Skip To Main Content

Biomarker Profiling Is Integral to Lung Cancer Characterization1,2

While many genetic alterations have been identified, knowing the presence of predictive biomarkers may lead to favorable patient outcomes1,2

Approximately 64% of patients with NSCLC have oncogenic driver mutations3

The National Comprehensive Cancer Network® (NCCN®) advises to consider testing for the indicated biomarkers1

Chart of biomarkers

AKT1=AKR mouse thymoma kinase; ALK=anaplastic lymphoma kinase; BRAF=v-raf murine sarcoma viral oncogene homolog B1; DDR2=discoidin domain receptor 2; EGFR=epidermal growth factor receptor; FGFR1=fibroblast growth factor receptor 1; HER2=human epidermal growth factor receptor 2; KRAS=Kirsten rat sarcoma 2 viral oncogene homolog; MEK1=mitogen-activated protein kinase kinase 1; MET=met proto-oncogene; NRAS=neuroblastoma rat; PIK3CA=phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit alpha; PD-L1=programmed death-ligand 1; PTEN=phosphatase and tensin homolog; RET=rearranged during transfection; ROS1=ROS proto-oncogene 1.

National Comprehensive Cancer Network® (NCCN®) and CAP/IASLC/AMP recommendations

NCCN Guidelines® for NSCLC recommend biomarker testing to identify driver mutations for which appropriate targeted therapy may be available1:

  • Test for EGFR mutations and ALK rearrangements (category 1 for metastatic nonsquamous NSCLC or NSCLC NOS), which are similar to the 2017 CAP/IASLC/AMP recommendations1,4,5
  • Test for ROS1* rearrangements and BRAF* mutations for nonsquamous NSCLC or NSCLC NOS1
  • Test for PD-L1 expression (category 1) and NTRK* gene fusions in patients with metastatic NSCLC1
Testing methodologies

*Recommendations are category 2A unless otherwise specified.1
The 2017 CAP/IASLC/AMP guidelines do not recommend testing for these genes as standalone assays. Appropriate validation of any sample prep and testing methodology is critical. The NCCN Guidelines also strongly advise broader molecular profiling to identify rare driver mutations.1,4
FDA=US Food and Drug Administration; NGS=next-generation sequencing; NOS=not otherwise specified; PCR=polymerase chain reaction.

Detecting ROS1+ NSCLC

NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) recommend testing for ROS1+ disease in all patients with nonsquamous metastatic NSCLC1

ROS1+ NSCLC can be detected with various testing methods7

Immunohistochemistry (IHC)

  • Uses specific monoclonal antibodies to detect overexpression of the ROS1 fusion protein7
  • Requires dedicated tissue and limits multiplexing8,9
  • Low specificity; molecular confirmation is necessary1

Fluorescence in situ hybridization (FISH)

  • Uses “break-apart” probes to show ROS1 gene rearrangement7
  • Requires dedicated tissue9,10
  • May underdetect the FIG-ROS1 variant1

Next-generation sequencing (NGS)

  • Multigene parallel sequencing assays7,9
  • Capable of detecting ROS1 fusions1
  • Provides information about multiple biomarkers, including rare and common mutations, and can typically use less tissue9,10
  • Turnaround time for results may be longer than with RT-PCR, IHC, or FISH11

Reverse transcriptase-polymerase chain reaction (RT-PCR)

  • Reverse transcription from extracted RNA12
  • Fast and sensitive detection of specific fusion variants12
  • May not detect fusions in novel partners1

NCCN=National Comprehensive Cancer Network; NSCLC=non-small cell lung cancer; ROS1=ROS proto-oncogene 1.

Detecting ALK+ NSCLC

ALK gene rearrangements are present in approximately 5% of patients with NSCLC13

NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) and CAP clinical guidelines recommend that patients with metastatic NSCLC be routinely tested for ALK+ disease.1,4

ALK may be detected by using a variety of methods14-16

Fluorescence in situ hybridization (FISH)

  • Uses fluorescently labeled DNA probes that bind to and localize specific regions in the tumor nuclei to detect ALK rearrangements and amplification14

Immunohistochemistry (IHC)

  • Uses specific monoclonal antibodies to detect overexpression of the ALK fusion protein15,17

Next-generation sequencing (NGS)

  • NGS allows massive parallel sequencing for the identification of potentially targetable genetic alterations in tumors18
  • Other testing methods are under investigation, including reverse transcriptase-polymerase chain reaction (RT-PCR)19,20

