlotinib reversibly occupy the ATP-binding site of the EGFR kinase domain and inhibit receptor phosphorylation and downstream signaling. Both agents have relatively high activity against EGFR exon 19 deletions and L858R mutations, but acquired resistance frequently develops during treatment.

(2) Second-Generation EGFR Inhibitors

Afatinib and Dacomitinib form irreversible covalent bonds with EGFR, HER2, and HER4 and inhibit signaling across multiple ErbB-family receptors. Second-generation inhibitors are active against classic EGFR sensitizing mutations, although their selectivity and tolerability remain limited for certain resistance mutations.

(3) Third-Generation EGFR Inhibitors

Osimertinib selectively inhibits classic sensitizing EGFR mutations and the T790M resistance mutation and has relatively favorable central nervous system activity. Compared with earlier agents, third-generation inhibitors exert weaker inhibition of wild-type EGFR, although new on-target mutations and bypass resistance mechanisms can still emerge after treatment.

2.4 Acquired Resistance to EGFR-Targeted Therapy

(1) On-Target Mutations

The T790M mutation may emerge after treatment with first- or second-generation EGFR inhibitors. This mutation alters the structure near the ATP-binding site and reduces drug inhibition. After Osimertinib treatment, abnormalities such as C797S may arise and disrupt covalent binding between the drug and EGFR.

(2) Bypass-Signaling Activation

MET amplification, HER2 amplification, ALK or RET fusion, and enhanced AXL signaling can reactivate PI3K-AKT or MAPK pathways after EGFR inhibition, reducing tumor-cell dependence on EGFR signaling.

(3) Downstream Pathway Alterations

Mutations in KRAS, BRAF, and PIK3CA can directly activate MAPK or PI3K-AKT signaling and maintain downstream proliferation and survival signals despite EGFR inhibition.

(4) Histological and Phenotypic Transformation

Some EGFR-mutant lung adenocarcinomas can transform into small-cell lung cancer or lung squamous cell carcinoma or undergo epithelial-mesenchymal transition. Histological transformation generally requires confirmation by post-progression tissue biopsy, because liquid biopsy alone cannot fully reflect changes in cellular lineage.

3 ALK Fusions and Targeted Intervention

3.1 Formation of ALK Fusion Proteins

Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase. The most common ALK abnormality in NSCLC is the EML4-ALK fusion generated by an inversion on chromosome 2. The fusion protein retains the ALK kinase domain, while EML4 or another fusion partner provides oligomerization capacity, resulting in constitutive ALK activation in the absence of ligand.

Figure 1 Formation of EML4–ALK fusion protein and activation of downstream signaling pathways

3.2 ALK Downstream Signaling

ALK fusion proteins activate the RAS-MAPK, PI3K-AKT, and JAK-STAT pathways and promote tumor-cell proliferation, survival, and migration. ALK fusions are generally mutually exclusive with classic EGFR or KRAS driver mutations, although coexisting abnormalities may occur in a small proportion of untreated tumors or in resistant subclones emerging after therapy.

3.3 ALK Inhibitors

Crizotinib inhibits ALK, ROS1, and MET but is limited by insufficient central nervous system exposure and acquired resistance. Ceritinib, Alectinib, and Brigatinib have greater ALK inhibitory activity and different levels of brain penetration. Lorlatinib can inhibit multiple resistance mutations within the ALK kinase domain. Different ALK inhibitors vary in their mutation coverage and central nervous system activity.

3.4 ALK Resistance Mechanisms

Resistance to ALK inhibitors may be mediated by kinase-domain mutations such as L1196M and G1202R, ALK gene amplification, activation of EGFR or MET bypass signaling, or reactivation of downstream pathways. Sequential treatment with different ALK inhibitors may also generate compound mutations in which multiple substitutions occur on the same ALK allele, further complicating drug selection.

4 ROS1 Fusions and Targeted Intervention

4.1 ROS1 Fusion Proteins

ROS1 is a receptor tyrosine kinase. ROS1 abnormalities in NSCLC are mainly gene fusions, with common partners including CD74, SLC34A2, EZR, SDC4, and TPM3. Fusion proteins retain the ROS1 kinase domain, while the fusion partner provides dimerization capacity. Different fusion partners may also affect subcellular localization, protein stability, and signaling intensity.

