Clinical Evaluation of Genetron TERT PCR Kit in Glioma Patients
Conditions: Glioma, Malignant
Sponsor: Genetron Health

 
Clinical Evaluation of Genetron TERT PCR Kit in  
Glioma Patients 
 
 
 
Study Document 
 
 
 
 
 
 
 
 
NTC number: NCT05100602 
Date: 02/01/2016 
 

Content 
1 Background................................................................................................................................... 1 
2 Test Purpose .................................................................................................................................. 3 
3 Trial Design ................................................................................................................................... 3 
3.1 Overall Design of the Trial .................................................................................................. 3 
3.2 Test design and study method selection ....................................................................... 4 
3.3 Study technology route .................................................................................................... 5 
3.4 Quality control method ..................................................................................................... 6 
4 Evaluation method .................................................................................................................... 6 
4.1 Evaluation method for reagents to be tested ............................................................... 6 
4.2 Evaluation method for nucleic acid sequence determination .................................... 7 
4.3 Evaluate the conformity between the kit and the reference method ........................ 7 
5 Statistical methods ................................................................................................................... 8 
6 Duration of clinical trials and its reasons ................................................................................... 8 
7 Inclusion criteria, exclusion criteria and rejection Criteria ........................................... 8 
7.1 Inclusion criteria ............................................................................................................... 8 
7.2 Exclusion criteria .............................................................................................................. 8 
7.3 Rejection Criteria ............................................................................................................. 9 
8 Provisions for amendments to clinical trial protocols .................................................... 9 
9 Ethical issues and explanations of clinical trials ............................................................. 9 
10 Data processing and record keeping .............................................................................. 10 
 

1 
1 Background  
Glioma is the most common primary intracranial tumor. According to the ranking of tumor mortality 
published by the World Health Organization in 1998, malignant glioma is the second leading cause 
of death among tumor patients under 34 years old and the third among patients between 35 and 54 
years old. In the past 30 years, the incidence of primary malignant brain tumors has been increasing 
year by year, with an annual growth rate of about 1.2%, especially in the elderly population. At 
present, WHO mainly relies on morphology for tumor classification and typing (Table 1). However, 
due to the heterogeneity of glioma and the subjective influence of pathologists, this method is highly 
subjective and one-sided. There is ample evidence that gliomas with the same or similar tissue 
characteristics can have different molecular genetic backgrounds. Molecular pathological 
classification based on tumor genetics can provide accurate and objective genetic information for 
tumor diagnosis, to help doctors understand the characteristics of each patient's tumor from a new 
perspective of molecular biology, so as to make more targeted treatment and rehabilitation programs. 
Table 1  WHO pathological classification and classification of glioma 
Tissue 
types 
Class 
Common subtype 
Glioma of 
the brain 
Class I 
Pilocytic astrocytoma, subependymal giant cell astrocytoma embryonal 
dysplasia of neuroepithelial tumors 
Class II 
Diffuse astrocytoma, mixed oligoastrocytomas, oligodendroglioma, 
pleomorphic xanthoastrocytoma, hairy myxoid astrocytoma 
Class III 
Anaplastic astrocytoma, anaplastic oligodendroglioma, anaplastic 
oligoastrocytomas 
Class IV Primary and secondary glioblastoma 
Telomerase is a kind of nucleic acid protein complex composed of two subunits of Telomerase RNA 
and Telomerase reverse transcriptase (TERT), which plays a key role in the maintenance of 
Telomerase length. TERT gene promoter mutation is one of the important mechanisms of abnormal 
telomerase expression in tumors. The most common mutation sites in glioma are C228T and C250T, 
which usually occur independently of each other. It has been proved that the mutation can enhance 
the transcriptional activity of TERT promoter and is a key cause of tumor occurrence and 
development. 

