Technical Considerations in Lung Cancer Biomarker Testing | Healthcare Professionals

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Overview of specimen types and detection techniques used for biomarker testing in NSCLC
Biomarker testing sample considerations
The clinical application of ctDNA testing in NSCLC
Specimen acquisition for biomarker testing in NSCLC
Methods of biomarker testing

Immune biomarkers (e.g. PD-L1) are distinct from molecular biomarkers and oncogenic mutations (e.g. EGFR mutations); each biomarker characterises different aspects of the tumour and is measured or tested for in different ways.^1,2

A variety of molecular and immunological tests are available for biomarker testing in NSCLC samples. However, it is essential that each method is appropriately quality-assured, both internally and externally, beforehand.²

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

*VENTANA PD-L1 (SP263) Assay with formalin-fixed paraffin-embedded fine-needle aspiration cell blocks only²⁸

ABBREVIATIONS:

ALK, anaplastic lymphoma kinase; BRAF, B-Raf proto-oncogene; ctDNA, circulating tumour DNA; EGFR, epidermal growth factor receptor; FISH, fluorescent in situ hybridisation; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; KRAS, Kirsten rat sarcoma virus; MET, MET proto-oncogene, receptor tyrosine kinase; NGS, next-generation sequencing; NTRK1/2/3, neurotrophic receptor tyrosine kinase 1/2/3; PD-L1, programmed cell death ligand-1; RET, rearranged during transfection; ROS1, ROS proto-oncogene 1; RT-PCR, real-time polymerase chain reaction

Additional testing considerations for 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.^2,11,29

Additional testing considerations for PD-L1 expression

The VENTANA PD-L1 (SP263) Assay has been validated for use with NSCLC FFPE fine-needle aspiration cell blocks for the assessment of tumour cell PD-L1 expression.²⁸ 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.³⁰

Evidence suggests that PD-L1 staining is concordant between fresh (<3 months old) and archival (<3 years old) tumour biopsies from the same patient.³¹ However, samples that are older than 3 years may lead to an underestimation of PD-L1 status.³² Therefore, both fresh and archival (<3 years old) tumour samples are suitable for PD-L1 testing.

PD-L1 testing on paired primary and metastatic lesions in NSCLC have shown high concordance, suggesting that tumour location does not influence PD-L1 expression.^31,33

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.³⁰ Instead, results are based on assay-specific cut-offs depending on the percentage of tumour and/or immune cells expressing PD-L1.³⁰

Biomarker testing sample considerations

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.

A tissue sample should contain at least 200–400 tumour cells.³⁴ Samples undergoing DNA sequencing should be comprised of at least 50% tumour cells,³⁵ however some highly sensitive methods may utilise tissue containing 10–20% tumour cells.^36,37

Overview of different specimens used for biomarker testing in NSCLC^{1,2,5,29,38–40}

Biomarker status can be determined using various sample types

Tumour tissue^{1,2,29,38–40}

Gold standard for all biomarker testing in NSCLC
Collect during surgical resection or biopsy
Sampling is invasive and could put patients at risk
Samples may be difficult to obtain, especially at disease progression
Single biopsy sampling may be subject to selection bias due to tumour heterogeneity

Cytology samples^1,2,38

Frequently used in absence of tumour biopsy in patients with NSCLC
Samples can be obtained using minimally invasive procedures, such as fine needle aspiration

Plasma ctDNA^2,5

Blood-based, minimally invasive
Alternative to tissue as a source of tumour DNA
In patients with EGFR mutations, can be used at diagnosis or progression when a tumour sample is not available
When plasma ctDNA is used for biomarker testing, a negative result should be followed with a confirmatory tumour tissue test where feasible

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.

Key considerations regarding specimens used for NSCLC biomarker testing^2,41–43

Tissue testing

Key considerations

Invasive approach that may not be appropriate for all patients⁴¹
Due to heterogeneity, some mutations may only appear in certain metastases or certain parts of the tumour⁴²

Plasma ctDNA testing

Key considerations

Not all tumours shed enough DNA to be detected in plasma⁴¹
Sensitivity varies by technology⁴³

cfDNA cell-free DNA and ctDNA circulating tumour DNA

cfDNA, cell-free DNA; ctDNA, circulating tumour DNA

The videos below provide further information on the clinical application and implementation of ctDNA testing in NSCLC.

