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Molecular diagnostics for RET inhibition in NSCLC and thyroid cancers

Molecular testing

Read time: 80 mins
Last updated:10th Sep 2021
Published:10th Sep 2021

Learn more about the molecular testing techniques used to identify RET alterations in patients.

  • Explore current diagnostic technology, such as next-generation sequencing (NGS), and understand the types of samples needed
  • Learn about the interpretation of results
  • Realise the strengths and limitations of different molecular testing techniques

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 alteration3,4.

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.

RET_T3_Fig_1_AUG2021.png

Figure 1. Molecular testing techniques suitable for the detection of RET alterations in medullary thyroid cancer and NSCLC (Adapted5–7). 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 PCR8,9

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 sequence10,11. This technique can be used to detect RET point mutations but has generally been superseded by NGS technologies5,11.

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 RNA5,12,13.

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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.

NSCLC sample requirements for RET alteration detection

Thyroid cancer sample requirements for RET alteration detection

1
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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.

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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 NGS11,29.

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.

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References

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  2. Salvatore D, Santoro M, Schlumberger M. The importance of the RET gene in thyroid cancer and therapeutic implications. Nature Reviews Endocrinology. 2021;17(5):296–306.
  3. Pralsetinib® Prescribing Information. https://www.accessdata.fda.gov/drugsatfda_docs/label/2020/214701s000lbl.pdf. Accessed 14 July 2021.
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  5. Belli C, Penault-Llorca F, Ladanyi M, Normanno N, Scoazec JY, Lacroix L, et al. ESMO recommendations on the standard methods to detect RET fusions and mutations in daily practice and clinical research. Annals of Oncology. 2021;32(3):337–350.
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  8. Subbiah V, Hu MI, Wirth LJ, Schuler M, Mansfield AS, Curigliano G, et al. Pralsetinib for patients with advanced or metastatic RET-altered thyroid cancer (ARROW): a multi-cohort, open-label, registrational, phase 1/2 study. The Lancet Diabetes and Endocrinology. 2021;9(8):491–501.
  9. Wirth LJ, Sherman E, Robinson B, Solomon B, Kang H, Lorch J, et al. Efficacy of Selpercatinib in RET-Altered Thyroid Cancers. New England Journal of Medicine. 2020;383(9):825–835.
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  17. NGS Basics for Cancer Research | Thermo Fisher Scientific - UK. https://www.thermofisher.com/uk/en/home/life-science/sequencing/sequencing-learning-center/next-generation-sequencing-information/cancer-research/basics.html. Accessed 27 July 2021.
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  24. Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine. 2015;17(5):405–424.
  25. HGVS recommendations: nomenclature for the description of sequence variants. https://www.hgvs.org/mutnomen/recs.html#intro. Accessed 9 August 2021.
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  1. Leukemia and Lymphoma Society: Cancer Molecular Profiling. https://www.lls.org/sites/default/files/National/USA/Pdf/Publications/FS31_Cancer_Molecular_Profiling.pdf. Accessed 22 July 2021.
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