Biomarker Testing
in Lung Cancer 


Why do I test?

Biomarker testing is used to drive treatment decisions in an approach known as personalised medicine, to ensure each patient receives the most appropriate treatment for their tumour. It has been shown that patients with biomarker results available at the time of their initial oncology consultation benefit from shorter times to treatment administration, compared with patients whose results are unavailable.1

Which biomarkers do I assess?

Tumours are identified by pathology and specific biological alterations known as biomarkers; targeted therapies are now available based on the results of biomarker testing.

Biomarkers identified as predictors of therapeutic efficacy in lung cancer include 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.2-4

ALK, anaplastic lymphoma kinase; BRAF, B-Raf proto-oncogene; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; PD-L1, programmed cell death ligand-1; ROS1, ROS proto-oncogene 1

Assessment of other emerging biomarkers, including human epidermal growth factor receptor 2 (HER2) mutations, Kristen rat sarcoma viral oncogene homolog (KRAS) mutations and MET amplifications, are also often considered when using extended genetic panels or when routine EGFR/ALK/ROS1/BRAF testing is negative.4, 5

The estimated biomarker prevalence ranges for patients with non-small cell lung cancer (NSCLC) are listed in the table below:

ALK, anaplastic lymphoma kinase; BRAF, B-Raf proto-oncogene; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer; PD-L1, programmed cell death ligand-1; ROS1, ROS proto-oncogene 1

EGFR mutation

EGFR mutations associated with NSCLC commonly occur between exons 18 and 21 of the EGFR gene. The two most prevalent EGFR mutations are an in-frame deletion of exon 19 (50% of patients’ samples) and L858R point mutation in exon 21 (40% of patients’ samples). Lung cancer tumours harbouring these sensitising mutations are sensitive to EGFR tyrosine kinase inhibitor (TKI) treatment.15

ALK fusions

ALK fusions occur when part of the ALK gene translocates to a partner gene, leading to the creation of a fusion oncogene. The most common partner gene implicated in NSCLC is EML4. Oncogenic EML4-ALK induces tumour formation in mice, which can be reduced after the administration of ALK-TKIs in vitro and in vivo.7

ROS1 rearrangements

ROS1 is a receptor tyrosine kinase of the insulin receptor family. Chromosomal rearrangements involving the ROS1 gene can lead to consecutive kinase activity and these mutations are associated with sensitivity to TKIs in vitro.8

BRAF mutations

BRAF is a protein kinase involved in the mitogen activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway, which is involved in cell growth, proliferation, survival and differentiation. Approximately 50% of BRAF mutations in NSCLC are due to a point mutation in exon 15 (V600E), with 50% due to non-V600E mutations.12 The V600E mutation results in oncogenic properties including enhanced ERK activity.12

PD-L1 expression

The programmed cell death-1 (PD-1)/PD-L1 pathway is an important checkpoint used by tumour cells to inhibit antitumour immune responses.16 In vitro assessment suggests that PD-L1 can be overexpressed by lung cancer cells through the actions of surrounding cytokines, which prevents T cell-mediated tumour cell elimination.16-18 PD-1/PD-L1 inhibitors are effective therapies in tumours with high PD-L1 expression since the interaction between immune cell PD-1 and tumour cell PD-L1 is prevented, thus promoting a natural immune response against the tumour.16

Mechanism of action of anti-PD-L1 therapy

PD-L1 expression leads to
immune cell evasion

Anti-PD-1/PD-L1 reverses
immune suppression

Figure adapted from Peters, et al. 201919
PD-1, programmed cell death-1; PD-L1, programmed cell death ligand-1

How PD-L1 testing can be useful for patients with NSCLC

Dr Federico Cappuzzo (Department of Oncology-Hematology, AUSL della Romagna, Ravenna, Italy) discusses how PD-L1 testing can be useful for patients with NSCLC.

Visit iD PD-L1 for more information on PD-L1 testing.

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When do I test?

It is recommended that biomarker testing in advanced/metastatic NSCLC should occur at the initial disease diagnosis and during disease progression.2, 4

At diagnosis of advanced/metastatic NSCLC, it is recommended that all sample types (resected tumours, small biopsies and cytology samples) undergo pathological analysis to confirm the presence of tumour biomarkers, in order to determine the most appropriate treatment.2 Circulating tumour DNA (ctDNA) from blood (plasma) may be considered if a tumour sample cannot be obtained.4

In some cases, such as patients with EGFR mutations, a tumour rebiopsy at disease progression is recommended in order to guide future treatment decisions.2

Two stages of disease management that use biomarker testing to inform treatment decision


Results of such biomarker tests inform treatment decisions, therefore notifying the patient of results should be done in a timely manner. For example, it is suggested that the turnaround time to get available results from a requested mutational test should be less than ten working days.25

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How to obtain tissue required for testing


Types of specimen

Quality tumour tissue acquisition is vitally important in the clinical management of NSCLC, allowing biomarker testing and disease staging to be performed. Adequate tumour tissue acquisition at first biopsy allows multiple tests to be performed without resampling at a later date.

