Targeted therapies for lung adenocarcinomas have advanced dramatically over the last 5 years. Many associated biomarkers have become essential for selecting appropriate treatment for patients. Until the end of 2020, biomarker testing was only recommended for patients with metastatic disease. The recent FDA approval (December 18, 2020) of the third generation EGFR inhibitor, osimertinib, as adjuvant therapy for patients with EGFR sensitizing mutations in earlier stages (stage IIB-IIIA and high-risk stage IB-IIA) is likely to change the landscape of biomarker testing significantly. Other targeted therapy agents will probably also become choices for adjuvant therapy soon with ongoing clinical trials (for example, ClinicalTrials.gov Identifier: NCT03456076). This report aims to summarize the important biomarkers associated with targeted therapies for practicing pathologists (please refer to the biomarkers associated with immunotherapy article by Dr. Walk for biomarkers associated with immunotherapy).
Besides adenocarcinomas, the following histology types of lung cancer are also indications for biomarker testing: adenosquamous carcinoma (mixed adenocarcinoma and squamous cell carcinoma components), large cell carcinoma and non-small cell lung cancer, not otherwise specified (NOS). For patients with squamous cell carcinoma who are light/never smokers, particularly those diagnosed with needle core biopsy, biomarker testing should be considered as well.
Tissue based next-generation sequencing (both DNA and RNA) is currently the preferred methodology for biomarker testing. This can be supplemented by fluorescence in-situ hybridization (FISH) and / or immunohistochemistry (IHC). Cell-free/circulating tumor DNA (cfDNA or ctDNA) testing can be considered in some clinical circumstances, such as patients who are medically unfit for invasive biopsy, or when the biopsy materials obtained are insufficient for molecular testing, or in the setting or relapsed/progressive disease with concern for emergence of resistance mutations which may occur with any targeted therapies (examples in Table 1). A few studies reported that cfDNA could identify a targetable biomarker when tissue-based analysis does not, due to tumor heterogeneity. Further studies are warranted to confirm these findings.
Various EGFR mutations have been described with different sensitivities to EGFR inhibitors. Therefore, the specific identity of mutations should be characterized using standardized HGVS/HUGO nomenclature in biomarker testing reports. Common clinically useful descriptors (e.g. “exon 19 deletion”) should be included as necessary. The most common and most therapeutically responsive mutations (sensitive to all generations of EFGR inhibitors), are exon 19 deletions and p.L858R single nucleotide variant (point mutation) in exon 21. Less common sensitizing mutations include, but are not limited to, exon 19 insertions, p.L861Q, p.G719X, and p.S768I).
EGFR p.T790M is most commonly observed as a resistance mutation after first- and second- generation EGFR inhibitor treatment. If p.T790M is observed in the absence of prior EGFR inhibitor treatment, genetic counseling and possible germline genetic testing are recommended. Third-generation EGFR inhibitors are typically efficacious in treating this mutation. Of note, resistance mutations, such as p.C797X, p.L292X, p.G796X, p.L718Q, and p.G724S, have been recently identified in patients receiving 3rd generation EGFR inhibitors. Additionally, the allelic context in which p.C797S is acquired has potential implications for treatment (for details, refer to Ref 5). Therefore, if phasing is not routine of the assay, communication with the laboratory and additional analysis may be necessary.
EGFR exon 20 insertions/duplications are generally associated with lack of response to EGFR inhibitors, with some exceptions: p.A763_Y764insFQEA is associated with sensitivity to EGFR inhibitors while p.A763_Y764insLQEA may be associated with sensitivity to EGFR inhibitors. Novel tyrosine kinase inhibitors are under clinical trials to target this class of mutation.
KRAS activating mutations lead to unregulated signaling through the MAP/ERK pathway. KRAS activating mutations are the most common driver mutations (about 25 to 30%) in lung adenocarcinoma and are most commonly seen at codon 12, followed by codons 13 and 61. KRAS mutation is associated with adverse prognosis. KRAS mutation is mutually exclusive with other targetable driver mutations. Therefore, in resource limited situations, KRAS mutations can be tested first and, when positive, further testing can be deferred. There are no currently fully approved targeted therapies for patients with KRAS mutations. Of note, the U.S. Food and Drug Administration (FDA) granted Breakthrough Therapy designation for Sotorasib in February 2021 for the treatment of patients with locally advanced or metastatic NSCLC with KRAS p.G12C mutation. Additional clinical trials are ongoing for inhibitors that specifically target KRAS p.G12C and other KRAS mutations (Table 1).
