Background
Thyroid cancer is the most common type of endocrine cancer, with an increasing overall incidence in recent decades [
1]. Based on the type of cells from which the cancer grows, thyroid cancer is generally divided into two categories: follicular cell-derived cancers, including papillary thyroid cancer (PTC), follicular thyroid cancer (FTC), poorly differentiated thyroid cancer (PDTC) and anaplastic thyroid cancer (ATC); and parafollicular C cell-derived medullary thyroid cancer (MTC). The two categories of thyroid cancers have different genetic background. Specifically, over half of the follicular cell-derived thyroid cancers are driven by
BRAF V600E,
TERT promoter mutations, and/or genetic alterations in the PI3K/AKT pathway, while the major genetic driver of MTC is germline or somatic rearranged during transfection (
RET) mutations [
2‐
5].
Interestingly, although
RET mutation is rarely observed in follicular cell-derived thyroid cancers,
RET fusion occurs frequently in PTC and PDTC [
6,
7], particular in the patients with young age and environmental radiation exposure [
8‐
12]. The most common breakpoint of
RET was observed in intron 11, and then it fused with coiled-coil domain containing 6 (
CCDC6), nuclear receptor co-activator 4 (
NCOA4), or other N-terminal partner genes [
13]. These rearrangements lead to constitutively ligand-independent RET tyrosine kinase domain (TKD) activation and act as oncogenic drivers in cancer progression [
14].
Major advanced were made recently in the field of targeted therapy for
RET-altered cancers [
15]. Based on efficacy data from clinical trials, two highly selective RET inhibitors selpercatinib and pralsetinib were approved by the FDA in the year 2020 for treating patients with metastatic
RET fusion-positive non-small cell lung cancer (NSCLC), advanced or metastatic
RET-mutant MTC and advanced or metastatic
RET fusion-positive thyroid cancer [
16‐
19]. To catch the right patients for prescribing selpercatinib or pralsetinib in the clinic, the first essential step is accurate detection of
RET mutation and fusions. Compared with conventional methods used for gene mutation or fusion detection, the droplet digital PCR (ddPCR) showed several advantages, including high sensitivity and accuracy [
20,
21]. The ddPCR for
RET mutation detection has been well established [
22,
23], but there is no report on how to detect
RET fusions by ddPCR. Herein, in this study we developed a ddPCR method for
RET fusion detection and compared its performance with qRT-PCR in clinical samples from 112 PTC patients.
Methods
Patients
The Cancer Genome Atlas (TCGA) database for PTC patients was downloaded, and the distribution of key driver genetic alterations and the frequency of
RET fusion subtypes were analyzed in 402 patients with whole exome sequencing data [
6]. A total of 112 patients (87 women and 25 men), with a median (interquartile range) age of 36 (33–39) years, who were diagnosed and treated for PTC at The First Affiliated Hospital of Sun Yat-sen University between 2017 and 2019, were enrolled for
RET fusion detection. This study was approved by the ethics committee of our hospital and informed patient consent to participate in this study was obtained where required.
RNA extraction and cDNA preparation
The total RNA from each tissue was extracted by TRIzol™ Reagent (cat#15,596,018, Invitrogen, Waltham, MA, USA) according to the user guide. Then 1 µg of isolated RNA was used to generate first strand cDNA using a RevertAid First Strand cDNA Synthesis Kit (cat#K1622, Thermo Fisher Scientific, Waltham, MA, USA). 1ug RNA, 1 μl of Oligo(dT)18 primer and nuclease-free water were mixed gently to a total volume of 12 μl. To reduce the influence of GC-rich or secondary structures of RNA, RNA solution was incubated at 65 °C for 5 min and chilled on ice. Then 2 μl of 10 mM dNTP mix, 4 μl of 5 × reaction buffer, 1 μl of RiboLock RNase inhibitor, 1 μl of RevertAid RT was added to each tube. This mixture was incubated at 42 °C for 60 min and at 70 °C for 5 min. Followed, the product of the first strand cDNA synthesis was diluted ten times with nuclease-free water (final concentration 5 ng/ul) then stored at − 80 °C until it was used.
Standard template construction
A plasmid containing
CCDC6 (Exon 1)::
RET (Exon 12) infusion fragment was constructed and linearized as the standard template to evaluate the performance of qRT-PCR and ddPCR. Synthetic DNA sequence was inserted into pUC57 vector. The plasmid was linearized with restriction endonuclease NotI (NEB, R3189S) and XhoI (NEB, R0146S), and frozen at − 80 °C. The gene copy number was estimated by calculation formula: copies/ul = con.(ng/ul)*(10
–9)*(6.02*10
23) / (DNA length*660) [
24].
ddPCR
The forward primer (5’- TGCAGCAAGAGAACAAGGTG -3’), reverse primer (5’- TGACCACTTTTCCAAATTCGCC-3’), and probe (5’-FAM- ATTCCCTCGGAAGAACTTG -MGB-3’) were purified with high-performance liquid chromatography (HPLC). Optimized reactions were performed in 20 ul of duplex ddPCR reaction mix that consisted of 1X Droplet PCR Supermix (cat#186–3024, Bio-Rad, München, Germany), forward and reverse primers (final concentration of 800 nmol/L for each primer), probe (final concentration of 200 nmol/L) and 1 ul of template cDNA. After well mixed, the mixture was partitioned into 20,000 nanoliter-sized water-in-oil droplets by QX200™ Droplet Generator (cat#1,864,002, Bio-Rad). After gently transferred to 96-well plate and sealed, the PCR reaction was carried out in a Thermocycler T100 (Bio-Rad) using the following program: 95 °C for 5 min, 40 cycles of 94 °C for 30 s and 62.5 °C for 60 s (ramp rate: 2.5 °C/sec), 1 cycle of 98 °C for 10 min and holding at 12 °C. Droplets were counted at room temperature using the QX200 Droplet Reader (Cat#1,864,003, Bio-Rad) and analyzed using the Quantasoft software. The total number of droplets detected by each reaction was equal or exceed 10,000.
