Technical
Considerations

             

How do I test?

Immune biomarkers (programmed cell death ligand-1 [PD-L1]) are distinct from molecular biomarkers and oncogenic mutations (epidermal growth factor receptor [EGFR]; anaplastic lymphoma kinase [ALK]; ROS proto-oncogene 1 [ROS1]; B-Raf proto-oncogene [BRAF]); each biomarker characterises different aspects of the tumour and is measured or tested for in different ways.1

A variety of molecular and immunological tests are available for biomarker testing in non-small cell lung cancer (NSCLC) samples. However, it is essential that each method is appropriately quality-assured, both internally and externally, beforehand.1

How do I test?

Immune biomarkers (programmed cell death ligand-1 [PD-L1]) are distinct from molecular biomarkers and oncogenic mutations (epidermal growth factor receptor [EGFR]; anaplastic lymphoma kinase [ALK]; ROS proto-oncogene 1 [ROS1]; B-Raf proto-oncogene [BRAF]); each biomarker characterises different aspects of the tumour and is measured or tested for in different ways.1

A variety of molecular and immunological tests are available for biomarker testing in non-small cell lung cancer (NSCLC) samples. However, it is essential that each method is appropriately quality-assured, both internally and externally, beforehand.1

Overview of specimen types and detection techniques used for biomarker testing in NSCLC

*VENTANA SP263 with formalin-fixed paraffin-embedded fine-needle aspiration cell blocks only18
ALK, anaplastic lymphoma kinase; BRAF, B-Raf proto-oncogene; ctDNA, circulating tumour DNA; EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridisation; IHC, immunohistochemistry; NGS, next-generation sequencing; PD-L1, programmed cell death ligand-1; ROS1, ROS proto-oncogene 1; RT-PCR, real-time polymerase chain reaction

Additional testing considerations for each biomarker can be found below.

EGFR mutations

Molecular determination of EGFR mutations should focus on exons 18–21.1, 11

IHC techniques with EGFR mutation-specific antibodies are not recommended for detecting mutations or when performing copy number analysis.11, 19

ctDNA testing is slightly less sensitive for EGFR T790M than exon 19 deletion and L858R.2, 5

ALK fusions

The use of cytological specimens has been validated in approaches using FISH.7

ROS1 rearrangements

For detecting ROS1 rearrangements, IHC is only recommended as a screening approach prior to confirmatory FISH analysis, due to the low specificity of IHC.1, 11, 20

BRAF mutations

IHC approaches are not recommended for BRAF mutation testing.11

PD-L1 expression

The VENTANA SP263 assay is validated for use with NSCLC formalin-fixed, paraffin-embedded (FFPE) fine-needle aspiration cell blocks for the assessment of tumour cell PD-L1 expression.18 Despite certain studies demonstrating concordance between cytological and tissue specimens, further validation is required before cytological samples are routinely used to determine PD-L1 expression.21

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.21 Instead, results are based on assay-specific cut-offs depending on the percentage of tumour and/or immune cells expressing PD-L1.21

Real-time polymerase chain reaction

Real-time polymerase chain reaction (PCR) is a highly targeted technique that is useful to detect point mutations, insertions and deletions in genes of interest.20, 22 Benefits of real-time PCR include the relatively short turnaround time (<4 days) and high sensitivity.22

Real-time PCR is commonly used to detect EGFR and BRAF mutations.4, 14 The cobas EGFR Mutation Test v2 (Roche Molecular Systems) and therascreen EGFR RGQ PCR Kit (Qiagen) are Food and Drug Administration (FDA)-approved companion real-time PCR assays for the determination of various EGFR mutations in NSCLC samples.23-25

Despite the use of real-time PCR for the detection of point mutations, this approach is not typically used to detect gene fusions (e.g. ALK) and rearrangements (e.g. ROS1).11, 20

Next-generation sequencing

Next-generation sequencing (NGS) enables a broader assessment of clinically relevant genes, genome or exome, compared with PCR-based approaches.22 Depending on the subtype of NGS used, it is possible to simultaneously detect point mutations, insertions, deletions copy number alterations and gene rearrangements.22 Multiplex sequencing is recommended over single-gene mutation tests in order to identify other mutations beyond EGFR, ALK and ROS1.1, 11, 20, 22

