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. The aim of biomarker testing is to inform treatment decisions to provide optimal outcomes for patients.
What biomarkers do I assess?
Tumours are identified by pathology and specific biological alterations known as biomarkers; targeted therapies are now available for these biomarkers.
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.1,2 The biomarker prevalence ranges for patients with NSCLC are listed below:
- EGFR mutations: ranges from approximately 10–21%, however, EGFR mutations are more common in never smokers, females and patients of East Asian ethnicity1,3–5
- ALK fusions: reported prevalence of 2–7% in adenocarcinoma cases1,6
- ROS1 rearrangements: ranges from 1–2% in adenocarcinoma cases7
- BRAF mutations: ranges from 2–4% in adenocarcinoma cases8–10
- PD-L1 expression: ranges from 13–49%, depending on the cut-offs used11,12
Diagnostic tests are available that look for the presence or absence of these biomarkers in tumour DNA:
- EGFR and BRAF mutations are commonly detected by real-time polymerase chain reaction (PCR), Sanger sequencing and next generation sequencing (NGS)1,2,13
- ALK fusions are commonly detected by fluorescence in situ hybridisation (FISH) and PCR1
- ROS1 rearrangements are commonly detected by FISH and immunohistochemistry (IHC)1
- PD-L1 expression is measured by IHC11
The results from these tests are used to inform treatment decisions, both at initial diagnosis and at disease progression.
When do I test?
At diagnosis it is recommended that pathological diagnosis of all sample types be made to confirm the presence of tumour biomarkers prior to treatment.1 This includes resected tumours, as well as small biopsy and cytology samples.1 However, circulating tumour DNA (ctDNA) obtained from blood (plasma) may be used at diagnosis to test for the presence of EGFR mutations if a tumour sample cannot be obtained, for example, because the patient's health has deteriorated.1,14
At disease progression, a tumour rebiopsy, or plasma sample if permitted, is recommended in patients where it may guide future treatment decisions.1
The two stages of disease management that use biomarker testing to inform treatment decision
Used to identify tumours with biomarkers that would make them more sensitive to certain treatments15
Patients with NSCLC harbouring a targetable biomarker may derive a greater benefit from targeted therapies than non-targeted therapies, such as chemotherapy15-18
Used to identify resistance biomarkers in patients who progress on treatment
Identification of resistance biomarkers at disease progression can inform the next treatment decision1,2
How do I test?
Immune biomarkers (such as PD-L1) are distinct from molecular biomarkers and oncogenic mutations (such as EGFR, ALK, or ROS1); each biomarker characterises different aspects of the tumour, and is measured or tested for in different ways.11,13,19 In patients with lung cancer, using a combination of DNA and histochemistry based tests provides a comprehensive assessment.2
BRAF mutation testing is now recommended in patients with non-squamous NSCLC to identify the BRAF V600E mutation as part of a larger testing panel strategy, either initially or when EGFR, ALK and ROS1 tests are negative.2,13,20
Real-time PCR and NGS are commonly used methods to assess BRAF mutations.2 IHC is also emerging as a tool for the rapid screening of BRAF mutations.1
Mutations in BRAF do not typically overlap with EGFR mutations or ALK rearrangements.2
It is recommended that all patients with advanced NSCLC and adenocarcinoma histology be tested for EGFR mutations.1,2 The presence of certain EGFR mutations, such as exon 19 deletion and L858R, are predictive of response to EGFR-TKIs.1,2 At the point of progression on first- or second-generation EGFR-TKIs, it is recommended to evaluate the EGFR mutation status with a new tumour biopsy or blood (plasma) sample for the presence of T790M resistance mutation.1,2
EGFR mutation testing should use validated methodology and be carried out by laboratories participating in external quality assurance schemes, and should have adequate coverage of mutations in exons 18–21.1
The two commonly used sample types for evaluating the EGFR mutation status are tumour biopsy and circulating tumour DNA (ctDNA) samples obtained from blood (plasma). When testing for EGFR mutations at diagnosis, and T790M at progression on a first- or second-generation EGFR-TKI, it is recommended that a tumour sample be used to confirm the presence of EGFR mutations prior to treatment.1 However, ctDNA obtained from blood (plasma) may be used at diagnosis and at progression when a tumour sample is not available, for example, if a patient's condition has deteriorated and they are no longer eligible for a biopsy.1,14 ctDNA obtained from a plasma sample offers a less invasive and potentially quicker alternative for EGFR mutation testing than tumour tissue testing; however ctDNA testing is slightly less sensitive for EGFR T790M than exon 19 deletion and L858R.22,34 When testing for T790M in patients with disease progression following first- or second-generation EGFR-TKI therapy, a negative plasma test should be followed up with a confirmatory tumour tissue test where feasible.22