4.2 ROS1 Downstream Signaling

ROS1 fusions constitutively activate the RAS-MAPK, PI3K-AKT, and JAK-STAT pathways and promote tumor-cell proliferation, survival, and migration. Because ROS1 fusions create a relatively well-defined oncogenic dependency, ROS1 fusion-positive NSCLC is generally considered a distinct molecular subtype.

4.3 ROS1 Inhibitors

Crizotinib inhibits ROS1 fusion kinases and is used in studies of ROS1 signaling and drug sensitivity. Ceritinib, Entrectinib, and other next-generation ROS1 inhibitors differ in kinase selectivity, central nervous system activity, and coverage of resistance mutations.

4.4 ROS1 Resistance Mechanisms

ROS1 G2032R is a representative solvent-front mutation that alters the drug-binding pocket and reduces the binding of multiple early-generation ROS1 inhibitors. Other resistance mechanisms include kinase-domain mutations such as D2033N and L2026M, ROS1 amplification, bypass-receptor activation, and restoration of downstream MAPK signaling.

5 KRAS and BRAF Mutations

5.1 KRAS Signaling Activation

KRAS is a small GTPase located downstream of receptor tyrosine kinases and normally cycles between a GTP-bound active state and a GDP-bound inactive state. Oncogenic mutations reduce GTP hydrolysis and maintain KRAS in an active conformation, thereby activating downstream pathways including RAF-MEK-ERK, PI3K-AKT, and RalGEF-Ral.

5.2 KRAS G12C Mutation

KRAS G12C is a relatively common KRAS mutation in NSCLC. The mutation introduces a cysteine residue at position 12, providing a binding site for covalent small-molecule inhibitors. Sotorasib and Adagrasib bind the Switch II pocket of KRAS G12C and stabilize the protein in its GDP-bound state. Resistance may subsequently arise through secondary KRAS mutations, gene amplification, feedback activation of receptor tyrosine kinases, or reactivation of MAPK signaling.

5.3 BRAF Mutations

BRAF is a serine/threonine protein kinase downstream of RAS. BRAF mutations can constitutively activate MEK-ERK signaling and promote tumor-cell proliferation. BRAF V600E exhibits strong RAS-independent monomeric kinase activity, whereas non-V600 mutations may involve kinase dimerization, reduced kinase activity, or RAS-dependent signaling. Different BRAF mutation classes should therefore be evaluated separately.

5.4 BRAF-Targeted Intervention

Dabrafenib and Vemurafenib inhibit BRAF V600-mutant proteins. BRAF inhibition alone may induce feedback restoration of MAPK signaling, so BRAF inhibitors are commonly combined with MEK inhibitors to further reduce ERK phosphorylation and delay pathway reactivation.

6 RET, MET, HER2, and PIK3CA Alterations

6.1 RET Fusions

RET is a receptor tyrosine kinase. Common RET fusions in NSCLC include KIF5B-RET and CCDC6-RET. Fusion partners mediate abnormal dimerization of the RET kinase domain and constitutively activate MAPK, PI3K-AKT, and JAK-STAT signaling. Vandetanib and Cabozantinib are multitarget inhibitors, whereas selective RET inhibitors more directly block RET fusion signaling and reduce nonspecific inhibition of other kinases.

6.2 MET Alterations

After binding hepatocyte growth factor, MET activates MAPK, PI3K-AKT, STAT, and SRC-FAK pathways and promotes tumor-cell proliferation, migration, invasion, and epithelial-mesenchymal transition. MET exon 14 skipping reduces receptor ubiquitination and degradation and prolongs MET signaling. MET amplification increases gene copy number and protein expression and thereby enhances signaling output. These alterations arise through different mechanisms and have distinct detection criteria and should be evaluated separately.

6.3 HER2 Alterations

HER2 belongs to the ErbB receptor family and can dimerize with EGFR, HER3, or HER4 to activate MAPK and PI3K-AKT signaling. HER2 abnormalities in NSCLC include kinase-domain mutations, gene amplification, and protein overexpression, with exon 20 insertion being a relatively common mutation type. HER2 amplification may also contribute to acquired resistance to EGFR inhibitors. However, HER2 mutation, amplification, and overexpression are distinct biomarkers and cannot substitute for one another.

6.4 PIK3CA Mutations

PIK3CA encodes the p110α catalytic subunit of class IA phosphatidylinositol 3-kinase. Activating mutations increase phosphatidylinositol-3,4,5-trisphosphate production and enhance AKT-mTOR signaling. In NSCLC, PIK3CA mutations may coexist with other driver alterations and may also participate in bypass resistance to EGFR inhibitors. Their function should be evaluated in the context of co-mutations, AKT and mTOR phosphorylation, and pathway-feedback changes.