qRT-PCR assay
The primers, probe, and cDNA used for qRT-PCR were same as the ddPCR. The reaction was performed using TaqMan® Fast Advanced Master Mix (#4,444,557, Applied Biosystems) and by the Applied Biosystems QuantStudio 5 Real-Time PCR System under the following program: preincubated at 50 °C for 10 min and 95 °C for 2 min; followed 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The results were analyzed by the statistical analysis system of the instrument.
PCR and Sanger sequencing
The PCR reaction was performed using OneTaq Hot Start DNA Polymerase (#M0481S, NEB) on the Applied Biosystems ProFlex PCR System under the following program: preincubated at 94 °C for 30 s; followed 45 cycles of 94 °C for 20 s, 60 °C for 30 s and 68 °C for 30 s; final extension at 68 °C for 10 min. The PCR products were separated by electrophoresis in a 2% agarose gel and recognized by Sanger sequencing.
Statistical analysis
The Oncoprinter from cBioPortal (
https://www.cbioportal.org/oncoprinter) was used to analyze and visualize the genetic alterations profiling [
25]. χ
2 test or Fisher’s exact test were selected for comparing differences between categorical variables by IBM SPSS (version 26.0). GraphPad Prism (version 7.0) was used to do the linear regression. And Probit regression analysis for LoD was done by MedCalc software (Version 20.121).
Discussion
RET fusion is a one of common genetic drivers in multiple human cancers, including PTC. Fluorescence in situ hybridization (FISH), qRT-PCR, and next-generation sequencing (NGS) were currently used for detecting
RET fusions. Although FISH is considered as the gold standard for fusion detection, it is time-consuming and requires experienced personnel [
26,
27]. Similarly, NGS is labor-intensive, time-consuming and expensive although it is one of the most comprehensive and sensitive methods for genetic analysis. The easy accessibility and high sensitivity of ddPCR makes it became a new trend for detecting specific genetic alteration [
28,
29]. In this study we developed a ddPCR method for detection of
CCDC6::
RET fusion, the most frequent subtype of
RET fusions. By optimizing the primer and probe concentrations and annealing temperature, the ideal condition for
CCDC6::
RET fusion detection was established.
Compared with the widely used qRT-PCR method, the LoD of our method is remarkably low, suggesting the sensitivity of this new method is superior to qRT-PCR. In support of this, when we applied these two methods in 112 PTC samples, all the fusion-positive cases identified by qRT-PCR were detectable in the ddPCR system, and ddPCR identified 4 additional
CCDC6::
RET fusion-positive samples. Importantly, although the copy number of
CCDC6::
RET is very low in the 4 samples, the fusion were successfully confirmed by Sanger sequencing except for the sample with the lowest copy number. This phenomenon is consistent with previous findings that ddPCR is more sensitive than Sanger sequencing for the detection of driver mutations [
30,
31], although we cannot exclude the possibility that the unconfirmed positive case was a false-positive result from ddPCR.
The frequency of RET fusion detected by qRT-PCR in the current study was in accordance with previous findings that the
RET fusion frequency was about 4–9% in sporadic PTC [
6,
32]. However, the ddPCR assay showed that the fusion frequency increased to 13.4%, suggesting that the incidence of
RET fusion in PTC might be underestimated. By analyzing the sequencing data of PTC from the TCGA cohort, we found that
RET fusions were mutually exclusive with somatic genetic alterations in
BRAF,
RAS,
EIF1AX and
TERT except in one sample, further indicating an oncogenic role of
RET fusion in PTC tumorigenesis. Moreover, although the relationship between
RET fusion and clinical behavior and outcome of PTC is controversial [
11,
33,
34], recent studies involving large sample numbers showed that
RET fusions were associated with more aggressive characteristics of PTC, including extrathyroidal extension, lymph node and distant metastases, radioiodine refractory, and worse prognosis [
12,
35,
36].
Advanced patients with
RET fusions can benefit from targeted therapy [
15]. A recent clinical trial showed that 79% of patients with previously treated
RET fusion- positive thyroid cancer had a response to
RET kinase specific inhibitor selpercatinib [
17]. Since the ddPCR system established in this study provides a sensitive method for
RET fusion detection, it would be definitely benefits more thyroid cancer patients in the clinic. In addition to
RET fusion-positive thyroid cancers, selpercatinib was also demonstrated durable and robust responses in
RET fusion-positive NSCLC and 12 other types of solid tumor [
18,
37,
38]. Since the ddPCR system established in this study provided a sensitive method for
CCDC6::
RET fusion detection, application of this method to these cancer types would be benefits more
RET fusion-positive patients in the clinic. It should be noted that the method reported here is designed for
CCDC6::
RET, but not for other subtypes of RET fusions, therefore multiplex ddPCR system for detecting all subtypes of RET fusion is needed to be established.
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