Key points

Introduction

Lung cancer is a heterogeneous genomic disease. Although smoking remains the primary cause of lung cancer, genetic susceptibility and environmental exposures are responsible for 10% to 15% of cases. Targeted therapies improved survival in patients with tumors with oncogenic drivers. Therefore, it is critical to expand our understanding of genetic alterations in non-small cell lung cancer (NSCLC) to increase the number of targeted therapies available for patients. Alterations beyond epidermal growth factor receptor ( EGFR ), ALK , and ROS1 exemplify lung cancer’s complexity and the need for greater investments in precision therapy to extend patient survival and outcomes. This article covers genetic targets outside of the big 3 ( BRAF , NTRK , KRAS , HER2 , RET , MET , NRG1 ), their novel agents, challenges, and future directions.

B-Raf proto-oncogene

The BRAF proto-oncogene encodes for the serine/threonine kinase that lies downstream of rat sarcoma ( RAS ) and leads to signaling through the RAS-rapidly accelerated fibrosarcoma (RAF)-mitogen-activated protein kinase (MAPK)-MAPK/extracellular-signal-regulated kinase (ERK) (MEK)-ERK signaling pathways, a key molecular cascade that regulates cell growth. After BRAF mutations were first described in melanoma, mutant BRAF was shown to mediate oncogenesis in lung adenocarcinoma. BRAF mutations represent 2% to 3% of lung adenocarcinomas, with 50% to 75% BRAF V600 E, and more frequently found in patients with a tobacco use history. ^,

Vemurafenib demonstrated efficacy in patients with BRAF V600 E mutated metastatic NSCLC. ^, Dabrafenib was evaluated in a phase II trial in patients with metastatic BRAF V600 E mutant NSCLC. The overall response rate (ORR) was 33%, and the median overall survival (OS) was 12.7 months. A combination of dabrafenib and trametinib was studied in another phase II trial of patients with BRAF V600 E mutant NSCLC. The combination therapy resulted in an increased ORR (63.2%) and has been approved by the European Medicines Agency and the US Food and Drug Administration for patients with stage IV NSCLC with BRAF V600 E mutation.

Neurotrophin tyrosine kinase receptor

Tropomyosin-related kinase ( TRK ) encodes the tyrosine kinase receptors for neurotrophins found in multiple tissues and associated with the nerve growth factor family. Three members of the family are proto-oncogenes encoded by NTRK1 , NTRK2 , and NTRK3 , which produce the proteins TrkA, TrkB, and TrkC, respectively, and activation leads to signaling in the MAPK and AKT pathways among others leading to cell proliferation, differentiation, and survival. Neurotrophin tyrosine kinase receptor ( NTRK ) rearrangements can occur in all 3 genes and have been identified in multiple cancers, including lung cancer. Fewer than 1% of NSCLC cases have NTRK fusions. NTRK fusions are found in men and women with various ages and smoking histories.

Multiple tyrosine kinase inhibitors (TKI) are being investigated for NTRK -altered cancers. The US Food and Drug Administration granted larotrectinib and entrectinib accelerated approval for adult and pediatric solid tumors positive for NTRK fusions, which will be the focus of the clinical data presented. The first patient with NTRK fusion to demonstrate tumor regression with a selective TRK inhibitor, larotrectinib, was reported in 2015; preclinical models confirmed tumor growth inhibition. A phase I trial investigated larotrectinib in adult and pediatric patients with NTRK fusions across tumor types. In the 55 patients enrolled across 13 tumor types, the most common fusions observed were NTRK3 (n = 29), followed by NTRK1 (n = 25) and NTRK2 (n = 1), with 14 unique fusion partners. The study demonstrated a 75% ORR in NTRK fusion-positive patients.

Early results from an entrectinib phase I study reported antitumor activity in a patient with an NTRK1 fusion NSCLC. An analysis of 3 early phase trials investigating entrectinib with NTRK or ROS1 positive tumors enrolled 54 patients with NTRK fusions with a 57% ORR. The median progression-free survival (PFS) was 11.2 months, and OS was 20.9 months. Additional TRK inhibitors are under investigation in the clinic.

