Molecular testing

Testing techniques

Professor Fernando Lopez-Rios, professor of Pathology and Molecular Pathology at San Pablo CEU University in Madrid, Spain, discusses molecular testing techniques that can detect rearranged during transfection (RET) gene alterations.

Molecular profiling identifies specific cancer biomarkers in patients and is an essential part of precision medicine¹

Cancer biomarkers include specific DNA, RNA or protein molecules that can inform diagnosis and prognosis and help guide treatment decisions¹. The proto-oncogene RET is one such biomarker. When altered by point mutation or fusion, RET gives rise to a hyperactive RET protein that triggers downstream signalling pathways implicated in certain cancers².

The RET inhibitors selpercatinib and pralsetinib are licenced for use in non-small cell lung cancer (NSCLC) and thyroid cancer patients harbouring a RET alteration^3,4.

Molecular testing is essential for identifying patients with NSCLC and thyroid cancer who harbour RET alterations and are eligible for targeted therapy with RET inhibitors

Numerous molecular profiling techniques have been used to detect RET alterations, including next-generation sequencing (NGS), fluorescence in situ hybridisation (FISH) and polymerase chain reaction (PCR) (Figure 1)⁵. The suitability of each test depends on factors such as the number and type of alterations (point mutation or fusion) to screen for, the type, amount and quality of sample, cost, and availability⁵. All tests have their own strengths and limitations, which should also be factored into decision-making when screening patients.

Figure 1. Molecular testing techniques suitable for the detection of RET alterations in medullary thyroid cancer and NSCLC (Adapted^5–7). FISH, fluorescence in situ hybridisation; MTC, medullary thyroid cancer; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; qPCR, quantitative polymerase chain reaction; RT-PCR, reverse transcription polymerase chain reaction.

During the ARROW and LIBRETTO-001 studies, patients with RET alterations were identified using a range of techniques, including DNA and RNA sequencing, FISH, and PCR^8,9

Sanger sequencing

DNA sequencing techniques are used to determine the sequence of nucleotide bases in a piece of DNA. Sanger sequencing was the first such technique to be developed and involves using the DNA molecule being sequenced as a template for DNA synthesis¹⁰. Fluorescently labelled nucleotides bind to complementary nucleotides on the template strand of DNA, indicating the genetic sequence^10,11. This technique can be used to detect RET point mutations but has generally been superseded by NGS technologies^5,11.

Next-generation sequencing

NGS refers to a collection of high-throughput technologies that can be used to detect mutations, copy number variations (where the number of copies of a gene varies between individuals) and gene fusions by sequencing DNA or RNA^5,12,13.

NGS involves sequencing millions of small fragments of nucleic acid (called ‘reads’) in parallel using fluorescently tagged nucleotides^12,14. The fragments are reassembled using bioinformatical analysis and compared to a human reference genome to identify regions that differ^12,14.

Different NGS platforms differ in the type of sample used and how much of the genome or transcriptome is sequenced (Table 1)¹⁵.

Table 1. Overview of different NGS techniques used in oncology (Adapted^15–17). DNA, deoxyribonucleic acid; NGS, next-generation sequencing; RNA, ribonucleic acid.

Numerous tests capable of screening for RET alterations are commercially available⁵. DNA-based NGS tests, particularly targeted panels, are useful for identifying RET point mutations in hotspot regions of the genome and can sometimes also identify gene fusions, if designed in the correct way⁵. Targeted RNA-based panels may also be used to identify RET point mutations and are the preferred option when screening for RET gene fusions⁵.

The European Society for Medical Oncology and the National Comprehensive Cancer Network recommend molecular testing for RET alterations in NSCLC and thyroid cancer patients, typically via next-generation sequencing^5,18

Quantitative polymerase chain reaction

Quantitative polymerase chain reaction (qPCR) is a technique that involves the amplification of small DNA sequences for quantification and analysis.

Reverse transcription polymerase chain reaction (RT-PCR) measures gene transcription by using an RNA strand to create a complementary DNA molecule, which is subsequently amplified and quantified¹³. This technique can indicate which genes are actively being transcribed in a patient.