Reverse transcriptase-polymerase chain reaction (RT-PCR)

  • RT-PCR is a technique that utilizes RNA extraction to detect ALK gene fusions, such as EML4-ALK19,20


Currently, FISH, IHC, and NGS are the only testing methods FDA approved for clinical application in detecting ALK+ NSCLC14-16

ALK=anaplastic lymphoma kinase; CAP=College of American Pathologists; DNA=deoxyribonucleic acid; EML4=echinoderm microtubule-associated protein-like 4; NCCN=National Comprehensive Cancer Network; NSCLC=non-small cell lung cancer; RNA=ribonucleic acid.

Detecting EGFR mutations in NSCLC

EGFR mutations play an important role in carcinogenesis21

  • The aberrant signaling influences several key aspects, including cell proliferation, apoptosis, migration, survival, and more complex processes, such as angiogenesis21
  • Different EGFR mutations have different signaling properties, but most mutations affect the adenosine triphosphate (ATP) binding cleft, where targeting TKIs compete for binding21

Ideal testing methods should cover a broad spectrum of EGFR mutations4

The 2017 CAP/IASLC/AMP guidelines suggest the following:

  • Detect all individual mutations that have been reported in at least 1% of EGFR-mutated lung adenocarcinomas4
  • Detect mutations in specimens with at least 20% cancer cells4,5
  • Use EGFR T790M mutational testing in patients harboring sensitizing EGFR mutations after treatment with EGFR-targeted therapies4,5
  • Total EGFR expression by IHC is not sufficient to select patients for EGFR-targeted therapies4,5
Biomarker testing

Note: These may not be all known mutations of EGFR.
Bp=base pair; IHC=immunohistochemistry.

    • Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Non-Small Cell Lung Cancer V1.2020. © National Comprehensive Cancer Network, Inc., 2019. All rights reserved. Accessed December 10, 2019. To view the most recent and complete version of the guideline, go online to NCCN.org. NCCN makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way.

      Referenced with permission from the NCCN Clinical Practice Guidelines in Oncology (NCCN Guidelines®) for Non-Small Cell Lung Cancer V1.2020. © National Comprehensive Cancer Network, Inc., 2019. All rights reserved. Accessed December 10, 2019. To view the most recent and complete version of the guideline, go online to NCCN.org. NCCN makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way.

    • My cancer genome. https://www.mycancergenome.org/content/disease/lung-cancer/. Accessed December 12, 2019.

      My cancer genome. https://www.mycancergenome.org/content/disease/lung-cancer/. Accessed December 12, 2019.

    • Kris MG, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA. 2014;311:1998-2006.

      Kris MG, et al. Using multiplexed assays of oncogenic drivers in lung cancers to select targeted drugs. JAMA. 2014;311:1998-2006.

    • Lindeman NI, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors. Arch Pathol Lab Med. 2018;142:321-346.

      Lindeman NI, et al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors. Arch Pathol Lab Med. 2018;142:321-346.

    • Kalemkerian GP, et al. Molecular testing guideline for the selection of patients with lung cancer for treatment with targeted 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 Clinical Practice Guideline update. J Clin Oncol. 2018;36:911-919.

      Kalemkerian GP, et al. Molecular testing guideline for the selection of patients with lung cancer for treatment with targeted 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 Clinical Practice Guideline update. J Clin Oncol. 2018;36:911-919.

    • Naidoo J, Drilon A. Molecular diagnostic testing in non-small cell lung cancer. Am J Hematol Oncol. 2014;10:4-11.

      Naidoo J, Drilon A. Molecular diagnostic testing in non-small cell lung cancer. Am J Hematol Oncol. 2014;10:4-11.

    • Bubendorf L, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch. 2016;469(5):489-503.

      Bubendorf L, et al. Testing for ROS1 in non-small cell lung cancer: a review with recommendations. Virchows Arch. 2016;469(5):489-503.

    • Sholl LM, et al. ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas. Am J Surg Pathol. 2013;37(9):1441-1449.

      Sholl LM, et al. ROS1 immunohistochemistry for detection of ROS1-rearranged lung adenocarcinomas. Am J Surg Pathol. 2013;37(9):1441-1449.

    • Su D, et al. High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods. J Exp Clin Cancer Res. 2017;36(1):121.