Kirsten rat sarcoma viral oncogene

Kirsten RAS viral oncogene ( KRAS ) is the most commonly mutated of the RAS family isoforms and occurs in 22% of tumors, one of the most common oncogenic driver mutations in cancer. Most KRAS mutations are found in exons 12 and 13 (39% G12C, 18%–21% G12V, and 17%–18% G12D). In advanced NSCLC, KRAS mutation is associated with poorer prognosis. Despite its early discovery, KRAS -mutant NSCLC is highly heterogeneous, and therapy targeting KRAS is just beginning to be understood.

KRAS is 1 of the 4 proteins encoded by RAS . Guanosine triphosphate binds to KRAS in the active state and guanosine diphosphate binds to KRAS in the inactive state ( Fig. 1 A). Activating point mutations in RAS proteins like KRAS typically confer tumorigenesis by loss of GTPase activity; this results in the active state and constitutive activation of downstream signaling pathways like phosphatidylinositol 3-kinase (PI3K) and MAPK, rendering them resistant to multiple standard therapies for NSCLC ( Fig. 1 B).

( A ) KRAS cycle in a KRAS wild-type cell—growth factors bind to receptor tyrosine kinases upstream of KRAS, which initiate signaling cascades via proteins with Src homology 2 (SH2). SH2 signaling domain proteins like growth factor receptor-bound protein 2 (GRB2) and Son of Sevenless (SOS) can then catalyze the KRAS guanosine diphosphate (GDP) → guanosine triphosphate (GTP) exchange via guanine nucleotide growth factor (GEF). GTP-bound KRAS can initiate signaling cascades like Raf/MEK/ERK. ERK can translocate into the nucleus activating a proliferative process. GTPase activating proteins (GAPs) mediate a GTP → GDP exchange resulting in inactivation of KRAS and homeostasis. ( B ) KRAS cycle in a mutant cell. The same upstream signaling pathways occurs in KRAS mutant cells. However, mutant KRAS is insensitive to GAPs, resulting in constitutive downstream action. * denotes possible treatment targets.

Previous attempts to target KRAS failed due to a lack of known allosteric binding sites, alternative pathways, and the protein’s high affinity for the active guanosine triphosphate–bound state. ^, Other attempts included combination therapy with MAPK kinase (MEK1/MEK2) inhibition. A phase II clinical trial in advanced KRAS -mutant NSCLC demonstrated efficacy at the cost of high toxicity. The selumetinib-docetaxel combination resulted in a 37% increase in ORR and 3.2-month PFS compared with patients who received docetaxel. The combination group experienced a 15% increase in grade 3 adverse events (AEs) with the most common grade 3 of 4 AEs being neutropenia, febrile neutropenia, and asthenia. A phase I/Ib study found that KRAS -mutant patients receiving trametinib and docetaxel had a 24% ORR, whereas those in the trametinib and pemetrexed arms had a 17% ORR.

The first-in-human, phase I study of small molecule AMG 510, which specifically and irreversibly inhibits KRAS G12C by locking it in its guanosine diphosphate-bound state, was presented with results from 22 patients with advanced KRAS G12C solid tumors. Six patients had NSCLC; 2 experienced partial response after 6 weeks, and 2 patients had stable disease. The median treatment duration was 9.7 weeks; treatment was well-tolerated. Two grade 3 AEs, namely, anemia and diarrhea, were reported; 68% of AEs were grade 1.