DNA-based qPCR is not usually suitable for identifying gene fusions but can be used to identify selected RET point mutations in hotspot regions⁵. In contrast, RT-PCR can be used to identify RET fusion transcripts along with the fusion partner, so long as the partner is known and the specific probe is available^5,6.

Fluorescence in situ hybridisation

Fluorescence in situ hybridisation (FISH) identifies a specific segment of DNA or RNA within tissue sections and can be used to identify gene deletions, amplifications, translocations and fusions^13,19. During FISH, the sample is mixed with a short fluorescently labelled probe¹⁹. The probe binds to the complementary sequence and the fluorescent label emits light when viewed under a fluorescent microscope to identify and localise the specific DNA or RNA¹⁹.

FISH can be used to identify RET fusions. Unlike RT-PCR, the fusion partner does not need to be known in order to detect a fusion alteration; however, FISH cannot identify what the fusion partner is⁶.

Table 2 summarises the different techniques available for detecting RET rearrangements in NSCLC and thyroid cancer.

Table 2. Summary of the techniques available for detecting RET rearrangements (Adapted⁵). DNA-seq NGS, DNA sequencing by next-generation sequencing; FISH, fluorescence in situ hybridisation; NGS, next-generation sequencing; RNA-seq NGS, RNA sequencing by next-generation sequencing; RT-PCR, reverse transcription polymerase chain reaction.

Limitations of alternative techniques

Immunohistochemistry (IHC) is a technique that has historically been used to screen for RET alterations⁵. IHC involves using an enzyme or fluorescently labelled antigen-specific antibody to visualise protein expression in tissue samples¹. As parafollicular C cells, but not follicular cells, express RET protein in healthy thyroid tissue, RET protein overexpression was assumed to indicate a RET alteration⁵. However, IHC has been found to have poor sensitivity and specificity in detecting RET alterations and is therefore not deemed a suitable screening tool to detect RET fusions or mutations in non-small cell lung cancer (NSCLC) or thyroid cancer patients⁵.

IHC is not recommended for detecting RET alterations in NSCLC or thyroid cancer patients⁵

Other traditional molecular techniques also have limitations. The utility of fluorescence in situ hybridisation (FISH) and quantitative polymerase chain reaction (qPCR) is limited by the restricted number of genes that can be interrogated in a single assay⁷. There is also limited scope for identifying novel mutations and fusion partners using these techniques^5,7.

In contrast, next-generation sequencing (NGS) is a high-throughput technique, allowing for simultaneous sequencing of the entire genome or transcriptome, or multiple targeted regions²⁰. Consequently, numerous alterations can be identified in parallel, in multiple samples, during the same assay²⁰. This is a clear advantage in patients with RET-altered cancers, where several actionable mutations may be present⁵. It also reduces the need for multiple tests, minimising turnaround time and the amount of sample required²⁰.

With regard to the different NGS platforms, each have their own strengths and weaknesses. For example, DNA-based sequencing can identify the exact breakpoint of a gene fusion but does not determine if an alteration (fusion or mutation) is transcribed^15,16. In contrast, RNA-based sequencing identifies only those alterations that are expressed¹⁶. However, as RET gene fusions primarily occur in introns, which are removed from RNA, RNA-based sequencing cannot detect the fusion breakpoint location^15,16.

RNA sequencing is more sensitive than DNA sequencing but requires high-quality RNA, which is less stable than DNA and susceptible to degradation, especially in formalin-fixed paraffin-embedded samples^15,16.

Targeted panels are generally more suitable in a clinical setting than genome or transcriptome-wide sequencing, owing to the lower sample requirements, faster turnaround time and less complex data interpretation¹⁵. However, whole genome or transcriptome sequencing may be appropriate when searching for novel alterations¹⁶.

Sample requirements

In the following videos, Professor Fernando Lopez-Rios details the journey of the clinical sample for molecular testing and sample requirements in both non-small cell lung cancer (NSCLC) and thyroid cancer.

Different molecular tests require different types of samples and preparation techniques (Figure 2).

