Clinical Evaluation of Genetron TERT/BRAF PCR Kit in Thyroid Cancer Patients
Conditions: Thyroid Cancer
Sponsor: Genetron Health

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Clinical Evaluation of Genetron TERT/BRAF PCR Kit in 
Thyroid Cancer Patients 
 
 
 
Study Document 
 
 
 
 
 
 
 
 
 
 
 
 
NTC number: NCT05395429 
Date: 05/30/2022 
 
 
 
 

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Content 
1 Background ............................................................................................................................ 3 
2 Study Purpose ......................................................................................................................... 5 
3 Study Design .......................................................................................................................... 5 
3.1 Overall Study Design ...................................................................................................... 5 
3.1.1 Detection performance validation of test reagent ..................................................... 5 
3.1.2 Performance validation of test reagent ..................................................................... 5 
3.2 Sample Screening ............................................................................................................ 6 
3.2.1 Basis to Determine Sample and Sample Size ........................................................... 6 
3.2.2 Inclusion, exclusion and elimination criteria ............................................................ 7 
3.2.4 Sample numbering .................................................................................................... 8 
3.3 Study Duration and Reasons for Determination .............................................................. 9 
4 Statistical Method ................................................................................................................... 9 
4.1 General Principles of Statistical Analysis ....................................................................... 9 
4.2 Basic clinical information of enrolled samples ............................................................... 9 
4.3 Positive, Negative and Total Coincidence Rates Between Test Reagent and Comparison 
Method ................................................................................................................................... 9 
4.4 Sensitivity, Specificity and Total Coincidence Rate of Test Reagent and Clinical 
Reference Standard .............................................................................................................. 10 
4.5 Kappa test ...................................................................................................................... 11 
4.6 Clinical Evaluation Standard ......................................................................................... 11 
5 Quality Control Method ....................................................................................................... 12 
6 Provisions for Protocol Amendment .................................................................................... 13 
7 Ethical issues and explanations involved in the clinical trial ............................................... 13 
8 Data Processing and Record Storage .................................................................................... 13 
9 Responsibilities of Involved Parties ..................................................................................... 13 
9.1 Sponsor’s responsibility ................................................................................................ 13 
9.2 Test facility’s responsibility .......................................................................................... 14 
10 Study Technology Roadmap .............................................................................................. 14 
 
 

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1 Background 
Thyroid cancer is the most common endocrine neoplasm malignant, which is mainly 
divided into papillary thyroid cancer (PTC, accounting for 80-85%), follicular thyroid 
cancer (FTC, accounting for 10-15%), poorly differentiated thyroid cancer (PDTC, 
accounting for 5-10%), anaplastic thyroid cancer (ATC, accounting for 2-3%) and 
medullary thyroid cancer (MTC, accounting for 2-3%) For different pathological 
types of thyroid cancer, their pathogenesis, biological behavior, histological 
morphology, clinical manifestations, treatments and prognosis vary greatly. PTC and 
FTC have mild biological behavior and good prognosis, ATC and PDTC have share 
higher degree of malignancy and lower survival rate, and the prognosis of MTC is 
intermediate. 
The molecular mechanism of thyroid carcinogenesis and progression is the abnormal 
activation of intracellular signal transduction induced by related gene changes. Among 
these, MAPK signaling pathway is one of the important signaling pathways for 
thyroid carcinogenesis and progression. The missense mutation of a single base in 
Exon 15 of BRAF gene (GTG>GAG) causes the replacement of valine (V) 
corresponding to codon 600 of translation protein by glutamate (E), which becomes 
activated protein kinase, and induces tumor formation by activating MAPK signaling 
pathway. TERT promoter mutation induces the abnormal activation of telomerase, 
which is a key step in thyroid carcinogenesis. It has been found that ETS 
(E-twenty-six) protein is a transcription factor family downstream of the 
BRAF-MAPK signaling pathway. When TERT gene promoter mutates, it can produce 
some special nucleotide sequences, which can bind to ETS protein, and cause 
tumorigenesis by enhancing TERT gene expression. Many studies have shown that 
BRAF gene V600E mutation and two mutations in TERT gene promoter region (1 295 
228C>T and 1 295 250C>T) are frequently observed in thyroid cancer, and the poor 
prognosis of thyroid cancer patients can be effectively evaluated. Further studies have 
confirmed that the coexistence of BRAF and TERT promoter mutations has a 
synergistic effect on the invasiveness of thyroid cancer, and the two mutated tumors 