Studies assessing KRAS co-mutations have demonstrated lower clinical response rates in KRAS -mutant lung adenocarcinomas with KEAP1 inactivation. This subset of programmed death ligand 1 inhibitor–resistant tumors demonstrates lower programmed death ligand 1 expression and inactivation of the tumor suppressor STK11 / LKB1 , resulting in the accumulation of tumor-associated neutrophils with suppressive effects on T cells. LKB1 is mutated in approximately 30% of somatic lung adenocarcinomas. A prior study showed NSCLC with LKB1 / KRAS co-mutations responded distinctly to targeted therapies. A murine study with LKB1 / KRAS mutations or p53 / KRAS mutations demonstrated a selective apoptotic response in LKB1 / KRAS -mutated NSCLC tumors to the metformin-analog metabolic drug phenformin. Apoptosis was observed in NSCLC cell lines with mutated LKB1 but not with KRAS WT. KRAS -mutated lung cancer is a rapidly evolving area of research in the search for providing more treatment options for these patients with unmet need.

Human epidermal growth factor receptor 2

Human epidermal growth factor receptor 2 (HER2), an erbB receptor tyrosine kinase family member, activates signaling through the PI3K–AKT and MEK–ERK pathways. HER2 is activated by homodimerization and heterodimerization with other members of the erbB family but does not have a known ligand. HER2 overexpression is observed in 13% to 20% of lung cancer cases and is more common in women, never smokers, and lung adenocarcinomas. HER2 mutations are oncogenic and result in constitutive HER2 phosphorylation and activation and EGFR pathway stimulation. HER2 amplification and mutations are uncommon, representing 9% and 2% to 3% of cases, respectively. HER2 mutations occur in exons 18 to 21, usually in exon 20 at codon 776 with a 12 base pair duplication/insertion of the YVMA amino acid sequence. It is unclear if HER2 -mutated tumors lead to worse patient outcomes compared with other variants.

A prospective study of the pan-HER TKI dacomitinib, which irreversibly binds HER2, HER1 (EGFR), and HER4, enrolled 30 patients with HER2 -mutated (n = 26) or HER2 -amplified (n = 4) NSCLC. The ORR was 12% for patients with HER2 mutation; no patients with amplification experienced tumor response. The median PFS was 3 months for all patients. In the HER2 -mutant cohort, the median PFS was also 3 months with a 1-year survival rate of 44%. The pan-HER TKI, afatinib, showed limited results for HER2 -mutated lung cancer. A single-arm phase II trial demonstrated a 12-week PFS of 53.8% and a medial PFS of 15.9 weeks with afatinib. The median OS was 56 weeks.

Other small molecule TKIs have been investigated. The median PFS for neratinib alone, an irreversible pan-HER inhibitor, was 2.9 months. The median PFS increased to 4 months with combined neratinib and temsirolimus treatment. A study showed that response to neratinib varied by cancer type, co-mutations, and concurrent pathway activation. Patients with HER2 -altered lung cancer (n = 26) had a low response rate, with 1 patient achieving an objective tumor response. For patients with HER2 -mutated disease, although not limited to lung cancer, patients who did not show clinical benefit were more likely to have co-mutations in TP53 and HER3 . RAS/RAF pathway activation and coincident cell cycle checkpoint aberrations were associated with worse outcomes and no clinical benefit.

Antibody-based drugs have showed efficacy against HER2 -mutated NSCLC. In a phase II trial, 18 patients with HER2 -mutant lung adenocarcinomas were treated with T-DM1, demonstrating a 44% partial response rate and a 5-month median PFS.

A retrospective study across European centers evaluated 101 patients with NSCLC with HER2 mutations treated with chemotherapy and/or HER2-targeted therapy. The median OS was 24 months for all patients, regardless of whether HER2-directed therapy was received. The ORR was highest for patients who received trastuzumab with or without chemotherapy or those who received T-DM1 at 50.9% with PFS 4.8 months.

The appropriate biomarker for selection remains elusive in this group of patients. At this time, mutation is the most predictive of response to HER2-directed therapy. Molecular aberrations in HER2- mutated lung cancers are heterogeneous, highlighted by varied effectiveness of HER2 kinase inhibitors. Important characteristics to consider are the type of mutation and the presence and degree of HER2 amplification, expression, and concurrent pathway activation.