The presence of an ALK rearrangement (fusion) is associated with responsiveness to oral ALK inhibitors. Various fusion partners have been reported with echinoderm microtubule-associated protein-like 4 (EML4) as the most common fusion partner. The critical component of the fusion is ALK, though, and rarer fusion partners such as KIF5B and even novel fusion partners should be considered candidates for targeted therapy.
ROS1 is a receptor tyrosine kinase and ROS1 fusions results in dysregulation and inappropriate signaling through the ROS1 kinase domain. ROS1 fusions are associated with responsiveness to ROS1 inhibitors. Many fusion partners have been reported including CD74, SLC34A2, CCDC6, and FIG. As with ALK fusions above, the key component of the fusion is ROS1, and even novel fusion partners may respond to therapy.
BRAF p.V600E Mutation
BRAF is a serine/threonine kinase and activating mutations in BRAF result in unregulated signaling through the MAP/ERK pathway. BRAF p.V600E mutation is by far the most common mutation and has been associated with response to combined inhibitors of BRAF and MEK. The clinical significance of other BRAF mutations remains unclear.
MET exon 14 Skipping Mutation
MET is a receptor tyrosine kinase. A variety of mutations result in loss of exon 14 leading to dysregulation and inappropriate signaling. MET exon skipping is associated with responsiveness to MET inhibitors.
RET is a receptor tyrosine kinase and RET fusions result in dysregulation and inappropriate signaling through the activation of RET kinase domain. Reported common fusion partners are KIF5B, NCOA4, and CCDC6; however, numerous other fusion partners have been identified. RET fusions, regardless of the fusion partner, are associated with responsiveness to RET inhibitors. The best methods to detect RET fusions are RNA NGS and FISH using RET break apart probes.
NTRK1/2/3 (neurotrophic tyrosine receptor kinase) Fusions
TRKA/B/C encoded by NTRK1/2/3 are tyrosine receptor kinases. NTRK1/2/3 genes are rarely rearranged in NSCLC. The fusion genes result in inappropriate signaling. Many fusion partners have been reported. Of note, point mutations in NTRK1/2/3 are usually non-activating and the clinical significance of point mutations remains uncertain.
Emergent Biomarkers (Table 1)
High-level MET Amplification (10 or higher copies)
This mutation may be associated with over-activating the MET pathway. Patients with this mutation have been shown to respond to crizotinib or capmatinib. Amplification of MET has also been seen as a secondary resistance mechanism that can emerge after treatment using other targeted agents.
ERBB2 (HER2) exon 20 Insertions and Point Mutations
These mutations lead to constitutive activation of ERBB2. Patients with these mutations have been shown to respond to Ado-trastuzumab emtansine.
- EJ Jordan et al, Prospective Comprehensive Molecular Characterization of Lung Adenocarcinomas for Efficient Patient Matching to Approved and Emerging Therapies, Cancer Discov; 7(6); 596–609, doi: 10.1158/2159-8290.CD-16-1337
- JM Sands et al, Next-generation sequencing informs diagnosis and identifies unexpected therapeutic targets in lung squamous cell carcinomas, Lung Cancer; 140; 35-41 doi: 10.1016/j.lungcan.2019.12.005
- J Wolf et al, Capmatinib in MET Exon 14–Mutated or MET-Amplified Non–Small-Cell Lung Cancer, N Engl J Med 2020; 383:944-957 DOI: 10.1056/NEJMoa2002787
- LT Bob et al, Ado-Trastuzumab Emtansine for Patients With HER2-Mutant Lung Cancers: Results From a Phase II Basket Trial doi: 10.1200/JCO.2018.77.9777
- A Leonetti et al. Resistance mechanisms to osimertinib in EGFR-mutated non-small cell lung cancer. Br J Cancer 2019; 121:725–737. DOI: 10.1038/s41416-019-0573-8
- NCCN Guideline, Non-small cell lung cancer, version 4.2021
- Mycancergenome https://www.mycancergenome.org/content/biomarkers/. Accessed 4/9/2021
Chung-Che (Jeff) Chang currently serves as Associate Editor for Archives of Pathology and Laboratory Medicine, and a member of Personized Healthcare Committee for College of American Pathologists. He was the Principal Investigator of several NIH/NCI grants to study myeloma, myelodysplastic syndromes, and lymphoma. Dr. Chang’s research interests include the use of next-generation sequencing technologies for clinical diagnostics and biomarker discovery.