      Su D, et al. High performance of targeted next generation sequencing on variance detection in clinical tumor specimens in comparison with current conventional methods. J Exp Clin Cancer Res. 2017;36(1):121.

    • Rogers TM, et al. Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer. Sci Rep. 2017;7:42259.

      Rogers TM, et al. Multiplexed transcriptome analysis to detect ALK, ROS1 and RET rearrangements in lung cancer. Sci Rep. 2017;7:42259.

    • Hechtman JF, et al. Pan-Trk immunohistochemistry is an efficient and reliable screen for the detection of NTRK fusions. Am J Surg Pathol. 2017;41(11):1547-1551.

      Hechtman JF, et al. Pan-Trk immunohistochemistry is an efficient and reliable screen for the detection of NTRK fusions. Am J Surg Pathol. 2017;41(11):1547-1551.

    • Shan L, et al. Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR. PLoS One. 2015;10(3):e0120422.

      Shan L, et al. Detection of ROS1 gene rearrangement in lung adenocarcinoma: comparison of IHC, FISH and real-time RT-PCR. PLoS One. 2015;10(3):e0120422.

    • Solomon B, Wilner KD, Shaw AT. Clin Pharmacol Ther. 2014;95:15-23.

      Solomon B, Wilner KD, Shaw AT. Clin Pharmacol Ther. 2014;95:15-23.

    • Weickhardt AJ, Aisner DL, Franklin WA, Varella-Garcia M, Doebele RC, Camidge DR. Diagnostic assays for identification of anaplastic lymphoma kinase–positive non–small cell lung cancer. Cancer. 2013;119:1467-1477.

      Weickhardt AJ, Aisner DL, Franklin WA, Varella-Garcia M, Doebele RC, Camidge DR. Diagnostic assays for identification of anaplastic lymphoma kinase–positive non–small cell lung cancer. Cancer. 2013;119:1467-1477.

    • Marchetti A, et al. ALK protein analysis by IHC staining after recent regulatory changes: a comparison of two widely used approaches, revision of the literature, and a new testing algorithm. J Thorac Oncol. 2016;11:487-495.

      Marchetti A, et al. ALK protein analysis by IHC staining after recent regulatory changes: a comparison of two widely used approaches, revision of the literature, and a new testing algorithm. J Thorac Oncol. 2016;11:487-495.

    • US Food and Drug Administration. https://www.accessdata.fda.gov/cdrh_docs/pdf17/P170019C.pdf. Accessed December 19, 2019.

      US Food and Drug Administration. https://www.accessdata.fda.gov/cdrh_docs/pdf17/P170019C.pdf. Accessed December 19, 2019.

    • Mino-Kenudson M, et al. Clin Cancer Res. 2010;16:1561-1571.

      Mino-Kenudson M, et al. Clin Cancer Res. 2010;16:1561-1571.

    • Vigneswaran J, et al. Comprehensive genetic testing identifies targetable genomic alterations in most patients with non-small cell lung cancer, specifically adenocarcinoma, single institute investigation. Oncotarget. 2016;7:18876-18886.

      Vigneswaran J, et al. Comprehensive genetic testing identifies targetable genomic alterations in most patients with non-small cell lung cancer, specifically adenocarcinoma, single institute investigation. Oncotarget. 2016;7:18876-18886.

    • Wang Y, et al. Feasibility of cytological specimens for ALK fusion detection in patients with advanced NSCLC using the method of RT-PCR. Lung Cancer. 2016;94:28-34.

      Wang Y, et al. Feasibility of cytological specimens for ALK fusion detection in patients with advanced NSCLC using the method of RT-PCR. Lung Cancer. 2016;94:28-34.

    • Soda M, et al. A prospective PCR-based screening for the EML4-ALK oncogene in non-small cell lung cancer. Clin Cancer Res. 2012;18:5682-5689.

      Soda M, et al. A prospective PCR-based screening for the EML4-ALK oncogene in non-small cell lung cancer. Clin Cancer Res. 2012;18:5682-5689.

    • Cheng L, et al. Molecular pathology of lung cancer: key to personalized medicine. Mod Pathol. 2012;25:347-369.

      Cheng L, et al. Molecular pathology of lung cancer: key to personalized medicine. Mod Pathol. 2012;25:347-369.

    Microscope icon

    Discover cancer biomarkers

    Learn more about the importance of oncologic biomarkers and what they can tell you.

    Molecular pathways icon

    Explore molecular pathways

    Learn more about how cancer avoids immune response.