RET proto-oncogene

RET is an receptor tyrosine kinase that mediates neural crest development; its activation causes cellular proliferation, migration, and differentiation. RET gene alterations are most common in thyroid and lung cancers. Activating RET rearrangements preserve the tyrosine kinase domain of the 3′ RET gene and have various upstream 5′ fusion partners. The most common fusion partner in NSCLC is KIF5B. RET fusions result in ligand-independent dimerization and downstream growth pathway activation.

RET fusions occur in approximately 1.4% of NSCLCs and 1.7% of adenocarcinomas and are present mostly in patients 60 years or older with adenocarcinoma and no smoking history. A study of more than 4800 patients with varied malignancies undergoing NGS found that RET gene status occurred in 1.8%; most cases had coexisting, actionable genomic alterations, suggesting that successful treatment would involve custom combination approaches.

Various multikinase TKIs have been tested on RET -rearranged NSCLC. A prospective phase II trial evaluating cabozantinib in 25 patients with RET -rearranged lung adenocarcinoma revealed a 28% ORR with a median PFS of 5.5 months and median OS of 9.9 months. A phase II study investigating vandetanib in 19 RET fusion-positive patients found a 53% ORR and a 4.7-month median PFS. A global, multicenter registry described treatment of 165 patients with RET -rearranged NSCLC, of which 53 had been treated with at least 1 RET inhibitor. Cabozantinib, sunitinib, and vandetanib had ORRs of 37%, 22%, and 18%, respectively, and lenvatinib and nintedanib also caused tumor responses. In all patients, the median PFS was 2.3 months and the median OS was 6.8 months. Although these trials suggest inhibitory activity in RET -rearranged NSCLC, response to these multitargeted TKIs is modest and short lived.

RET-specific inhibitors are being evaluated to hopefully overcome the limitations of multikinase inhibitors. A report in patients with RET -altered malignancies showed that the potent RET inhibitor, LOXO-292, resulted in an ORR of 65% in 26 RET -altered patients with NSCLC. BLU-667, another selective RET inhibitor, demonstrated preclinical activity and clinical responses in patients with RET -altered NSCLC. The study demonstrated an ORR of 58% for all patients (n = 48). BLU-667 was effective in patients with various RET fusion partners and against intracranial metastases.

Mesenchymal-epithelial transition

Mesenchymal-epithelial transition ( MET ) is a proto-oncogene that encodes for the transmembrane MET TKI. The binding of its ligand, hepatocyte growth factor, activates signaling pathways such as PI3K/AKT, MAPK, nuclear factor kappa B, and signal transducer and activator of transcription proteins, which promotes cell proliferation and invasion, blocking apoptosis, and increasing cell motility. MET alterations are found in many cancers, including lung cancers, and induce tumor growth via protein overexpression and phosphorylation, gene amplification, rearrangement, and mutations.

Protein overexpression and phosphorylation are the most common forms of MET-positive NSCLC, whereas MET amplification is relatively rare and observed in approximately 2.2% of newly diagnosed adenocarcinoma and up to 7% of NSCLC cases. ^, Increased MET gene copy number is a negative prognosis factor in surgically resected NSCLC, with OS of 25.5 versus 47.5 months for patients with MET of 5 or more copies per cell and MET of less than 5 copies per cell, respectively. Although the KIF5B-MET fusion has been reported in lung adenocarcinoma, MET rearrangements are rare.

MET exon 14 alterations (4% of lung adenocarcinomas ) are diverse and drive tumorigenesis. They are associated with older age and significant smoking history. Base substitutions or indels (usually deletions) in MET that disrupt the 3′ splice site or branch point of intron 13, or the 5′ splice site of intron 14, can lead to MET exon 14 skipping. Exon 14 skipping causes decreased MET ubiquitination by E3 ubiquitin-protein ligase CBL and MET degradation, leading to increased MET levels and downstream signaling, producing oncogenesis. MET exon 14 alterations vary widely. A study found 126 different variants in 223 distinct exon 14 aberrations.