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Recently, important studies have found that TERT mutations are commonly high in primary 
glioblastoma and oligodendroglioma, but low in astrocytomas. The researchers also detected the 
TERT gene promoter and IDH gene together. According to the mutation status of the two genes 
simultaneously or independently, the most typical subtypes of glioma could be further distinguished 
significantly: TERT mutations were found in 74.2% of primary glioblastoma patients among 473 
adult gliomas, but only in 18.2% of class II-III astrocytomas. In contrast, IDH mutations were 
observed in 78.4% of class II-III astrocytomas, but were infrequent in primary glioblastoma. TERT 
promoter mutations co-occurred with IDH mutations in 79% of oligodendrogliomas (Figure 1). 
Subsequently, several important multi-center, large-scale clinical trials fully supported this 
conclusion. 
 
A. Glioblastomas (n=240)
B. Astrocytomas (n=88)
C. Oligoastrocytomas (n=58)
D. Oligodendrogliomas (n=87)
TERT
IDH1/2
TERT
IDH1/2
TERT
IDH1/2
TERT
IDH1/2
 
Figure 1: The distribution of TERT gene promoter and IDH1/2 gene mutations in 473 adult glioma 
patients. 
The gene mutations are indicated by color, red indicating the TERT gene promoter mutation, green 
indicating the IDH1/2 gene mutation, and light gray indicating that neither gene has mutation. 
Therefore, for adult glioblastoma class III-IV patients, if TERT mutations is present alone, primary 
glioblastoma may be indicated, if IdH1/2 mutation is present alone, astrocytoma may be indicated, 
and if TERT and IDH1/2 mutations are present at the same time, oligodendroglioma may be 
indicated. These findings provide a unique diagnostic basis for promoting accurate classification of 
brain cancer. 

3 
The standard method for clinical detection of common gene promoter mutations is nucleic acid 
sequence determination, but this method is not sensitive enough, result judgment is time-consuming 
and laborious, and the operation process is complex, so this technology is difficult to be widely 
applied in clinical practice. 
The kit to be tested was developed by Genetron Health (Beijing) Co., Ltd. It is mainly used for the 
detection of TERT gene promoter mutation in glioma patient cells. The tested product series of the 
kit is prepared in a clean production workshop that meets the requirements of the "Detailed Rules 
for the Production of In Vitro Diagnostic Reagents", and passed the self-test. 
At present, there is no similar product on the market in China, so no similar kit can be used as a 
clinical trial control. The gold standard method for detecting common gene mutations is nucleic acid 
sequencing. In order to evaluate the clinical safety and effectiveness of this product, this clinical 
trial uses the qualified product provided by the manufacturer to retrospectively examine the tissue 
biopsy samples confirmed by pathology. The pathological types include gliomas, other brain tumors, 
and normal brain tissues, and the results of the nucleic acid sequence determination of the 
corresponding samples are used as controls for comparison. 
2 Test Purpose 
By using this product to detect the TERT gene promoter mutation in the cancer cells of glioma 
patients and comparing with the gene sequencing results, the safety and effectiveness of this product 
are confirmed and evaluated in combination with clinicopathological grading. 
3 Trial Design 
3.1 Overall Design of the Trial 
This study followed the principle of synchronous blind method. Enrolled cases were coded and 
compared with the nucleic acid sequence determination of the control method (gold standard). 
Results were determined independently according to the critical value or interpretation requirements 
provided by each method, and relevant statistics were conducted. Combined with the results of 
clinicopathological classification, the incidence of C228T and C250T mutations in different 
subtypes of TERT gene promoters was calculated to evaluate the clinical application performance 
for clinical research kits.  

4 
The total number of samples for clinical research should be no less than 1,000 cases, and clinical 
trials should be carried out in no less than 3 (including 3) provincial health care institutions or 
specialized hospitals to ensure that the results are statistically significant. All samples should have 
corresponding basic clinical information, including: patient visiting No. / medical record 
number/specimen number, age, sex, pathological diagnosis result, etc. 
3.2 Test design and study method selection 
3.2.1 Sample collection 
Tissue sections fixed in neutral formaldehyde and embedded in paraffin were detected. 
3.2.2 Sample data 
(1) Sample information 
A) Patient visiting No. / medical record No. / specimen No., age, sex, and pathological 
diagnosis result 
B) HE staining results of samples, indicating tumor area. 
(2) Pathological examination confirmed glioma, other brain tumors or normal tissues. 
(3) Number of samples: each sample contains 10 slices with a thickness of 10µm, and the tumor 
content is not less than 50%. 
3.2.3 Sample storage 
FFPE samples should be stored at room temperature. 
3.2.4 Kit source:  
(1) The kits were provided by Genetron Health (Beijing) Co., Ltd. 
(2) Test instrument: BIO-RAD: CFX96 Real-Time PCR Detection System or LIFE: 7500 Fast Dx 
Real Time PCR Instrument.  
3.2.5 Reference method 
Sanger sequencing was used as the control method. Sequencing fragments cover the target nucleic 
acid segments, sites and corresponding types amplified by the assessment reagent. After the 
sequencing reaction is over, submit a representative sample sequencing map and result analysis data. 