The three commonly used specimen types for biomarker testing are:

  • Tumour tissue biopsy
  • Cytology
  • Plasma ctDNA

Overview of different specimens used for biomarker testing in NSCLC2, 4, 26-29

Biomarker status can be determined using various sample types

ctDNA, circulating tumour DNA; EGFR, epidermal growth factor receptor; NSCLC, non-small cell lung cancer

Determining the type of specimen to obtain depends on various factors such as the health of the patient and the biomarker test required. Some key considerations of tumour tissue and ctDNA specimens are presented in the figure below.

Overview of different specimens used for biomarker testing in NSCLC2,26,30,31

ctDNA, circulating tumour DNA

Techniques for acquiring specimens for biomarker testing

Tumour or cytology biopsies can be acquired through surgical and non-surgical techniques.

Surgical techniques

  • Video-assisted thoracoscopic surgery (VATS): A minimally invasive surgical procedure in which a thoracoscope is inserted into the chest via small incisions32
  • Mediastinoscopy: A surgical procedure used to examine the mediastinum by the insertion of a mediastinoscope33

Non-surgical techniques

  • Bronchial brushing via bronchoscopy: A small brush inserted via the mouth or nose that allows the removal of tumour cells from the lungs34
  • Endoscopic ultrasound-guided fine needle aspiration (EUS-FNA): An endoscope with an ultrasound probe and biopsy needle that is inserted into the oesophagus and allows guided biopsy35
  • Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA): A transbronchial approach involving FNA of mediastinal lesions using an endobronchial ultrasound probe36

EBUS guide for interventional bronchoscopists

The instructional animation below provides expert guidance on safely and optimally performing EBUS-TBNA.

ctDNA, part of the circulating cell-free DNA coming from tumour cells, can be extracted from plasma samples for molecular testing.37

Please refer to the Technical Considerations page to learn more about the methodologies and techniques used to test for biomarkers in NSCLC.

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Biomarker testing and treatment guidelines

NSCLC biomarker testing and treatment guidelines, including those from the American Society of Clinical Oncology (ASCO), College of American Pathologists (CAP)-International Association for the Study of Lung Cancer (IASLC)-Association for Molecular Pathology (AMP), Chinese Society of Oncology (CSCO)-European Society for Medical Oncology (ESMO), ESMO, National Comprehensive Cancer Network (NCCN) and Spanish Society of Medical Oncology (SEOM)-Spanish Society of Pathology (SEAP) suggest that all patients with advanced/metastatic NSCLC should be tested for biomarkers.2, 4, 5, 25, 38, 39 The PD-L1 immunohistochemistry test is now mandatory for all patients diagnosed with advanced NSCLC.2

Treatment recommendations for a patient with advanced NSCLC reflect the benefit of targeting the genetic abnormality driving the disease, where any is detected.2, 4 It has been shown that some tumours harbouring genetic mutations also express PD-L1 and treatment with PD-1/PD-L1 inhibitors in these patients is associated with low response rates.40 Therefore, it is recommended that targeted therapy against the genetic abnormality should take precedence over treatment with PD-1/PD-L1 therapies.4 Establishing the status of all relevant lung biomarkers before initiating treatment is essential.

The importance of obtaining all biomarker test results before starting treatment for NSCLC

In the interview below, Dr Federico Cappuzzo (Department of Oncology-Hematology, AUSL della Romagna, Ravenna, Italy) discusses the importance of obtaining all biomarker test results before starting treatment for NSCLC.

Please refer to your local guidelines for guidance on treatment and management options in NSCLC.

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  13. Dietel M, Savelov N, Salanova R, et al. Real-world prevalence of programmed death ligand 1 expression in locally advanced or metastatic non-small-cell lung cancer: the global, multicenter EXPRESS study. Lung Cancer 2019;134:174–179
  14. Aggarwal C, Abreu DR, Felip E, et al. Prevalence of PD-L1 expression in patients with non-small cell lung cancer screened for enrollment in KEYNOTE-001, -010, and -024. Ann Oncol 2016;27(supplement 6):vi359–vi378
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  19. Peters S, Cho BC, Reinmuth N, et al. Abstract CT074: tumor mutational burden (TMB) as a biomarker of survival in metastatic non-small cell lung cancer (mNSCLC): blood and tissue TMB analysis from MYSTIC, a phase III study of first-line durvalumab ± tremelimumab vs chemotherapy.  AACR Annual Meeting 2019; Atlanta, GA, USA 2019. CT074–CT074
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