Retsevmo® ▼ (selpercatinib): RET Testing

RET-Altered Cancers

Molecular tumor testing is performed to

• facilitate a diagnosis by identifying tumor characteristics

• identify predictive and prognostic biomarkers that can be used when looking at possible clinical outcomes

• identify appropriate patients for clinical trials, and

• identify appropriate targeted or systemic therapies.¹

Molecular testing may also identify actionable mutations, defined as mutations that a current or investigational product directly or indirectly targets.²

RET-altered cancers is a broad term that encompasses 2 sets of genetic changes including RET fusions and RET mutations.^3,4

These alterations activate downstream RET signaling pathways promoting cell proliferation and survival in cancers.³

Molecular Tumor Testing

The most appropriate molecular testing method depends on the alteration type. Table 1 provides an overview of recommended testing methods.

Table 1. Testing Methods by Alteration Type

Testing Method
Alteration Type	Allele-Specific PCR5	FISH6-11	IHC6,11-13	Microarray14	NGS3,15,16	RT-PCR17	Sanger Sequencing18
Single nucleotide substitutions and indels	√	NA	NA	NA	√	NA	√
Copy number amplifications	NA	√	NA	√	√	NA	NA
Rearrangements and fusions	NA	√	NA	NA	√	√	NA
Protein level expression	NA	NA	√	NA	NA	NA	NA

Abbreviations: FISH = fluorescence in situ hybridization; IHC = immunohistochemistry; NA = not applicable; NGS = next-generation sequencing; PCR = polymerase chain reaction; RT = reverse transcription.

There are several testing methods appropriate for diagnosing RET-mutations.

Next generation sequencing is a broad, comprehensive testing method that analyzes DNA and/or RNA to detect RET.⁶ This method allows for multiplex testing on a small amount of tissue and detection of both common and rare cancer-related biomarkers.¹⁵ The NGS platform

• allows for accurate and sensitive detection of gene fusions, mutations, and amplifications

• affords the potential identification of RET fusions as in-frame events, and

• allows the detection of upstream partners and concurrent genomic alterations that involve genes other than RET.^3,6,11,15

Next generation sequencing is suitable for formalin-fixed, paraffin-embedded samples, and can be adapted to analyze liquid biopsy specimens.⁶

Although it is more invasive for the patient, tissue testing is preferred because

• it is possible that a RET alteration that is found from a biopsy may not be found in the blood through liquid biopsy, and

• up to 30% of RET alterations may be missed if only ctDNA is tested.^19,20

Table 2 provides an overview of RET testing methods in order of preference.

Table 2. RET Testing Methods

Testing Methoda	Description	Advantage	Disadvantage	Appropriate for RET Testing	Time Needed to Complete Test
NGS3,15,16	Allows for multiplex testing on a small amount of tissue Detects rare and common cancer-related biomarkers	Highly sensitive and specific Simultaneous query for potentially actionable targets Detects known and novel fusions	No standardized model or guidelines regarding NGS in clinical practice May identify low frequency variants with unknown interpretation on significance	Yes	2-4 weeks21
DNA-based NGS15,22-24		RET identification/ mutation information	Poor coverage of some intronic areas	Yes
RNA-based NGS6,15,17		Unbiased fusion information; no intron coverage issues	Affected by quality of RNA	Yes
RT-PCR11,25-29	Uses RNA from tissue to make cDNA and uses separate probes for each 5’ partner	Fast Relatively inexpensive Specific at identifying known RET fusion partner and will not detect novel RET rearrangements	PCRs are designed for the predominant fusions and the actual frequency of RET fusions are underestimated	Yes	1-2 days21
FISH6-11,30	Locates the positions of specific DNA sequences on chromosomes	Highly sensitive, definitive standard for detecting a RET rearrangement, regardless of fusion partner	Lack of standard cutoffs for defining RET fusion positivity results in poor specificity High rate of FP/FN Unable to detect mutation information	Rare circumstances	2-3 days21
IHC6,11-13	Uses antibodies to find tissue antigens that are expressed with certain cancers	Widely available	Unreliable at detecting RET rearrangements Low sensitivity Variable specificity Significant FP/FN	No	2 days31

Abbreviations: cDNA = complementary deoxyribonucleic acid; FISH = fluorescence in situ hybridization; FN = false-negative; FP = false-positive; IHC = immunohistochemistry; NGS = next-generation sequencing; PCR = polymerase chain reaction; RET = rearranged during transfection; RT = reverse transcription.

^a Listed in descending order of acceptability.

Clinical Guidelines for Testing

Non-Small Cell Lung Cancer

In general, IASLC, CAP, and AMP recommend that patients with adenocarcinoma be tested for EGFR, ALK, and ROS1 variants. It is also recommended that BRAF could be included in a broader panel.^25,26

In addition to these recommendations, the National Comprehensive Cancer Network^® (NCCN^®) also recommend testing for MET exon 14 skipping mutations and NTRK gene fusions.³²

An overview of clinical guidelines for RET testing in lung cancer is provided in Table 3.