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are related to the high recurrence risk and increased disease-specific mortality of 
differentiated thyroid cancer, as well as a higher mortality trend in ATC. Therefore, 
TERT promoter mutation and BRAF mutation can be used as molecular markers for 
thyroid diagnosis. 
TERT promoter mutation and BRAF mutation are closely related to clinical 
evaluations such as preoperative diagnosis and prognostic risk assessment of thyroid 
cancer, which have been clearly described in relevant guidelines. The NCCN Clinical 
Guidelines (Thyroid Cancer) - 2018 V3 Edition, the World Health Organization 
(WHO) Endocrine Tumors 2017 V4 Edition, the ATA Management Guidelines for 
Thyroid Nodules and Anaplastic Thyroid Cancer 2015 Edition, the Expert Consensus 
on Diagnosis and Treatment of Thyroid Micropapillary Carcinoma, and the Chinese 
Society of Clinical Oncology (CSCO) Guidelines for Diagnosis and Treatment of 
Persistent/Recurrent and Metastatic Thyroid Cancer in 2018 V1 Edition pointed out 
that, TERT promoter mutation or BRAF gene mutation showed high mutation 
frequency in thyroid tissue, and predicted benign and malignant tumors and adverse 
prognosis of patients. Among these, the ATA Management Guidelines for Thyroid 
Nodules and Anaplastic Thyroid Cancer 2015 Edition and the Expert Consensus on 
Diagnosis and Treatment of Thyroid Micropapillary Carcinoma clearly pointed out 
that, benign and malignant thyroid tumors cannot be determined by fine needle biopsy, 
and the detection of BRAF mutation and TERT promoter mutation gene can be 
carried out on punctured specimens, which is helpful to improve the diagnosis rate. In 
addition, CSCO guidelines point outed that, BRAF gene mutation and TERT gene 
promoter mutation are not only poor prognostic indicators for patients with thyroid 
cancer, but also have a greater impact on the absorption of radioactivity I in PTC 
patients. For PTC patients with distant metastasis, the pre-detection of BRAF V600E 
and other gene mutations is helpful for predicting the 131I uptake, therapeutic effect 
and prognosis of patients with distant metastasis. Moreover, the targeted drug 
dabrafenib plus trametinib can effectively alleviate ATC with BRAF mutation, while 
for 131I-resistant patients with BRAF mutation, dabrafenib plus131I can restore the 
absorption of 131I in some patients. Therefore, the detection of TERT promoter and 

5 
BRAF gene mutation is helpful for the diagnosis and clinical prognosis prediction of 
thyroid cancer, and convenient for making individualized diagnosis and treatment 
options. 
The Genetron TERT/BRAF PCR Kit is used to qualitatively detect two mutations in 
the TERT gene promoter region (1295 2228C > T and 1295 250C > T) and BRAF 
gene V600E mutation (GTG > GAG) within the DNA of thyroid nodule histiocytes 
obtained by fine needle aspiration (FNA). Assisted fine needle biopsy is used in the 
diagnosis of benign and malignant thyroid nodules. 
2 Study Purpose 
The purpose of this study is to evaluate the detection performance of the Genetron 
TERT/BRAF PCR Kit (PCR-fluorescence probe method) by comparing the results of 
this kit to those of the simultaneous detection with Sanger sequencing method. 
Meanwhile, the clinical diagnosis performance of the Genetron TERT/BRAF PCR Kit 
(PCR-fluorescence probe method) will also be evaluated by comparing the results of 
this kit to the clinical reference standard (pathological findings or clinical diagnosis 
conclusions).  By combining both evaluations, the intended use of the kit and the 
detection accuracy of the analyte can be comprehensively evaluated. 
3 Study Design 
3.1 Overall Study Design 
3.1.1 Detection performance validation of test reagent 
This trial will be conducted in accordance with the principles of the synchronous blind 
method. The selected cases will be coded, and the selected samples will be detected 
with the test reagent and comparison method (Sanger sequencing). When the 
comparison results are inconsistent with the detection results of Sanger sequencing 
method, the detection results of sanger will be acceptable. 
3.1.2 Performance validation of test reagent 
Among the enrolled patients, postoperative pathological results will be obtained from 
those who underwent surgery subsequently. Those with pathologically confirmed 
thyroid cancer will be considered as positive, while those without pathologically 

