Testing for
Oncologists

 

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

Initial Testing (Diagnosis)

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

Testing on Progression

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

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Mutations

BRAF mutations

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

EGFR mutations

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

Test for EGFR T790M

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Gene fusions and rearrangements

ALK fusions

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

ROS1 rearrangements

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

 

Guidelines

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.