Figure 2. Sample requirements for different molecular testing techniques for the identification of RET alterations in NSCLC and thyroid cancer (Adapted^5,17). DNA, deoxyribonucleic acid; FISH, fluorescence in situ hybridisation; NGS, next-generation sequencing; qPCR, quantitative polymerase chain reaction; RNA, ribonucleic acid; RT-PCR, reverse transcription polymerase chain reaction.

Testing for germline RET mutations is performed in blood or sputum samples, while screening for somatic RET mutations requires a tissue sample or liquid biopsy⁵

For a diagnosis of rearranged during transfection (RET)-altered cancer, next-generation sequencing (NGS) or quantitative polymerase chain reaction (qPCR) is typically performed on biopsy-derived tissue samples^5,17,19. Similarly, fluorescence in situ hybridisation (FISH) is performed on tissue samples that have been cut into very thin sections and attached to a glass slide. Tissue samples are preserved, usually by freezing them or by fixing in formalin before paraffin-embedding^13,19. Fresh frozen tissue is preferrable when performing DNA or RNA analysis but requires tissue to be frozen in liquid nitrogen soon after resection, and samples need to be kept at very low temperatures for long-term storage²¹.

Formalin-fixed and paraffin-embedded (FFPE) samples can be stored at room temperature for years and are routinely used in clinical practice; however, fixation can degrade RNA, which may have implications when performing NGS and RT-PCR on FFPE samples, where sample purity is imperative^5,21.

Molecular testing of tissue samples is often not possible, due to lack of availability or poor quality of biopsy samples⁷. An alternative is a liquid biopsy, which measures cell-free DNA (cfDNA) shed from tumour cells in a peripheral blood sample^7,13. Liquid biopsies are well-validated and approved by the Food and Drug Administration (FDA) and can be used to identify RET alterations in NSCLC and thyroid cancer patients^2,7. Blood samples are easily obtained and less invasive than tissue biopsies. They also allow repeated screening for acquired mutations associated with treatment resistance^7,13.

The appropriate test for identifying patients eligible for treatment with RET inhibitors depends on the type of malignancy and the sample available⁵

Diagnostic algorithms, such as those provided by the European Society for Medical Oncology (ESMO) can assist physicians in performing molecular assessments in patients suspected of having RET-altered cancer, including NSCLC and thyroid cancer⁵.

Patients with NSCLC or non-medullary thyroid cancer (MTC) should be tested for RET fusions (Figure 3). If FFPE samples are available, these should be screened using NGS. If NGS is not available, FISH or reverse transcription polymerase chain reaction (RT-PCR) may be used, but if the result is negative, an NGS panel is recommended⁵.

If FFPE samples are not available, a liquid biopsy can be performed and analysed for RET fusions using NGS or RT-PCR⁵. However, if a RET alteration is not detected in the liquid biopsy sample, analysis of a tumour biopsy sample will still be required to exclude the possibility of a RET fusion⁵.

Figure 3. Diagnostic algorithm for identifying RET fusions in patients with NSCLC and non-MTC (Adapted⁵). FFPE, formalin-fixed paraffin-embedded; FISH, fluorescent in situ hybridisation; MTC, medullary thyroid cancer; NGS, next-generation sequencing; NSCLC, non-small cell lung cancer; Q-PCR, quantitative polymerase chain reaction; RET, rearranged during transfection; RT-PCR, reverse transcription polymerase chain reaction.

For patients with MTC, germline RET mutations can be identified in sputum or blood samples via qPCR or NGS. Alternatively, known hereditary mutations may be identified using Sanger sequencing in blood leukocyte DNA (Figure 4)⁵. If no germline RET mutations are identified in MTC patients, and the disease metastasises, FFPE samples from the metastatic site should be acquired and analysed using qPCR or NGS⁵.

Figure 4 . Diagnostic algorithm for identifying RET alterations in patients with MTC (Adapted⁵). FFPE, formalin-fixed paraffin-embedded; MTC, medullary thyroid cancer; NGS, next-generation sequencing; Q-PCR, quantitative polymerase chain reaction; RET, rearranged during transfection.