7 Immune Checkpoints and Immune Evasion in NSCLC

7.1 PD-1 and PD-L1

Programmed cell death protein 1 (PD-1) is mainly expressed on activated or functionally exhausted T cells, whereas PD-L1 can be expressed on tumor cells and multiple immune-cell populations. Binding of PD-1 to PD-L1 suppresses T-cell receptor and costimulatory signaling, reduces T-cell proliferation, cytokine release, and cytotoxicity, and thereby promotes tumor immune escape.

7.2 CTLA-4

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) competes with CD28 for binding to CD80 and CD86 on antigen-presenting cells and thereby limits early T-cell activation. CTLA-4 blockade can enhance T-cell priming and clonal expansion but may also increase immune-related adverse effects.

7.3 Immune Checkpoint Inhibitors

PD-1 inhibitors include Pembrolizumab, Nivolumab, and Cemiplimab. PD-L1 inhibitors include Atezolizumab, Durvalumab, and Avelumab. CTLA-4 inhibitors include Ipilimumab. PD-L1 expression can be used for immunotherapy stratification but cannot independently predict treatment response. Driver-gene status, tumor mutational burden, antigen-presentation capacity, and the immune microenvironment can all affect therapeutic outcomes.

8 Resistance Mechanisms and Experimental Evaluation of NSCLC-Targeted Therapy

8.1 Classification of Resistance Mechanisms

The major resistance mechanisms to targeted therapy in NSCLC include on-target mutations, amplification of the target or bypass genes, activation of alternative receptors, restoration of downstream signaling, histological transformation, and insufficient drug distribution within tissues. Different resistance mechanisms may coexist in different lesions or subclones. Therefore, results from a single lesion or a single time point may not fully represent tumor heterogeneity.

Table 2 Major Resistance Mechanisms in NSCLC-Targeted Therapy

8.2 Molecular Testing

(1) DNA and RNA Testing

DNA sequencing is suitable for detecting point mutations, short insertions or deletions, and certain copy-number alterations involving EGFR, KRAS, BRAF, PIK3CA, and related genes. RNA sequencing directly detects abnormal fusion transcripts and is more suitable for analysis of ALK, ROS1, RET, NTRK, and other gene fusions.

(2) Protein- and Chromosome-Level Testing

Immunohistochemistry can be used to evaluate protein expression of ALK, ROS1, PD-L1, MET, and related targets. Fluorescence in situ hybridization can assist in confirming gene rearrangements and amplifications. Because these methods detect different events at the DNA, RNA, protein, or chromosomal level, discordant results should be interpreted according to sample quality and assay principles.

8.3 Tissue Biopsy and Liquid Biopsy

(1) Tissue Biopsy

Tissue biopsy can simultaneously evaluate molecular alterations, protein expression, and histological transformation and is important for confirming transformation into small-cell lung cancer, squamous cell carcinoma, and other morphological changes. However, tissue biopsy is affected by lesion location, sample volume, and spatial tumor heterogeneity.

(2) Liquid Biopsy

Plasma circulating tumor DNA testing is suitable for dynamic monitoring of mutation profiles, fusion events, and resistant clones. A negative plasma result does not completely exclude an alteration, because low tumor burden, disease confined to the thorax, and central nervous system lesions may all reduce circulating tumor DNA detection.

8.4 Experimental Evaluation Indicators

(1) In Vitro Evaluation

In vitro studies can assess target-protein phosphorylation, ERK and AKT activity, cell proliferation, apoptosis, migration, invasion, and colony formation. Resistance studies should also compare mutation profiles, protein expression, and pathway changes between parental and resistant cells.

(2) In Vivo Evaluation

In vivo studies should evaluate drug exposure, duration of target inhibition, central nervous system distribution, control of brain metastases, and emergence of resistant subclones in addition to tumor volume and tumor-growth inhibition. Short-term tumor shrinkage alone is insufficient to fully evaluate delayed resistance and long-term therapeutic effects.

9 Products Related to NSCLC Molecular Targets, Signaling-Pathway Inhibitors, and Immune Checkpoint Antibodies

Table 3 Products Related to EGFR, ALK, and ROS1 Inhibition

Table 4 Products Related to KRAS-MAPK, RET, MET, HER2, and PI3K-AKT-mTOR Pathways

Table 5 NSCLC Immune Checkpoint Antibody Products

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