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3.2.6 Testing process 
The test samples were tested in strict accordance with the kit operation instructions. 
3.3 Study technology route 
Samples were enrolled and 
randomly coded
Samples were screened 
according to pathological 
diagnosis results
DNA extraction & 
concentration 
determination
Tumor tissue was 
scraped according to 
staining results
Genetron Health kit 
detection
Reference method: Sanger test
Data analysis
Data analysis
Statistical result
Success
Failure
Invalid sample 
rejestion
HE staining (or providing 
previous information on the 
same area)
 

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3.4 Quality control method 
(1) The clinical verification institutions are all three-level medical institutions above the provincial 
level with the ability to conduct the clinical research technology and equipment to ensure the 
quality of clinical diagnosis. 
(2) Each clinical trial institution has 1 project director, and 1 to 3 fixed project members. Strictly 
follow the requirements of the clinical trial protocol. The reporting institution appoints 1 
inspector to monitor and cooperate with the team leader institution and the trail institution at 
any time. The technical personnel of the team leader institution keep close contact with each 
trail center at any time. 
(3) The test kit detection and reference method detection are carried out in accordance with the 
blind method. 
(4) Hospital laboratories participating in clinical trials should establish standard operating 
procedures and quality control procedures for experimental observation indicators. The test 
report must be complete, which must include date, test items, test results and their normal range. 
The person concerned should sign. Each test is set up control, and strictly in accordance with 
the requirements of the manual. 
(5) Training: Before the start of the experiment, conduct protocol training for all personnel 
participating in the experiment, and conduct training and assessment for clinical research staff, 
so that clinical trial personnel able to quickly become familiar with and master the fluorescence 
PCR technology and the applicable instruments, operating methods, and technical performance 
etc. of the kit. 
(6) This protocol has been determined through repeated communication and research between the 
experimental institution and the clinical institution, and must be strictly implemented. Any 
changes in the plan must be approved by the team leader (master) institution. 
4 Evaluation method 
4.1 Evaluation method for reagents to be tested 
1) 
The Ct value of FAM channel of positive control substance was ≤18, and the amplification 
curve had obvious exponential growth period. 

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(2) The blank control FAM channel had no amplification curve, or the amplification curve was 
slightly oblique without significant exponential growth, and the Ct value of FAM channel was 
≥24. 
(3) Result determination: After determining Ct value and △Ct value according to the above steps, 
determine the result according to the following table. 
Table 2 Result determination 
Mutation 
type 
Negative 
Negative 
Positive 
C228T 
There was no obvious amplification of FAM 
channels 
Or the Ct value is not displayed 
Ct value < 
25 
△ 
Ct > 9 
Ct 
value < 
25 
△Ct 
≤ 9 
C250T 
There was no obvious amplification of FAM 
channels 
Or the Ct value is not displayed 
Ct value < 
25 
△ 
Ct > 9 
Ct 
value < 
25 
△Ct 
≤ 9 
4.2 Evaluation method for nucleic acid sequence determination 
(1) The results of the positive reference substance tested on the machine for nucleic acid sequence 
determination were positive, and the results of the blank reference substance should be in line 
with expectations without mutation detection. 
(2) Determination of reagent results: the sequencing software Mutation Surveyor was used to 
interpret the results. When 6695 G>GA, corresponding mutation is indicated; otherwise, no 
mutation is indicated. 
4.3 Evaluate the conformity between the kit and the reference method 
(1) If the results to be tested and the reference results are both positive or negative, it is regarded 
as in conformity. 
(2) In the event that the result to be tested is positive and the reference result is negative, or the 
result to be tested is negative and the reference result is positive, it is recorded as non-
conformity. 