Gene fusions and rearrangements
It is recommended that routine testing for ALK fusions be carried out in the same patient group as EGFR mutations, i.e. all patients with advanced NSCLC and adenocarcinoma histology.1,13 The presence of ALK fusions is predictive of response to ALK-TKIs.1,24-26
ALK FISH testing is commonly used to detect ALK fusions and select patients for ALK-TKI therapy; multiplex PCR and NGS can also be used.1 If validated, IHC is an alternative to ALK FISH, and it is being increasingly used in Europe to select patients for ALK-TKIs.1,13
In addition to EGFR mutation and ALK fusion testing, it is recommended that patients with advanced NSCLC and adenocarcinoma histology also receive routine testing for ROS1 rearrangements, irrespective of clinical characteristics.2,13
Similar to ALK fusions, ROS1 rearrangements can be detected using IHC and FISH. ROS1 IHC may be used to screen patients for targeted therapy; positive ROS1 IHC results should be confirmed by a molecular or cytogenetic method.1,2,13,27 NGS can also be used if appropriately validated.1,2,27
Protein expression – PD-L1
Immune biomarkers such as PD-L1 characterise the immune activity in the tumour microenvironment. PD-L1 expression is upregulated on tumour cells due to increased cytokine release in the tumour cell’s surroundings.28
IHC is commonly used to detect PD-L1 expression.11 Interestingly, PD-L1 expression does not provide a binary result, such as positive/negative or mutated/wild type, like genetic alterations or mutation diagnostic biomarkers in lung cancer.11 Instead, results are based on a range of the percentage of tumour cells expressing the biomarker: from low to high.11
Many guidelines include eligibility criteria for biomarker (EGFR, ALK, ROS1, BRAF, and PD-L1) testing in patients with lung cancer. Guidelines recommend that biomarker testing is done in all patients with adenocarcinoma.1,2,13,30
Results of such tests inform treatment decisions, therefore notifying the patient of results should be done in a timely manner, for example: the turnaround time from a mutation test being requested to a result being available should be less than ten working days.31
Treatment recommendations for a patient with advanced NSCLC reflect the benefit of targeting the genetic abnormality driving the disease, where any is detected.2,32 Although PD-L1 testing results are often reported earlier than other molecular analyses, it is possible that tumours harbouring genetic mutations also express PD-L1.11,33 Treating a patient based on PD-L1 expression alone could prevent the patient from receiving appropriate standard of care treatment.2 It is important to establish the status of all relevant lung biomarkers before initiating treatment.
Check your local guidelines for guidance on treatment options.
- Novello S, Barlesi F, Califano R, et al. Metastatic non-small-cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2016;27(suppl 5):v1-v27.
- NCCN. NCCN Clinical Practice Guidelines in Oncology NSCLC (version 2.2018), 2018. Available at: https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf. Accessed 15 February 2018.
- Douillard JY, Ostoros G, Cobo M, et al. First-line gefitinib in Caucasian EGFR mutation-positive NSCLC patients: a phase-IV, open-label, single-arm study. Br J Cancer 2014;110(1):55-62.
- Shigematsu H, Lin L, Takahashi T, et al. Clinical and biological features associated with epidermal growth factor receptor gene mutations in lung cancers. J Natl Cancer Inst 2005;97(5):339-346.
- Rosell R , Moran T, Queralt C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med 2009;361:958–967.
- Kerr K. ALK in Lung Cancer: ESMO Biomarker Factsheet. Available from: http://oncologypro.esmo.org/Education-Library/Factsheets-on-Biomarkers/ALK-in-Lung-Cancer#eztoc1208888_0_0_3. Accessed 05 March 2018.
- Kerr K. ROS1 in Lung Cancer: ESMO Biomarker Factsheet. Available from: http://oncologypro.esmo.org/Education-Library/Factsheets-on-Biomarkers/ROS1-in-Lung-Cancer. Accessed 05 March 2018.
- Cardarella S, Ogino A, Nishino M, et al. Clinical, pathologic, and biologic features associated with BRAF mutations in non-small cell lung cancer. Clin Cancer Res 2013;19(16):4532-4540.
- Paik PK, Arcila ME, Fara M, et al. Clinical characteristics of patients with lung adenocarcinomas harboring BRAF mutations. J Clin Oncol. 2011;29(15):2046-2051.
- Pratilas CA1, Hanrahan AJ, Halilovic E, et al. Genetic predictors of MEK dependence in non-small cell lung cancer. Cancer Res. 2008;68(22):9375-9383.
- Kerr KM, Hirsch FR. Programmed Death Ligand-1 Immunohistochemistry: Friend or Foe? Arch Pathol Lab Med. 2016;140:326-331.
- Huynh TG1, Morales-Oyarvide V2, Campo MJ, et al. Programmed Cell Death Ligand 1 Expression in Resected Lung Adenocarcinomas: Association with Immune Microenvironment. J Thorac Oncol 2016;11(11):1869-1878.
- Lindeman NI, Cagle PT, Aisner DL, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. J Thorac Oncol 2017;13(3):323-358.