Overview of ctDNA testing techniques: current and future clinical utility of ctDNA

Veeva ID: Z4-61364 | Date of preparation: January 2024

Application: current and future clinical utility of ctDNA

Dr Stephen Finn (Associate Professor Consultant, Histopathology and Principal Investigator, The University of Dublin, Trinity College and St James’ Hospital, Dublin, Ireland) and Chris Abbosh (Senior Director, Translational Medicine, AstraZeneca) provide an overview of ctDNA testing techniques, and discuss when ctDNA testing can be used to inform clinical decision making for patients with NSCLC.

Play video

Discussion on how ctDNA and tissue can be used in a complementary fashion - the Yin and Yang of ctDNA and tissue

Veeva ID: Z4-61365 | Date of preparation: January 2024

Implementation:
the Yin and Yang of ctDNA and tissue in the management of patients with NSCLC

Dr Phillipe Taniére (Consultant Histopathologist/Molecular Pathologist, University Hospitals Birmingham NHS Foundation Trust, UK) and Prof. Sanjay Popat (Consultant Medical Oncologist at The Royal Marsden and Professor of Thoracic Oncology, The Institute of Cancer Research, UK) discuss how ctDNA and tissue can be used in a complementary fashion, and comment on the technical challenges associated with sample acquisition in NSCLC.

Play video

Discusión on the interpretation of molecular test reports generated from ctDNA tests

Veeva ID: Z4-61366 | Date of preparation: January 2024

Implementation:
optimising practise variables for ctDNA testing in NSCLC

Dr Michelle Shiller (Associate Medical Director, Genomic and Molecular Pathology Services, Path Group and Medical Director, Baylor Sammons Cancer Center, US) shares her perspective on the management of analytical and pre-analytical variables associated with ctDNA testing. Using four case studies, Dr Tracy Stockley (Head, Division of Clinical Laboratory Genetics, Princess Margaret Cancer Centre, University Health Network, Toronto, and Professor, Department of Lab Medicine and Pathobiology, University of Toronto, Canada) discusses the interpretation of molecular test reports generated from ctDNA tests.

Play video

Veeva ID: Z4-61367 | Date of preparation: January 2024

Implementation:
establishing ctDNA testing in your institution

Prof. Paul Hofman (Professor of Pathology and Head of the Laboratory of Clinical and Experimental Pathology, Pasteur Hospital, Nice Sophia Antipolis University, Nice, France) and Prof. Umberto Malapelle (Department of Public Health, University Federico II of Naples, Napes, Italy) recount their experience of introducing liquid biopsy into their respective institutions, and discuss the variables that their teams considered when developing their testing methodologies.

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Techniques for acquiring specimens for biomarker testing
Tumour or cytology biopsies can be acquired through surgical and non-surgical techniques.

Surgical techniques
VATS:	a minimally invasive surgical procedure in which a thoracoscope is inserted into the chest via small incisions⁴⁴
Mediastinoscopy:	a surgical procedure used to examine the mediastinum by the insertion of a mediastinoscope⁴⁵

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 airways⁴⁶
EUS-FNA:	an endoscope with an ultrasound probe and biopsy needle that is inserted into the oesophagus and allows guided biopsy⁴⁷
EBUS-TBNA:	a transbronchial approach involving FNA of mediastinal lesions using an endobronchial ultrasound probe⁴⁸
Plasma sample:	ctDNA, part of the circulating cell-free DNA coming from tumour cells, can be extracted from plasma samples for molecular testing⁴⁹

EBUS guide for interventional bronchoscopists

The instructional animations below provide expert guidance from Dr Neal Navani (Consultant Respiratory Physician, University College London Hospital, London, UK)
on safely and optimally performing EBUS-TBNA.