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confirmed thyroid cancer whose clinical diagnosis is non-cancer without surgery will 
be considered as negative. The results of the test reagents will be compared and the 
clinical sensitivity and specificity of the test reagents will be calculated. 
3.2 Sample Screening 
3.2.1 Basis to Determine Sample and Sample Size 
3.2.1.1 Sample Size of Test Performance Verification 
𝑛=
[𝑍1−𝛼/2√𝑃0(1 −𝑃0) + 𝑍1−𝛽√𝑃𝑇(1 −𝑃𝑇)]
2
(𝑃𝑇−𝑃0)2
 
Where, n represents the sample size; Z1-α/2 and Z1-β are fractions of standard 
normal distribution, P0 is the target value of the evaluation indicator, and PT is 
the expected value of the evaluation indicator for in vitro diagnostic reagents. 
（1） BRAF gene: According to the results of preclinical studies, the target value of 
positive coincidence rate between test reagent and contrast reagent is up to 85% 
with the expected value up to 95%, the minimum sample size of positive 
control group is estimated to be 78; the target value of negative coincidence 
rate is up to 85% with the expected value up to 90%, the minimum sample size 
of negative control group is estimated to be 363 and the minimum overall 
sample size is 441. 
（2） TERT gene: According to the results of preclinical studies, the target value of 
positive coincidence rate between test reagent and contrast reagent is up to 85% 
with the expected value up to 98%, the minimum sample size of positive 
control group is estimated to be 40; the target value of negative coincidence 
rate is up to 85% with the expected value up to 90%, the minimum sample size 
of negative control group is estimated to be 363 and the minimum overall 
sample size is 403. 
3.2.1.2 Sample Size of Diagnostic Performance Verification 
𝑛=
[𝑍1−𝛼/2√𝑃0(1 −𝑃0) + 𝑍1−𝛽√𝑃𝑇(1 −𝑃𝑇)]
2
(𝑃𝑇−𝑃0)2
 
Where, n represents the sample size; Z1-α/2 and Z1-β are fractions of standard 
normal distribution, P0 is the target value of the evaluation indicator, and PT is 

7 
the expected value of the evaluation indicator for in vitro diagnostic reagents. 
Samples with BRAF or TERT mutation detected by the test reagent are considered as 
positive, and samples without mutation are considered as negative. 
The results of pathological examination and clinical diagnosis will be used for 
comparison method. Those with pathologically confirmed thyroid cancer will be 
considered as positive, while those without pathologically confirmed thyroid cancer 
whose clinical diagnosis is non-cancer without surgery will be considered as negative. 
According to relevant literature reports and preclinical study results, if the target value 
of sensitivity for the test reagent and comparison method is up to 50% with the 
expected value up to 60%, the minimum sample size of positive control group is 
estimated to be 194. According to previous experience, the proportion of positive 
diagnosis upon postoperative pathological examination is generally more than 70%. If 
estimated as 70%, the overall sample size of prospectively enrolled patients is 278. 
The target specificity value is 80% with the expected value of 90%, the minimum 
sample size of negative control group is estimated to be 108. If the proportion of 
negative results obtained from the postoperative pathological examination is estimated 
as 30%, the overall sample size of prospectively enrolled patients is 360. For the final 
overall sample size calculated based on sensitivity and specificity, the larger one (360 
cases) shall prevail. Considering the estimated dropout rate of 10%, the final actual 
overall sample size is 400. 
It is planned to carry out the trial in at least three clinical sites, with the enrollment of 
no less than 300 samples at each site. In case of objective reasons, when the sample 
size is insufficient, appropriate adjustments can be made among the sites to ensure the 
overall sample size. If the relevant laws and regulations change during the trial, the 
latest laws and regulations shall prevail, and the number of samples should be 
statistically significant. 
3.2.2 Inclusion, exclusion and elimination criteria 
3.2.2.1 Inclusion criteria 
（1） Patients with clinically suspected of thyroid cancer who underwent fine needle 
aspiration (FNA) 

