Non-Small Cell Lung Cancer - Non-Small Cell Lung Cancer Molecular Markers

Lung cancer is the leading cause of cancer-related mortality in the United States. Diagnosis typically involves a combination of imaging studies, cytologic or histopathologic specimen evaluation, and subsequent immunohistochemistry (IHC) and genetic analysis. More than 80% of lung cancer cases are classified as non-small cell lung cancers (NSCLCs), and adenocarcinoma is the most common NSCLC subtype in nonsmokers. Adenocarcinoma is characterized by a prevalence of oncogenic driver genetic alterations (in EGFR, ERBB2, ALK, ROS1, BRAF, MET, KRAS, NTRK1/2/3, and RET) that may influence prognosis and predict response to targeted therapies if present. Guidelines recommend molecular analysis to identify actionable targets and guide subsequent therapy selection.

Quick Answers for Clinicians

Is BRAF testing recommended as routine biomarker testing in patients with non-small cell lung cancer?

Yes. The American Society of Clinical Oncology (ASCO) and the National Comprehensive Cancer Network (NCCN) recommend BRAF testing in all patients with advanced lung adenocarcinoma, irrespective of clinical characteristics.

What is the role of cell-free DNA testing in non-small cell lung cancer?

Although cell-free/circulating tumor DNA testing has generally high specificity, it has low sensitivity (with a false-negative rate of up to 30%) and should not be used in place of tissue-based testing if tissue is available.

Cell-free/circulating tumor DNA testing is appropriate when invasive tissue sampling is not an option for a given patient or when the tissue sample is insufficient for molecular analysis. Negative cell-free/circulating tumor DNA testing results should be confirmed by tissue-based analysis whenever possible.

Which biomarkers are emerging as predictive for non-small cell lung cancer?

High-level MET amplification is an emerging predictive biomarker. Currently, evaluating this biomarker is not considered part of routine care. However, broad molecular profiling to identify rare targets for which effective drugs may be available is strongly advised, and in that context, including this biomarker is appropriate and encouraged.

Indications for Testing

Individuals with advanced or metastatic disease should undergo:

Biomarker testing to identify oncogenic driver variants for which effective drugs may be available
Programmed death-ligand 1 (PD-L1) IHC testing to assess whether PD-L1 inhibitor therapy is an option

Laboratory Testing

Predictive and Prognostic Biomarker Testing

Several oncogenic driver alterations, including alterations in EGFR, ALK, ROS1, BRAF, MET, KRAS, NTRK1/2/3, ERBB2, and RET genes, may inform treatment selection in lung cancer and are considered predictive biomarkers. KRAS oncogenic variants are also prognostic biomarkers, confer a poor prognosis, and predict a lack of benefit from EGFR tyrosine kinase inhibitor (TKI) therapy.

Genetic testing to detect the following somatic alterations is recommended for adenocarcinoma, large cell carcinoma, and NSCLC not otherwise specified (NOS) and should be considered for squamous cell carcinoma in patients with advanced or metastatic disease:

EGFR mutations
BRAF mutations
KRAS G12C mutation
ALK rearrangements
ROS1 rearrangements
MET exon 14 skipping mutations
RET rearrangements
NTRK1/2/3 rearrangements
ERBB2 (HER2) mutations

The use of broad molecular profiling is strongly recommended. Broad molecular profiling includes all the biomarkers listed here, preferably in conjunction with any emerging biomarkers (ie, high-level MET amplifications). These variants may be detected by a single assay or by a combination of assays. A tiered approach may be used because typically only one oncogenic variant is present in an individual patient. However, in rare cases, EGFR variants, KRAS variants, or RET rearrangements may overlap.

PD-L1 expression by IHC is recommended for all histologic subtypes to guide immunotherapy selection.

Testing for Oncogenic Driver Variants
Genes/Gene Mutations	Clinical Significance	Preferred Method(s) for Testing
ALK rearrangements	Correlates with responsiveness to ALK TKI therapy Associated with resistance to EGFR TKIs	FISH IHC (used as a screening test or, if FDA approved, as a standalone test) Sequencing (if appropriately designed) PCR (useful in some settings)
BRAF (V600E)	Associated with responsiveness to combined therapy with oral BRAF and MET inhibitors	Real-time PCR Sequencing (eg, NGS)
EGFR^a	Generally predictive of responsiveness to EGFR TKI therapy	Real-time PCR Sequencing (eg, NGS)
ERBB2 (HER2)	Consider treatment with fam-trastuzumab deruxtecan-nxki in those who have received previous systemic therapy	Sequencing (eg, NGS)
KRAS	Suggests poor survival Associated with reduced responsiveness to EGFR TKI therapy KRAS p.G12C associated with responsiveness to oral KRAS G12C inhibitors	Real-time PCR Sequencing (eg, NGS)
MET (exon 14 skipping variants)	Associated with responsiveness to oral kinase inhibitors that target MET	Sequencing (eg, NGS)
NTRK1/2/3 gene rearrangements	Associated with responsiveness to oral TRK inhibitors	FISH IHC PCR Sequencing (eg, NGS)
RET rearrangements	Correlated with responsiveness to oral kinase inhibitors that target RET, regardless of the fusion partner	Sequencing (RNA-based sequencing tests are preferred over DNA-based tests) FISH (may underdetect some fusions) Real-time reverse transcription PCR (in some situations)
ROS1 rearrangements	Correlates with responsiveness to ROS1 TKIs	FISH (may underdetect FIG-ROS1 variants) Sequencing (DNA-based NGS assays may underdetect ROS1 fusions) IHC (positive result must be confirmed with additional testing) Real-time PCR (useful in some settings)
^aExon 19 deletions and the exon 21 missense variant L858R account for approximately 90% of EGFR variants in patients with NSCLC. FDA, U.S. Food and Drug Administration; FISH, fluorescence in situ hybridization; NGS, next generation sequencing; PCR, polymerase chain reaction Source: NCCN, 2022

PD-L1 Expression Testing

Testing for PD-L1 expression levels by IHC is recommended before first-line treatment in patients with metastatic NSCLC to assess whether PD-1 or PD-L1 inhibitors are a treatment option. Refer to the ARUP Consult PD-L1 Testing topic for the most up-to-date testing recommendations.

Therapy Resistance Testing

Patients can develop resistance to therapy. For example, the EGFR T790M variant is associated with acquired resistance to EGFR TKI therapy. Therefore, patients with an underlying EGFR sensitizing variant who have been treated with older-generation EGFR TKIs should undergo high-sensitivity testing for EGFR T790M. The presence of a T790M variant suggests that a patient may benefit from third-generation EGFR TKI therapy. If there is no evidence of a T790M variant, testing for alternate mechanisms of resistance, such as MET or ERBB2 amplification, may be considered to direct patients to alternative therapies. There is currently insufficient evidence to support routine secondary ALK variant testing in patients who have relapsed after an initial response to ALK inhibitor therapy. Broad genomic profiling at multiple points during treatment may be the most informative approach to assess mechanisms of resistance.