Multitargeted TKIs have been used against MET in lung cancer, as have TKIs with increased MET sensitivity. In addition, monoclonal antibodies are being investigated in patients with MET-driven tumors. The dual MET/ALK inhibitor crizotinib showed antitumor responses in MET amplified and MET exon 14 mutated NSCLC. ^, In addition, crizotinib and cabozantinib have demonstrated antitumor response in patients with exon 14-altered lung adenocarcinoma. A phase I study found that patients with high levels of MET amplification (MET/CEP7 <SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='≥’>≥≥
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4) demonstrated antitumor activity with crizotinib, with median PFS of 6.7 months.

Other studies have examined highly specific MET inhibitors for MET exon 14 mutated NSCLC. A phase II study investigated tepotinib for patients with NSCLC with MET exon 14 skipping mutations. For patients with the variant detected with liquid biopsy, preliminary results demonstrated an ORR of 50.0% and a median PFS of 9.5 months. For patients with the variant detected by tissue biopsy, preliminary results demonstrated an ORR of 45.1% and a median PFS of 10.8 months.

Another phase II study investigated MET-specific inhibitor capmatinib in advanced NSCLC with MET exon 14 skipping mutations. Preliminary data from this study reported an ORR of 40.6% and a PFS of 5.42 months. Patients without prior therapies had an ORR of 67.9% and a PFS of 9.69 months. Capmatinib also demonstrated a response against intracranial metastases, and patients tolerated the drug well.

It has been reported that MET amplification can increase to 5% to 22% after treatment with EGFR TKI therapy (erlotinib, gefitinib, osimertinib), ^, MET amplification is an alternative mechanism of resistance to EGFR TKIs in patients with EGFR mutation positive NSCLC. Multiple combinations of MET and EGFR therapies are being evaluated in patients with resistant EGFR mutant NSCLC.

Neuregulin 1

Neuregulin 1 ( NRG1 ) codes for the neuregulin protein. In contrast to other NSCLC fusions, NRG1 codes for a HER3 and HER4 tyrosine kinase receptor ligand. In these fusions, NRG1 is the 3′ partner; other genes such as CD74 , RBPMS , WRN , and SDC4 are the 5′ partners. The EGF domain of NRG1, located in the carboxy-terminal region and essential for receptor interaction, is preserved. NRG1 fusions in lung cancer samples are found without other known driver mutations, and CD74-NRG1 fusions represent 1.7% of lung adenocarcinomas and occur most commonly in invasive mucinous adenocarcinoma, an NSCLC subtype that represents 2% to 10% of all lung adenocarcinoma cases. This fusion causes PI3K-AKT pathway activation, which induces tumorigenesis.

Although there are few data available, an in vitro study showed that lapatinib and afatinib suppressed HER2, HER3, and ERK phosphorylation produced by CD74-NERG1 fusions. Two cases of patients with NRG1 fusions showed response to afatinib, a HER2 inhibitor. A SLC3A2-NRG1 and a CD74-NRG1 fusion demonstrated 12 months and 10 months PFS, respectively. In addition, a recent study reported a patient’s CD74-NRG1 fusion-positive NSCLC responded for 19 months to an investigational anti-HER3 monoclonal antibody.

Summary

Lung cancer represents a heterogenous group of thoracic tumors with distinct biologic and genomic characteristics. Clinical studies and molecular genotyping delineate appropriate therapy for many patients with NSCLC that represent precision treatment for the defined alteration. The list of genomic alterations is growing and broad molecular profiling for patients with advanced NSCLC is essential. Molecular selection defines specific populations that derive enhanced benefit from targeted treatment, and provide insights into potential mechanisms of resistance. Despite the progress that has been made, work is necessary to untangle the complex causes for primary and secondary resistance to therapy to make a dramatic impact on survival. Furthermore, interactions within the tumor microenvironment and with immune cells is becoming increasingly important, leading to exploration of combination therapies.