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（2） Samples should have appropriate basic clinical information, including: The 
patient's unique traceable number, age, sex, pathological diagnosis results, etc. 
（3） Subjects who can provide a complete FNA sample 
（4） Subjects who agreed to provide follow-up information 
3.2.2.2 Exclusion criteria 
Those who do not meet any of the above inclusion criteria will be excluded. 
3.2.2.3 Elimination criteria 
（1） The investigators consider it inappropriate to continue clinical trials, such as 
samples prepared without following the required procedures. 
（2） Sample with incomplete sample information. 
（3） Samples with DNA extraction not enough to complete the testing of test 
reagent or comparison method. 
3.2.3 Sample collection, storage and transportation 
Sample collection 
Meet the hospital-based FNA puncture standard 
Sample storage and transportation 
Immediately after puncture, specimens should be placed in RNAlater protective 
solution. It is recommended that solid substances be isolated and tested immediately. 
Otherwise, it should be stored at 2 - 8 °C for no more than one month; for long-term 
storage, it is recommended that the isolated solid substances be stored at a 
temperature no higher than-70 °C for no more than one year. 
3.2.4 Sample numbering 
In principle, only one sample should be collected from one subject, and each enrolled 
sample of the two detection methods should have a unique clinical trial number 
(secondary code). After sample enrollment, before sample detection, the blinding 
personnel will mark the secondary code outside the DNA sample tubes according to 
the blinding table, prepare the Blinding Records and carefully record the comparison 
table between the primary code (subject enrollment number) and the secondary code 
as the blind codes. Blinding documents shall be kept independently by the blinding 

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personnel until breaking of blindness. 
During experimental procedures, only the secondary code of samples is reflected. 
3.3 Study Duration and Reasons for Determination 
This clinical study will last about 12 - 36 months from drafting protocol, project 
establishment, ethical review, agreement signing, specimen collection, detection until 
the end of experiment, report completion and stamping. If samples are collected 
smoothly and the sample size meets the trial requirements, the trial duration can be 
shortened; if it doesn't go smoothly, the duration can be extended appropriately.  
4 Statistical Method 
4.1 General Principles of Statistical Analysis 
SAS 9.4 or above software will be used for statistical analysis. The sample size is 
calculated with PASS16. The statistical analysis of the validity and accuracy of test 
results is based on full analysis set (FAS) and per protocol set (PPS), and the safety 
analysis is based on safety set (SS). All statistical tests will be conducted on both sides 
and P ≤ 0.05 will be considered statistically significant. (Unless otherwise specified)  
The quantitative variables are expressed as mean, standard deviation, median, 
minimum, maximum, lower quartile (Q1) and upper quartile (Q3), while the 
categorical variables are expressed as the number and percentage of each category. 
4.2 Basic clinical information of enrolled samples 
Descriptive statistics is collected for the basic information and data of the population 
corresponding to included samples. The descriptive statistics of age, sex and disease 
follows the general principle. 
4.3 Positive, Negative and Total Coincidence Rates Between Test Reagent and 
Comparison Method 
For each genetic locus, fourfold tables will be used to calculate the positive and 
negative coincidence rates, positive and negative predictive values, total coincidence 
rate and their 95% confidence intervals. Using statistical software, Kappa test is used 
to determine statistical significance of the two methods. 
Table 1 Result Summary of Clinical Trial Comparing Test Reagent and Comparison Method 