Veeva ID: Z4-60055 | Date of preparation: January 2024

EBUS guide for interventional bronchoscopists

Play video

EBUS Guide Part 1 Video Series - Advancing Lung Cancer Diagnostics with EBUS-TBNA

Veeva ID: Z4-61362 | Date of preparation: January 2024

EBUS guide for interventional bronchoscopists Part 1

Play video

EBUS Guide Part 2 Video Series – Procedural tips for Enhancing the yield of EBUS-TBNA

Veeva ID: Z4-61363 | Date of preparation: January 2024

EBUS guide for interventional bronchoscopists Part 2

Play video

Methods of biomarker testing

RT-PCR

RT-PCR is a highly targeted technique that is useful to detect point mutations, insertions and deletions in genes of interest.^29,50 Benefits of RT-PCR include the relatively short turnaround time (<4 days) and high sensitivity.⁵⁰

RT-PCR is commonly used to detect genetic mutations.^7,17 The cobas EGFR Mutation Test v2 (Roche Molecular Systems) and therascreen EGFR

RGQ PCR Kit (Qiagen) are FDA-approved companion RT-PCR assays for the determination of various EGFR mutations in NSCLC samples.^51–53

Despite the use of RT-PCR for the detection of point mutations, this approach is not typically used to detect gene fusions and rearrangements.^11,29

NGS

NGS enables a broader assessment of clinically relevant genes, genome or exome, compared with
PCR-based approaches.⁵⁰ Depending on the subtype of NGS used, it is possible to simultaneously detect point mutations, insertions, deletions, copy number alterations and gene rearrangements.²⁹ Multiplex sequencing is recommended over single-gene mutation tests in order to identify other mutations beyond those routinely screened for.^2,11,29,50

NGS panels are commonly amplicon- or hybrid capture-based.¹¹ 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.¹¹

Examples of NGS panels that are FDA-approved companion diagnostic devices for use in NSCLC include FoundationOne CDx (Foundation Medicine) and Oncomine Dx Target Test (Life Technologies).⁵¹

FISH

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.⁵⁴

Gene fusions and rearrangements in NSCLC samples are commonly detected through FISH assays.^2,9–11,16

IHC

IHC utilises antigen-antibody interactions to visualise the quantity and distribution of a protein of interest in patient samples.⁵⁵

PD-L1 expression can only be determined by IHC.^28,56

An overview of the different steps involved in PD-L1 IHC can be seen below. Briefly, samples are initially processed and mounted onto microscope slides. Sample sections are then stained for PD-L1 by using a commercial IHC assay with an automated staining platform. Stained samples are then quality control checked by a trained pathologist.

Steps performed during PD-L1 testing

Sections cut

Sections mounted

Slides dried

Slides labelled

Automated IHC stainer

Staining quality checked

ABBREVIATION:
IHC, immunohistochemistry

Before PD-L1 expression can be assessed, samples must be appropriately processed in line with the recommended PD-L1 IHC assay recommendations.

Standard tissue sample preparation and processing procedure for PD-L1 testing^*28,57–59

Formalin fixation

10% (v/v) neutral-buffered formalin
Optimal fixation times vary between assays

Paraffin embedding

Embed tumour sample in a paraffin block

Sectioning

Cut 4–5µm sections onto microscope slides
Dry slides at 58 ± 2ºC for 1 hour
Store at 2–8ºC or room temperature, to protect from light

*The preparation instructions differ for each PD-L1 assay. For further details, refer to specific package insert for individual assays.

There are five IHC assays, either commercially available or in development, which can be used to determine PD-L1 expression. The choice of PD-L1 IHC assay and the staining procedure is used in line with specific immunotherapies according to their respective diagnostic labels.

Table of specific immunotherapies according to their respective diagnostic labels

*Based on the FDA and CE-IVD diagnostic regulatory approval⁵¹

ABBREVIATIONS:
BMS, Bristol Myers Squibb; CE-IVD, Conformité Européenne-in vitro diagnostic; Dx, diagnostic; FDA, Food and Drug Administration; IHC, immunohistochemistry; MSD, Merck Sharp & Dohme; N/A, not applicable; PD-L1, programmed cell death ligand-1

