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
|
|
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
|
|
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.
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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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