NGS panels are commonly amplicon- or hybrid capture-based.22 The former relies on a PCR approach to amplify and sequence restricted genomic regions of interest. This provides an enhanced analytical sensitivity for key genes; however, there is a loss of gene fusion and copy number alteration detection. Hybrid capture NGS panels capture a larger genomic region than amplicon approaches, thus enabling a broader assessment of genetic variants.22

There are two NGS panels that are FDA-approved companion diagnostic devices for use in NSCLC: FoundationOne CDx (Foundation Medicine) and Oncomine Dx Target Test (Life Technologies).23

Fluorescence in situ hybridisation

Fluorescence in situ hybridisation (FISH) is a cytogenetic technique that uses fluorescently-labelled nucleic acid probes to target complementary DNA or RNA sequences. This approach enables the detection and visualisation of chromosomal regions of interest, including translocations, insertions and deletions.26

ALK fusions and ROS1 rearrangements in NSCLC samples are commonly detected through FISH assays.1, 6, 7, 11, 12

The Vysis ALK Break Apart FISH Probe Kit (Abbott Molecular) is the only FISH FDA-approved companion diagnostic test to select patients with ALK positive NSCLC for treatment with the tyrosine kinase inhibitor (TKI) crizotinib.23, 27

Immunohistochemistry

Immunohistochemistry (IHC) utilises antigen-antibody interactions to visualise the quantity and distribution of a protein of interest in patient samples.28

In NSCLC, IHC has been accepted as an equivalent alternative to FISH for ALK testing.1, 11 The FDA-approved VENTANA ALK (D5F3) CDx Assay (Ventana Medical Systems) is available to detect ALK protein expression in FFPE NSCLC tissues prior to TKI treatment.23, 29

PD-L1 expression can only be determined by IHC.1, 19, 20 There are four (VENTANA SP142, VENTANA SP263, PD-L1 IHC 28-8 pharmDx and PD-L1 IHC 22C3 pharmDx) commercially available IHC assays for the quantification of PD-L1 in FFPE NSCLC tissues.21 Each assay requires different automated staining equipment, detection system and algorithms to determine the PD-L1 expression of specimens.21

  1. Planchard D, Popat S, Kerr K, et al. Metastatic non-small cell lung cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2018;29:iv192–iv237
  2. Jenkins S, Yang JC, Ramalingam SS, et al. Plasma ctDNA analysis for detection of the EGFR T790M mutation in patients with advanced non-small cell lung cancer. J Thorac Oncol 2017;12:1061–1070
  3. Ellison G, Zhu G, Moulis A, et al. EGFR mutation testing in lung cancer: a review of available methods and their use for analysis of tumour tissue and cytology samples. J Clin Pathol 2013;66:79–89
  4. Wang S, Yu B, Ng CC, et al. The suitability of small biopsy and cytology specimens for EGFR and other mutation testing in non-small cell lung cancer. Transl Lung Cancer Res 2015;4:119–125
  5. 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;34:3375–3382
  6. Du X, Shao Y, Qin H-F, et al. ALK-rearrangement in non-small-cell lung cancer (NSCLC). Thorac Cancer 2018;9:423–430
  7. Minca EC, Lanigan CP, Reynolds JP, et al. ALK status testing in non-small-cell lung carcinoma by FISH on ThinPrep slides with cytology material. J Thorac Oncol 2014;9:464–468
  8. McCoach CE, Blakely CM, Banks KC, et al. Clinical utility of cell-free DNA for the detection of ALK fusions and genomic mechanisms of ALK inhibitor resistance in non-small cell lung cancer. Clin Cancer Res 2018;24:2758–2770
  9. Mezquita L, Jovelet C, Lacroix L, et al. An amplicon-based liquid biopsy for detecting ALK and ROS1 fusions and resistance mutations in advanced non-small cell lung cancer (NSCLC) patients. J Clin Oncol 2018;36:9095
  10. Inivata. InVision First Lung. 2020. Available from: https://www.inivata.com/invisionfirst-lung/. Accessed: 11 June 2020
  11. 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. Arch Pathol Lab Med 2018;142:321–346
  12. 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;11:1029–1039
  13. Zhang L, Wang Y, Zhao C, et al. High feasibility of cytological specimens for detection of ROS1 fusion by reverse transcriptase PCR in Chinese patients with advanced non-small-cell lung cancer. Onco Targets Ther 2019;12:3305–3311
  14. Brustugun OT, Khattak AM, Tromborg AK, et al. BRAF-mutations in non-small cell lung cancer. Lung Cancer 2014;84:36–38
  15. Lin Q, Zhang H, Ding H, et al. The association between BRAF mutation class and clinical features in BRAF-mutant Chinese non-small cell lung cancer patients. J Transl Med 2019;17:298–298