An LDT is an alternative approach for those with limited or no access to commercially developed PD-L1 assays. In this regard, LDTs are PD-L1 IHC assays that contain components from a variety of sources and apply custom procedures (de-novo LDT); or are based on the components from a regulatory-approved assay, but either apply a custom procedure to detect PD-L1 or for a different purpose (kit-derived LDT).⁷⁰

The use of LDTs is associated with less reliable capture of PD-L1 positivity in NSCLC samples, when compared with approved PD-L1 assays.^71,72

It is important that LDTs are adequately validated before their use on patient samples.⁷⁰

ABBREVIATIONS:
ALK, anaplastic lymphoma kinase; BRAF, B-Raf proto-oncogene; CDx, companion diagnostic; CE-IVD, Conformité Européenne-in vitro diagnostic; ctDNA, circulating tumour DNA; Dx, diagnostic; (EBUS)-TBNA, (endobronchial ultrasound-)guided transbronchial needle aspiration; EGFR, epidermal growth factor receptor; EUS-FNA, endoscopic ultrasound-guided fine needle aspiration; FDA, US Food and Drug Administration; FFPE, formalin-fixed, paraffin-embedded; FISH, fluorescent in situ hybridisation; HER2, human epidermal growth factor receptor 2; IHC, immunohistochemistry; KRAS, Kirsten rat sarcoma virus; LDT, laboratory-developed test; MET, MET proto-oncogene, receptor tyrosine kinase; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; NTRK1/2/3, neurotrophic receptor tyrosine kinase 1/2/3; PCR, polymerase chain reaction; PD-L1, programmed cell death ligand-1; RET, rearranged during transfection; RGQ, Rotor-Gene Q; ROS1, ROS proto-oncogene 1; RT-PCR, real-time polymerase chain reaction; VATS, video-assisted thoracoscopic surgery

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Technical Considerations

How do I test?

Jump to section:

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

ABBREVIATIONS:

Additional testing considerations for ROS1 rearrangements

Additional testing considerations for PD-L1 expression

Biomarker testing sample considerations

Overview of different specimens used for biomarker testing in NSCLC1,2,5,29,38–40

Biomarker status can be determined using various sample types

Tumour tissue1,2,29,38–40

Cytology samples1,2,38

Plasma ctDNA2,5

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.

Key considerations regarding specimens used for NSCLC biomarker testing2,41–43

Tissue testing

Key considerations

Plasma ctDNA testing

Key considerations

The videos below provide further information on the clinical application and implementation of ctDNA testing in NSCLC.

Application: current and future clinical utility of ctDNA

Implementation: the Yin and Yang of ctDNA and tissue in the management of patients with NSCLC

Implementation: optimising practise variables for ctDNA testing in NSCLC

Implementation: establishing ctDNA testing in your institution

Techniques for acquiring specimens for biomarker testing

Surgical techniques

Non-surgical techniques

EBUS guide for interventional bronchoscopists

EBUS guide for interventional bronchoscopists

EBUS guide for interventional bronchoscopists Part 1

EBUS guide for interventional bronchoscopists Part 2

Methods of biomarker testing

RT-PCR

NGS

FISH

IHC

Steps performed during PD-L1 testing

Before PD-L1 expression can be assessed, samples must be appropriately processed in line with the recommended PD-L1 IHC assay recommendations.

Standard tissue sample preparation and processing procedure for PD-L1 testing*28,57–59

Formalin fixation

Paraffin embedding

Sectioning

There are five IHC assays, either commercially available or in development, which can be used to determine PD-L1 expression. The choice of PD-L1 IHC assay and the staining procedure is used in line with specific immunotherapies according to their respective diagnostic labels.

REFERENCES:

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Technical
Considerations

Overview of different specimens used for biomarker testing in NSCLC^{1,2,5,29,38–40}

Tumour tissue^{1,2,29,38–40}

Cytology samples^1,2,38

Plasma ctDNA^2,5

Key considerations regarding specimens used for NSCLC biomarker testing^2,41–43

Implementation:
the Yin and Yang of ctDNA and tissue in the management of patients with NSCLC

Implementation:
optimising practise variables for ctDNA testing in NSCLC

Implementation:
establishing ctDNA testing in your institution

Standard tissue sample preparation and processing procedure for PD-L1 testing^*28,57–59