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, ALK, ROS1, BRAF, MET, and RET) that may influence prognosis and predict response to targeted therapies if present. Guidelines recommend IHC and/or molecular analysis to identify actionable targets and guide subsequent therapy selection.

Quick Answers for Clinicians

Is BRAF testing recommended as routine biomarker testing in 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 emerging biomarkers are on the horizon for non-small cell lung cancer?

ERBB2 mutations, high-level MET amplifications, and tumor mutational burden (TMB) are emerging predictive biomarkers. Currently, evaluating these biomarkers is not considered part of routine care. However, broad molecular profiling to identify rare targets (such as these) for which effective drugs may be available is strongly advised, and in that context, including these biomarkers is appropriate and encouraged.

Testing for NTRK gene fusions can be considered to detect rare driver alterations to determine the potential for effective therapy; NTRK gene fusions can be detected by fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), next generation sequencing (NGS), and polymerase chain reaction (PCR) assays.

Indications for Testing

Individuals with advanced or metastatic disease should undergo:

Biomarker testing to identify oncogenic driver mutations for which effective drugs may be available
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, and RET genes, may inform treatment selection in lung cancer and are considered predictive biomarkers. KRAS oncogenetic mutations are prognostic biomarkers and confer a poor prognosis and predict lack of benefit from EGFR tyrosine kinase inhibitor (TKI) therapy.

Testing for the presence of genetic mutations or alterations is recommended for the following NSCLCs: adenocarcinoma, large cell, and NSCLC not otherwise specified (NOS). Such testing should also be considered for squamous cell carcinoma. At a minimum, the National Comprehensive Cancer Network (NCCN) recommends that EGFR, BRAF, ALK, ROS1, MET exon 14 skipping, and RET testing be performed, but also encourages the use of broad molecular profiling when available. This method of testing may identify rare driver mutations, such as NTRK gene fusions, for which effective therapy might be available. PD-L1 testing should also be performed to guide immunotherapy selection.

Minimum^a Recommended Testing Based on Histologic Subtype
Disease(s)	Recommended Testing
Adenocarcinoma, large cell lung cancer, and NSCLC NOS	EGFR, ALK, ROS1, BRAF, MET exon 14 skipping, RET PD‑L1 expression by IHC
Squamous cell carcinoma	Consider EGFR and ALK testing in individuals who have never smoked, in small biopsy specimens, or in cases of mixed histology Consider ROS1, BRAF, MET exon 14 skipping, and RET testing in small biopsy specimens or in cases of mixed histology PD-L1 expression by IHC
^aBroad molecular profiling is strongly encouraged to identify other driver mutations for which effective therapy might be available. Source: NCCN, 2020

EGFR Mutations

Epidermal growth factor receptor (EGFR) mutations occur in approximately 10% of NSCLC adenocarcinomas in the U.S. and are observed more frequently in nonsmokers. The two most common EGFR mutations, exon 19 deletions and exon 21 point mutations (L858R), account for approximately 90% of EGFR-mutated NSCLC cases and are associated with responsiveness to EGFR TKI therapy. However, there are many other less common EGFR mutations that are also sensitizing, and patients should be tested for these, as well. The NCCN recommends EGFR testing for patients with metastatic nonsquamous NSCLC or NSCLC NOS. DNA mutational analysis is the preferred method for determining EGFR status; IHC is not recommended.

ALK Gene Rearrangements

Anaplastic lymphoma kinase (ALK) gene rearrangements are present in roughly 5% of patients with NSCLC. The presence of an ALK gene rearrangement correlates with responsiveness to ALK TKIs. The NCCN recommends ALK testing in patients with metastatic nonsquamous NSCLC. The U.S. Food and Drug Administration (FDA)-approved IHC assay (D5F3 clone) is an adequate standalone test to detect ALK alterations, although secondary confirmation is encouraged. Fluorescence in situ hybridization (FISH) is also widely used for ALK gene rearrangement detection. Next generation sequencing (NGS) methodologies may detect ALK rearrangements, if appropriately designed.

ROS1 Gene Rearrangements

ROS proto-oncogene 1 (ROS1) gene rearrangements are more common in patients who are negative for EGFR mutations, KRAS mutations, and ALK rearrangements. The presence of a ROS1 gene rearrangement correlates with responsiveness to ROS1 TKIs. The NCCN recommends ROS1 testing in patients with metastatic nonsquamous NSCLC or NSCLC NOS. FISH is often used to detect ROS1 rearrangements, but may not detect the FIG-ROS1 variant. IHC testing is also available but has lower specificity than other methodologies. Therefore, positive IHC results should be confirmed molecularly or cytogenetically. NGS methodologies may detect ROS1 rearrangements, if appropriately designed.

BRAF Mutations

BRAF mutations at amino acid position 600 (BRAF V600E) have been associated with responsiveness to combined therapy with oral inhibitors of BRAF and MEK. The American Society of Clinical Oncology (ASCO) and NCCN recommend BRAF testing in all patients with advanced lung adenocarcinoma, irrespective of clinical characteristics. Testing methodologies include polymerase chain reaction (PCR), NGS, and Sanger sequencing.

MET Exon 14 Skipping Mutations

MET exon 14 skipping mutations, which include deletions and base substitutions, are more common in patients with adenocarcinomas than in patients with other NSCLC histologies. MET exon 14 skipping mutations have been associated with responsiveness to oral kinase inhibitors that target MET. MET alterations do not usually overlap with EGFR, KRAS, ROS1, and ALK mutations, but MET amplification and MET exon 14 skipping mutations may overlap. The NCCN recommends testing MET exon 14 skipping mutations in patients with metastatic NSCLC. Testing methodologies include NGS and reverse transcriptase PCR (RT-PCR).

RET Mutations

RET rearrangements occur between the RET gene and other genes, which may result in RET protein overexpression. The presence of RET rearrangements is correlated with responsiveness to oral kinase inhibitors that target RET. RET rearrangements do not usually overlap with EGFR, KRAS, ROS1, ALK, and MET exon 14 skipping mutations, although evidence suggests that they may rarely overlap with EGFR and KRAS mutations. The NCCN recommends testing for RET rearrangements in patients with metastatic NSCLC. Testing methodologies include FISH, NGS, and RT-PCR.

KRAS Mutations

The presence of a KRAS mutation, considered a prognostic biomarker, suggests poor survival for patients with NSCLC and is associated with reduced responsiveness to EGFR TKI therapy. EGFR, KRAS, ROS1, and ALK genetic alterations do not usually overlap. Therefore, the presence of a KRAS mutation suggests that patients may not benefit from further testing. Targeted therapy is not currently available for patients with KRAS mutations, although immune checkpoint inhibitors appear to be effective.

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 mutation is associated with acquired resistance to EGFR TKI therapy. Therefore, patients with an underlying EGFR sensitizing mutation who have been treated with an EGFR TKI should undergo high-sensitivity testing for EGFR T790M. The presence of a T790M mutation suggests that a patient may benefit from third-generation EGFR TKI therapy. If there is no evidence of a T790M mutation, 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 mutation testing in patients who have relapsed after initial response to ALK inhibitor therapy.