Table 3. Clinical Guidelines for RET Testing in Lung Cancer

	NCCN®32a	IASLC/CAP/AMP25,26	ESMO33
RET testing	Category 2A recommendation as part of routine biomarker screening.bc	Consider testing Not recommended as a routine stand-alone assay Appropriate as part of a larger panel either initially or if negative for EGFR, ALK, and ROS1	Targeting/testing not currently routinely recommended
Other recommendations	Molecular testing and advise broad molecular profilingin effort to identify rare driver mutations for which therapy is currently available or clinical trials are ongoing.b	Broad multiplex/NGS panels preferred over single-gene testing to identify wider range of actionable alterations	Recruitment into open clinical trials encouraged

Abbreviations: ALK = anaplastic lymphoma kinase; AMP = Association for Molecular Pathology; CAP = College of American Pathologists; EGFR = epidermal growth factor receptor; ESMO = European Society for Medical Oncology; IASLC = International Association for the Study of Lung Cancer; NCCN = National Comprehensive Cancer Network; NGS = next generation sequencing; RET = rearranged during transfection.

^a  NCCN^® makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way.

^b The NCCN^® Guidelines for NSCLC provide recommendations for individual biomarkers that should be tested and recommend testing techniques but do not endorse any specific commercially available biomarker assays.

^c Category 2A: Based on lower-level evidence, there is uniform NCCN consensus that the intervention is appropriate.

Thyroid

Molecular testing in thyroid cancer may help in available treatment options, as well as identify patients eligible for clinical trials.³⁴

Medullary thyroid cancer is a subset of approximately 1% to 2% of all thyroid cancer cases.³⁵ Approximately 75% of MTC cases are sporadic and 25% are hereditary.³⁵ Molecular testing can classify somatic MTC as MEN2a (which includes associated conjugated linoleic acid or Hirschsprung’s Disease) or MEN2b. Guidelines vary depending on how somatic MTC is classified.³⁶

Molecular testing can also determine the presence of RET. Most patients with MEN2A or MEN2B and familial MTC have RET germline mutations and approximately 50% of patients with sporadic MTC have somatic RET mutations.³⁶

Approximately 15% to 30% of thyroid nodules are classified as cytologically indeterminate by FNA at the time of diagnosis. Molecular testing has been developed to aid in diagnosis and appropriate management of these lesions and to

• avoid unnecessary surgery for benign thyroid nodules

• identify high-risk cancers for possible total thyroidectomy, and

• identify premalignant nodules with low-to-intermediate risk for possible lobectomy.³⁷

An overview of RET testing guidelines in thyroid cancer is provided in Table 4.

Table 4. Clinical Guidelines for RET Testing in Medullary Thyroid Cancer

	ATA38 (at Diagnosis)	ATA36 (Somatic Testing of Tumor for Prognostic and Treatment Decisions)	ESMO39	NCCN®ab34
RET testing	Consider testing in suspected cytology with a 7-gene mutation panel	Test in: MTC sporadic MTC 1st-degree relatives of patients with MTC patients with CLA parents of infants with classic phenotype MEN2B infants/children with HD and exon 10 RET germline mutations adults with MEN2A and exon 10 mutations with symptoms of HD	Molecular testing, including RET for thyroid nodules	Test in: MTC on FNA or after thyroid surgery locally recurrent advanced and/or metastatic disease not amenable to RAI therapy newly diagnosed sporatic MTC germline WT or unknown with recurrent or persistent MTC germline mutationc MEN2A MTC MEN2B MTC family  members
Other recommendations	NA	Parents who do not want prenatal RET testing should be offered genetic counseling	NA	Consider testing in FNA samples that are indeterminate

Abbreviations: ATA = American Thyroid Association; CLA = conjugated linoleic acid; ESMO = European Society for Medical Oncology; FNA = fine needle aspiration; HD = Hirschsprung’s Disease; MEN = multiple endocrine neoplasia; MTC = medullary thyroid carcinoma; NA = not applicable; NCCN = National Comprehensive Cancer Network; RAI = radioactive iodine; RET = rearranged during transfection; WT = wild type. 

^a Molecular markers should be interpreted with caution, and in the context of clinical, radiologic, and cytologic features of each individual patient.

^b NCCN^® makes no warranties of any kind whatsoever regarding their content, use or application and disclaims any responsibility for their application or use in any way.

^c Specific mutation testing and genetic counseling in family members should be ensued.

References

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19. Hou H, Yang X, Zhang J, et al. Discovery of targetable genetic alterations in advanced non-small cell lung cancer using a next-generation sequencing-based circulating tumor DNA assay. Sci Rep. 2017;7(6):14605. https://doi.org/10.1038/s41598-017-14962-0

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Glossary

ALK = anaplastic lymphoma kinase 

AMP = Association for Molecular Pathology

BRAF = B-rapidly accelerated fibrosarcoma

CAP = College of American Pathologists

ctDNA = circulating tumor DNA

EGFR = epidermal growth factor receptor

FNA = fine needle aspiration

IASLC = International Association for the Study of Lung Cancer

MEN = multiple endocrine neoplasia

MET = MET proto-oncogene

MTC = medullary thyroid cancer

NCCN = National Comprehensive Cancer Network

NTRK = neurotrophic tropomyosin receptor kinase

NGS = next-generation sequencing

RET = rearranged during transfection

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