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Test Reagent 
Comparison Method 
Total 
Positive 
Negative 
Positive 
A 
B 
A+B 
Negative 
C 
D 
C+D 
Total 
A+C 
B+D 
A+B+C+D 
Samples with mutant genotype detected are considered as positive, and samples 
without mutant genotype are considered as negative. 
The calculation formula is: 
Positive coincidence rate = A/(A+C)×100% 
Negative compliance rate = D/(B+D) ×100% 
Positive predictive value (PPV) = A/(A+B)×100% 
Negative predictive value (NPV) = D/(C+D)×100% 
Total coincidence rate = (A+D)/(A+B+C+D)×100% 
The calculation formula of 95% confidence interval: p±1.96×[ p(1-p)/n]1/2 (where P 
represents positive and negative coincidence rates, positive and negative predictive 
values and total coincidence rate, n presents the sample size. If p>0.9, Wilson score 
method will be used for correction). 
4.4 Sensitivity, Specificity and Total Coincidence Rate of Test Reagent and 
Clinical Reference Standard 
Table 2 Result Summary of Test Reagent and Clinical Reference Standard 
Test Reagent 
Gold standard 
Total 
Positive 
Negative 
Positive 
A (true-positive) 
B (false-positive) 
A+B 
Negative 
C (false-negative) 
D (true-negative) 
C+D 
Total 
A+C 
B+D 
A+B+C+D 
Those with pathologically confirmed thyroid cancer will be considered as positive, 
while those without pathologically confirmed thyroid cancer whose FNA result is 
non-cancer without surgery will be considered as negative. 
The test reagent and comparison method will be compared, but patients who fail to 
provide follow-up pathological results will not be included in the comparative 

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statistics of test reagent and clinical reference standard. 
The calculation formula is: 
Sensitivity = A/(A+C)×100% 
Specificity = D/(B+D) ×100% 
Total coincidence rate = (A+D)/(A+B+C+D)×100% 
Positive predictive value (PPV) = A/(A+B)×100% 
Negative predictive value (NPV) = D/(C+D)×100% 
The calculation formula of 95% confidence interval: p±1.96×[ p(1-p)/n]1/2 (where P 
represents sensitivity, specificity, positive and negative predictive values and total 
coincidence rate, n presents the sample size. If p>0.9, Wilson score method will be 
used for correction.) 
4.5 Kappa test 
Kappa test will be used to analyze the equivalence of test reagent and comparison 
method (clinical reference standard). Using statistical software, Kappa test will be 
used to compare the results of test reagent and the comparison method (clinical 
reference standard), and k value will be calculated. When k>0.75, the investigational 
reagent and comparison method show good consistency; when k>0.5, the 
investigational reagent and clinical reference standard show general consistency. 
1
a
c
c
P
P
Kappa
P



, where Pa= 
A
D
N

, PC = 
(
)(
)
(
)(
)
A
B
A
C
B
D C
D
N
N
N





 
 
4.6 Clinical Evaluation Standard 
1) Coincidence rate: Compared to the comparison method, if the lower limit of 
two-sided 95% confidence interval for both positive and negative coincidence rates of 
BRAF and TERT genes are greater than the 85% of preset target value, the statistical 
hypothesis is valid and in good agreement with the comparison method; otherwise, the 
statistical hypothesis is invalid, and the agreement with comparison method is not 
acceptable. 
2) Sensitivity and specificity: Compared to the clinical reference method, if the lower 