 

  1. Reck M, Rodríguez-Abreu D, Robinson AG, et al. Pembrolizumab versus chemotherapy for PD-L1-positive non-small-cell lung cancer. N Engl J Med 2016;375:1823–1833
  2. Heymann JJ, Bulman WA, Swinarski D, et al. PD-L1 expression in non-small cell lung carcinoma: Comparison among cytology, small biopsy, and surgical resection specimens. Cancer Cytopathol 2017;125:896–907
  3. Roche. VENTANA PD-L1 (SP263) Assay package insert. 2020. Available from: https://pim-eservices.roche.com/eLD/api/downloads/4b0ce998-2fb6-ea11-fc90-005056a71a5d?countryIsoCode=gb. Accessed: 8 October 2020
  4. Garrido P, Conde E, de Castro J, et al. Updated guidelines for predictive biomarker testing in advanced non-small-cell lung cancer: a national consensus of the Spanish Society of Pathology and the Spanish Society of Medical Oncology. Clin Transl Oncol 2019;[Online ahead of print]
  5. NCCN. Clinical Practice Guidelines in Oncology Non-Small Cell Lung Cancer Version 3.2020. 2020. Available from: https://www.nccn.org/professionals/physician_gls/pdf/nscl.pdf. Accessed: 11 June 2020
  6. Kim H, Chung JH. PD-L1 testing in non-small cell lung cancer: past, present, and future. J Pathol Transl Med 2019;53:199–206
  7. Pennell NA, Arcila ME, Gandara DR, et al. Biomarker testing for patients with advanced non-small cell lung cancer: real-world issues and tough choices. Am Soc Clin Oncol Educ Book 2019;39:531–542
  8. FDA. List of Cleared or Approved Companion Diagnostic Devices (In Vitro and Imaging Tools). 2020. Available from: https://www.fda.gov/medical-devices/vitro-diagnostics/list-cleared-or-approved-companion-diagnostic-devices-vitro-and-imaging-tools. Accessed: 20 May 2020
  9. Qiagen. therascreen EGFR RGQ PCR Kit version 2. 2020. Available from: https://www.qiagen.com/au/products/diagnostics-and-clinical-research/oncology/therascreen-solid-tumor/therascreen-egfr-rgq-pcr-kit-v2/#orderinginformation. Accessed: 11 June 2020
  10. Roche. cobas® EGFR Mutation Test v2. 2020. Available from: https://diagnostics.roche.com/global/en/products/params/cobas-egfr-mutation-test-v2.html. Accessed: 11 June 2020
  11. Huber D, Voith von Voithenberg L, Kaigala GV. Fluorescence in situ hybridization (FISH): history, limitations and what to expect from micro-scale FISH? Micro and Nano Engineering 2018;1:15–24
  12. FDA. XALKORI® (crizotinib) Prescribing Information. 2017. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/label/2017/202570s021lbl.pdf. Accessed: 11 June 2020
  13. Kim S-W, Roh J, Park C-S. Immunohistochemistry for pathologists: protocols, pitfalls, and tips. J Pathol Transl Med 2016;50:411–418
  14. Roche. VENTANA ALK (D5F3) CDx Assay. 2015. Available from: https://www.accessdata.fda.gov/cdrh_docs/pdf14/P140025c.pdf. Accessed: 11 June 2020