- EMA. IRESSA® (gefitinib) Summary of Product Characteristics. 2017; http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_Product_Information/human/001016/WC500036358.pdf. Accessed 15 February 2018.
- Sequist LV, Yang JC, Yamamoto N, et al. Phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations. J Clin Oncol 2013;31(27):3327-3334.
- Maemondo M, Inoue A, Kobayashi K, et al. Gefitinib or chemotherapy for non-small-cell lung cancer with mutated EGFR. N Engl J Med 2010;362(25):2380-2388.
- Mitsudomi T, Morita S, Yatabe Y, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol 2010;11(2):121-128.
- Wu YL, Zhou C, Hu CP, et al. Afatinib versus cisplatin plus gemcitabine for first-line treatment of Asian patients with advanced non-small-cell lung cancer harbouring EGFR mutations (LUX-Lung 6): an open-label, randomised phase 3 trial. Lancet Oncol 2014;15(2):213-222.
- Korpanty GJ, Graham DM, Vincent MD, Leighl NB. Biomarkers That Currently Affect Clinical Practice in Lung Cancer: EGFR, ALK, MET, ROS-1, and KRAS. Front Oncol 2014;4:204.
- Planchard D, Besse B, Groen HJM, et al. Dabrafenib plus trametinib in patients with previously treated BRAF(V600E)-mutant metastatic non-small cell lung cancer: an open-label, multicentre phase 2 trial. Lancet Oncol 2016;17(7):984-993.
- Schwaederle M, Husain H, Fanta PT, et al. Detection rate of actionable mutations in diverse cancers using a biopsy-free (blood) circulating tumor cell DNA assay. Oncotarget 2016;7(9):9707-9717.
- Jenkins S, Yang J C-H, Ramalingam SS, et al. Plasma ctDNA analysis for detection of the EGFR T790M mutation in patients with advanced non-small cell cancer. J Thorac Oncol 2017;12(7):1061-1070.
- Thress KS, Brant R, Carr TH, et al. EGFR mutation detection in ctDNA from NSCLC patient plasma: A cross-platform comparison of leading technologies to support the clinical development of AZD9291. Lung Cancer 2015;90(3):509-515.
- Solomon BJ, Mok T, Kim DW, et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med 2014;371(23):2167-2177.
- Soria JC, Tan DSW, Chiari R, et al. First-line ceritinib versus platinum-based chemotherapy in advanced ALK-rearranged non-small-cell lung cancer (ASCEND-4): a randomised, open-label, phase 3 study. Lancet 2017;389(10072):917-929.
- Peters S, Camidge DR, Shaw ATN, et al. Alectinib versus Crizotinib in Untreated ALK-Positive Non-Small-Cell Lung Cancer. N Engl J Med. 2017;377(9):829-838.
- Viola P, Maurya M, Croud J, et al. A Validation Study for the Use of ROS1 Immunohistochemical Staining in Screening for ROS1 Translocations in Lung Cancer. J Thorac Oncol 2016;11(7):1029-1039.
- Kerr KM, Nicolson MC. Non–Small Cell Lung Cancer, PD-L1, and the Pathologist. Arch Pathol Lab Med. 2016;140(3):249-254.
- Roche. cobas® EGFR Mutation Test v2. 2016; https://www.accessdata.fda.gov/cdrh_docs/pdf15/P150047c.pdf. Accessed 16 May 2018.
- Leighl NB, Rekhtman N, Biermann WA, et al. Molecular testing for selection of patients with lung cancer for epidermal growth factor receptor and anaplastic lymphoma kinase tyrosine kinase inhibitors: American Society of Clinical Oncology endorsement of the College of American Pathologists/International Association for the study of lung cancer/association for molecular pathology guideline. J Clin Oncol 2014;32(32):3673-3679.
- Lindeman NL, Cagle PT, Beasley MB, et al. Molecular testing guideline for selection of lung cancer patients for EGFR and ALK tyrosine kinase inhibitors: guideline from the College of American Pathologists, International Association for the Study of Lung Cancer, and Association for Molecular Pathology. J Thorac Oncol 2013;8(7):823-859.
- Bubendorf L, Lantuejoul S, De Langen AJ, et al. Nonsmall cell lung carcinoma: diagnostic difficulties in small biopsies and cytological specimens. Eur Respir Rev 2017;26(144):170007.
- Gainor JF, Shaw AT, Sequist LV, et al. EGFR mutation and ALK rearrangements are associated with low response rates to PD-1 pathway blockade in non-small cell lung cancer: a retrospective analysis. Clin Can Res 2016;22(18):4585-93.
- Oxnard GR, Thress KS, Alden RS, et al. Association between plasma genotyping and outcomes of
treatment with osimertinib (AZD9291) in advanced non–small-cell lung cancer. J Clin Oncol 2016;34(28):3375-3382.