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limit of two-sided 95% confidence interval for the sensitivity of BRAF or TERT gene 
pooled detection is greater than 50% of the preset target value and the lower limit of 
two-sided 95% confidence interval for the sensitivity of BRAF or TERT gene pooled 
detection is greater than 80% of the preset target value, the statistical hypothesis is 
valid and the diagnostic accuracy is acceptable; otherwise the statistical hypothesis is 
invalid, and the diagnostic accuracy is not acceptable. 
5 Quality Control Method 
Quality control should be applied at every stage of the clinical trial to ensure that all 
data are reliable and correct. The quality control method is as follows: 
 (1) Qualifications of researchers involved in the study Researchers involved in the 
clinical trial must have professional expertise, qualifications and competences in the 
clinical trial, and pass the qualification examination, and staffing should be relatively 
fixed. 
(2) Training and preliminary tests: The sponsor is responsible for the training of 
researchers before the start of clinical trial, so as to help clinical researchers fully 
understand the clinical trial protocol, the detection of investigational products, the 
filling of original records and forms, etc. 
(3) Laboratory quality control: Clinical laboratories should be strictly in accordance 
with the Measures for the Administration of Clinical Gene Amplification Testing 
Laboratories in Medical Institutions (MOH Office MA [2010] No. 194 or the current 
effective version) and other relevant administrative regulations of molecular biology 
laboratories and clinical gene amplification laboratories. 
(4) Only when the quality control of investigational products meets relevant 
requirements can the test data be valid. 
(5) Clinical trial monitoring: The monitor shall make a complete monitoring plan 
before the start of the clinical trial, and monitor the clinical trial according to the 
monitoring plan to ensure that the clinical trial is carried out according to the clinical 
protocol, and ensure the intactness and accuracy of the original test data and records. 

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6 Provisions for Protocol Amendment 
Any modification of the protocol during the trial shall be explained, and the time, 
reason, process and filing of the change shall be elaborated in detail, and its influence 
on the evaluation of the overall study results shall be demonstrated. If the 
investigators modify the protocol and case report form during the trial, it must be 
approved by the ethics committee before continuing the clinical trial. 
7 Ethical issues and explanations involved in the clinical trial 
The purpose of this clinical trial is to evaluate the performance of human 
TERT/BRAF Gene Mutation Pooled Detection Kit (PCR-fluorescence probe method) 
in clinical application. All the samples used in this clinical trial are prospective 
samples, which are specially collected for this clinical trial and shall be accompanied 
by a copy of signed informed consent form from the patient. 
8 Data Processing and Record Storage 
Investigators should record all the items on the clinical trial case report form truthfully, 
in detail and carefully according to the filling requirements, so as to ensure that the 
contents in the form are complete, true and reliable. All observations and findings 
obtained from the clinical trial should be verified to ensure the reliability of data and 
ensure that all conclusions in clinical trials come from original data. The clinical trial 
personnel should truly fill in relevant clinical information and the test results of 
clinical trials, and have them reviewed and signed by a specially-assigned person. 
All original data records generated by this clinical trial shall be filed and preserved by 
the clinical sites, and the retention period shall be determined by the sites, but shall 
not be less than 5 years upon the completion of the clinical trial. 
9 Responsibilities of Involved Parties 
9.1 Sponsor’s responsibility 
（1） Work with medical institutions to design and formulate clinical trial 
protocols, and sign clinical trial protocols and contracts agreed by both parties; 
（2） Provide medical institutions with tested products and required contrast 

14 
reagents free of charge, and provide necessary test equipment; 
（3） Provide training for study staff. 
9.2 Test facility’s responsibility 
（1） Should be familiar with the relevant information provided by the 
implementing party and familiar with the use of the tested products; 
（2） Work with the implementing party to design and formulate clinical trial 
protocols, and sign the clinical trial protocols between both parties; 
（3） Propose clinical trial reports and be responsible for the correctness and 
reliability of the reports; 
（4） The information provided by the implementing party shall be kept 
confidential, and the test results and process shall not be disclosed to any third party. 
10 Study Technology Roadmap 
 
 

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Patient Screening 
Signing Informed Consent 
Form 
Sample Enrollment 
Complianc
Obtaining 
Detection by Test 
Reagent 
Detection by 
Comparison Method 
Receiving subsequent therapy 
according to doctor's advice 
Non-surgical Patients 
Surgical Patients 
Pathologic findings 
Clinical diagnosis 
conclusion 
Result Comparison 
Result Comparison 
Statistical Analysis 
Clinical Report 
Ethics Committee Approval 
Singing Clinical Trial 
Agreement 
Clinical trial Filing 
Clinical Trial Initiation