Testing for


Biomarker Testing in Lung Cancer

Robust, reliable, high-quality biomarker testing is key to making informed treatment decisions for patients with advanced lung cancer.

It is recommended that patients with metastatic adenocarcinoma non-small cell lung cancer (NSCLC) and all non-squamous NSCLC are tested for epidermal growth factor receptor (EGFR) mutations, anaplastic lymphoma kinase (ALK) gene fusions, ROS proto-oncogene 1 (ROS1) gene rearrangements, B-Raf proto-oncogene (BRAF) mutations and programmed cell death-ligand 1 (PD-L1) expression levels.1,2

Molecular biomarkers have historically been identified from tumour tissue samples; however, techniques to identify these biomarkers in liquid biopsy samples are being increasingly used.3-5 The percentage of tumour cells showing PD-L1 positive staining have to be assessed using tissue immunohistochemistry (IHC) testing.1

Mutation testing

In order to determine the biomarker status of a lung tumour, various diagnostic tests can be utilised to establish whether or not biomarkers are present in the tumour DNA.

These tests are commonly conducted using the following specimen types:4,6

  • Tumour tissue
  • Cytology
  • Plasma circulating tumour DNA (ctDNA)

The biomarker testing process should be well-validated and aligned with standard diagnostic practices for patients with advanced NSCLC.




Available tests

Real-time polymerase chain reaction (PCR)

Real-time PCR is a highly targeted, mutation specific test, however, only the alternations that are targeted by the specific assay will be detected.1

Real-time PCR is commonly used to assess EGFR and BRAF mutations.1 PCR testing is unlikely to detect gene rearrangements, and is therefore therefore only utilised in some settings for ALK fusion or ROS1 rearrangement testing; these can be detected using fluorescence in situ hybridisation (FISH).1

The cobas® EGFR mutation test is a real-time PCR test for the detection of EGFR mutations in exons 18–21. The test uses DNA derived from formalin-fixed paraffin-embedded (FFPET) tumour tissue samples or ctDNA from blood (plasma) samples from patients with NSCLC. Results from the test can be used to guide EGFR-TKI treatment decisions.7

Next generation sequencing (NGS)

NGS is increasingly used in clinical laboratories to facilitate multiple gene mutations and gene fusion testing.2 It can be used to detect small mutations, as well as gene fusions/rearrangements and copy number changes in the targeted genes, when designed with these alterations in mind.8

As NGS is primer dependent, the panel of genes detected will depend on the design of the NGS platform used.1 Two different types of NGS assays are used in molecular oncology: amplicon based and hybrid capture based.8

It is most commonly used to detect EGFR mutations, but can also be used for the detection of BRAF mutations, ALK fusions and ROS1 rearrangements.1,2,8

The first companion diagnostic NGS test for gene mutations associated with NSCLC, the Oncomine™ Dx Target test, has recently been approved by the FDA (June 2017).1,9,10 The Oncomine™ Dx Target test (Thermo Fisher Scientific Inc.) can simultaneously test for EGFR sensitising mutations (L858R and exon 19 deletions), BRAF V600E, and ROS1 rearrangements; the test does not detect ALK fusions.9

Fluorescence in situ hybridisation (FISH)

FISH can be used to examine copy number, amplification and gene rearrangements in the targeted genes.1 FISH testing remains the standard test for the detection of ALK fusions in NSCLC, and is used to assist with selecting patients for treatment with ALK-TKIs.1,2,11,12 FISH testing is also commonly used to detect ROS1 rearrangements.1,2

The FISH assay (Vysis LSI ALK Break Apart Rearrangement Probe Kit; Abbott Molecular) is an FDA-approved companion diagnostic test for the detection of ALK fusions in NSCLC, and aids in selecting patients for treatment with crizotinib.12,13 Using FFPE tissue samples, the test uses break-apart probes to label the fusion breakpoint.12

Immunohistochemistry (IHC)

IHC is a valid alternative to FISH for ALK testing, as it has a high positive and negative predictive value for ALK fusions. It is widely used to identify patients suitable for confirmatory ALK FISH testing, and it is being increasingly adopted in Europe as the primary test for prescribing ALK-TKIs.2,8

IHC testing for ROS1 can be used as a screening test for patients with lung adenocarcinoma, however, it is recommended that a positive result be confirmed by a molecular or cytogenic method.8 Due to the high sensitivity of IHC, tumours lacking any clear ROS1 staining can be interpreted as a negative result.8

IHC testing is not appropriate for EGFR mutational testing, and should not be used to select patients for targeted EGFR-TKI therapy.1,8

IHC is commonly used to assess PD-L1 expression. PD-L1 testing first requires a tissue sample to undergo FFPE preparation and processing.14 A Haematoxylin and Eosin stained section should be examined to determine whether the sample has at least 100 viable tumour cells.14 IHC staining can then be conducted for detecting the presence of PD-L1. Following the staining procedure, scoring algorithms are used to classify whether a sample is PD-L1 high or low, making the treatment decision more personalised.15,16

Representation of PD-L1 expression localised in a tumour sample

Tumour sample

For more information on testing for PD-L1, please visit ID.PD-L1.