Interpreting results

Professor Fernando Lopez-Rios, Dr Alexander Drilon and Professor Lori Wirth discuss how to interpret molecular testing results in non-small cell lung cancer (NSCLC) and thyroid cancers.

The data provided by different molecular tests can vary in quantity and complexity. For example, analysis of fluorescence in situ hybridisation (FISH) data is relatively simple, with samples deemed positive if a signal is demonstrated by ≥15% of cells⁶. In contrast, next-generation sequencing (NGS) produces extremely large amounts of complex data that require expertise in bioinformatics for analysis¹¹. Untargeted NGS tests can identify millions of points of difference between the patient and reference genomes²². The majority of these variants may have little or no clinical significance and so data must be filtered to identify data pertinent to patient care, such as RET alterations in patients with certain types of cancer²².

Clinical testing laboratories typically categorise germline or somatic alterations based on their pathogenicity and impact on clinical management, respectively, in accordance with published guidelines^23,24. It is recommended that genetic variants are annotated and reported according to Human Genome Variation Society (HGVS)/Human Genome Organisation (HUGO) nomenclature, alongside the colloquial terminology^23,24. Reporting of RET gene fusions should include the fusion gene partner (for example, RET/KIF5B)²³. Figure 5 provides an example of how a point gene mutation may be reported, according to the recommended nomenclature and a breakdown of the meaning of each section.

Figure 5. Example RET point mutation reported according to HGVS/HUGO nomenclature (Adapted²⁵). cDNA, coding DNA; DNA, deoxyribonucleic acid; RET, rearranged during transfection.

The multidisciplinary team involvement

Molecular test results are often discussed initially amongst pathologists before a wider multidisciplinary team (MDT) meeting is convened. This team meeting is a collaborative effort among the MDT to provide a comprehensive package of care for the patient²⁶.

Indeed, the advent of precision medicine has seen multidisciplinary care of cancer patients extend to include those with expertise in molecular profiling in the form of a molecular tumour board (MTB)²⁷. A MTB typically comprises oncologists, research scientists, bioinformaticians, pathologists, medical geneticists, genetic counsellors and genomicists (Figure 6) with the expertise to interpret complex molecular data and support physicians in applying it to treatment decisions²⁸.

Figure 6. Members of a molecular tumour board involved in the interpretation of the results of molecular tests²⁸. MTB, molecular tumour board.

Germline testing should be performed in patients with a family history of RET mutations or familial MTC caused by an unknown RET alteration. Genetic counselling is recommended for patients found to harbour a germline RET mutation^5,23

Although MTBs are not currently universal, increased use of molecular testing has been seen among physicians who have this service available to them²⁸. MTBs are likely to become more widespread as the field of precision medicine grows.

Access to testing

Despite the advent of next-generation sequencing (NGS), traditional techniques such as fluorescence in situ hybridisation (FISH) and quantitative polymerase chain reaction (qPCR) are still widely used in the clinic¹⁶. While the cost of sequencing has declined considerably in recent years, NGS remains a costly technique that is not available in all healthcare systems²⁸. Much of the cost associated with NGS is due to the infrastructure required. For example, high performance computational facilities, plus the personnel to operate them, are required to store and analyse the vast amounts of data produced by NGS^11,29.

Health care providers need to ensure that molecular testing facilities are accessible to all patients to provide optimal care³⁰

Systems must also be put in place to align the various teams involved in the sequencing workflow³⁰. Firstly, a commitment from care providers to access molecular diagnostic facilities is required³⁰. Molecular diagnostic facilities must keep abreast of developments in precision oncology to ensure that they are operating in line with the most up-to-date evidence-based guidelines³⁰. Finally, coordination of experts from a variety of fields is required to provide a multidisciplinary approach when acting upon molecular data³⁰.

As molecular testing is vital to support targeted therapy in patients with RET-altered non-small cell lung cancer (NSCLC) and thyroid cancer, alternatives must be considered if NGS is not accessible. FISH, qPCR or reverse transcription polymerase chain reaction (RT-PCR) can all be used to detect RET aberrations, depending on the type of malignancy and alteration in question⁵.

References

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