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(3) If the result to be tested fails and the reference method has confirmed results, it shall be 
recorded as non-conformity. The reasons and results of the non-conformity shall be truthfully 
recorded, and the comparison results shall be repeatedly confirmed when necessary. 
(4) Complete subsequent statistical analysis based on the above statistical results. 
5 Statistical methods 
Statistical analysis of clinical study data: Kappa test was used for statistical analysis of the 
comparison between the kit test results and gene sequencing test results, and then the difference 
between the results obtained by the two methods was compared. If Kappa > 0.6, it was proved that 
there was no statistical difference between the two methods in detecting TERT gene promoter 
mutations in glioma samples. 
6 Duration of clinical trials and its reasons  
The clinical study lasted about 6-8 months from the drafting of the protocol, project approval, ethical 
review, agreement signing, specimen collection, the beginning of the test to the end of the trial, and 
the report and seal are completed. If the sample collection is smooth and the number of samples 
meets the test requirements, the clinical trial time can be shortened; if it is not smooth, the time can 
be appropriately extended. 
7 Inclusion criteria, exclusion criteria and rejection Criteria 
7.1 Inclusion criteria 
(1) Be able to provide samples timely according to the requirements of the protocol. 
(2) The pathological examination conforms to the type of tissue sample listed in Table 1, and the 
tumor content is not less than 50%.  
(3) The sample should have corresponding basic clinical information, including: the patient's 
visiting No. / medical record No./specimen No., age, sex, and pathological diagnosis result.  
7.2 Exclusion criteria 
Those who do not meet any of the above conditions are excluded. 
Rationale: Included glioma, other brain tumors, and normal samples should be statistically 
significant and the conclusions obtained are scientifically valid. The selection of subjects for this 

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study, while excluding research-related influencing factors, has no adverse effect on the health of 
the subjects. 
7.3 Rejection Criteria 
(1) Samples considered by the researcher to be unsuitable for continuing clinical trials, such as 
samples that have not been prepared in accordance with the required steps. 
(2) Samples with unqualified genomic DNA quality, such as: 
① DNA concentration is less than 10ng/µL; 
② Genomic DNA was severely degraded or had insufficient purity, and OD260/OD280 
exceeded the range of 1.6~2.0. 
(3) Samples whose repeated test results of reference methods failed. 
(4) Samples with incomplete clinical basic information (patients' visiting No. / medical record 
No./specimen No., age, sex, pathological diagnosis result). 
8 Provisions for amendments to clinical trial protocols 
Any modification to the protocol during the test should be explained, and the time, reason, process 
and record of the modification should be elaborated in detail, and its impact on the evaluation of the 
whole study results should be demonstrated. 
9 Ethical issues and explanations of clinical trials 
This clinical trial verification test uses the patient’s pathological specimens in the previous treatment 
process. It is limited to in vitro diagnosis, does not directly contact the patient, does not cause any 
harm to the patient, and only compares the performance of the reagent with the control reagent. The 
results are only used for study and analysis related to this test, and will not guide the diagnosis and 
treatment of patients based on this, so it will not have any adverse effects on the human body. 
This clinical trial strictly complies with the "Declaration of Helsinki", and this clinical verification 
trial involves relevant patient data. Such as: Patient visiting No. / medical record No. / specimen 
No., age, sex, pathological diagnosis result, etc. The hospital and Genetron Health (Beijing) Co., 
Ltd. will jointly undertake the confidentiality of the patient information, and the clinical trial 

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operator and clinical validation unit promise not to disclose the content related to the subject 
specimen. 
Therefore, the clinical trial was verified to submit an informed consent exemption to the ETHICS 
Committee. 
10 Data processing and record keeping 
It is required to keep all experimental records during the clinical trial, the results should be objective, 
timely and serious, and the operator and operation time should be recorded. Medical institutions 
shall keep the clinical trial data for five years after the termination of the trial.