Clinical Evaluation of Genetron IDH1 PCR Kit in Glioma Patients
Conditions: Glioma, Malignant, Glioma, Mixed
Sponsor: Genetron Health

 
Clinical Evaluation of Genetron IDH1 PCR Kit in  
Glioma Patients 
 
 
 
Study Document 
 
 
 
 
 
 
 
 
NTC number: NCT05100173 
Date: 02/01/2016 
 

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

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 
Isocitrate dehydrogenase (IDH), located in the long arm of chromosome 2 (2q33), is a key rate-
limiting enzyme in the tricarboxylic acid cycle, providing energy for cell metabolism and precursors 
of biosynthesis. There are three isomerases in the IDH gene family (IDHl, IDH2 and IDH3). IDH1 
or IDH2 mutations lead to the accumulation of 2-hydroxyglutaric acid, another new metabolite, in 
cells, which can not only lead to abnormal histone modification, but also increase the activity of 
VEGF pathway, thereby inducing tumorigenesis. The data showed that IDH1/2 gene mutation was 
an early genetic mutation in the development of glioma. It usually occurs in young adults and 

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adolescents with diffuse glioma. IDH1/2 mutation had a certain distribution pattern corresponding 
to WHO classification, which was mainly distributed in grade II~III oligodendroglioma (69.2%), 
astrocytoma (79.8%), oligoastrocytomas (75.5%) and grade IV secondary glioblastoma (73%), but 
rare in primary glioblastoma (<5%) and childhood glioblastoma. 
IDH1 mutations accounted for more than 90% of IDH1 and IDH2 gene mutations, among which 
IDH1 R132H (CGT>CAT, arginine replaced by histidine) mutations were the most common, 
accounting for about 90% of IDH1 mutations. 
The National Comprehensive Cancer Network (NCCN) Clinical Practice Guidelines for Central 
Nervous System Cancer (V1, 2015) include the presence or absence of IDH1 or IDH2 gene 
mutations as one of the indicators to assess the risk type of patients with low-class glioma. The 
Chinese Guidelines for molecular Diagnosis and Treatment of glioma (2014) also included IDH1 or 
IDH2 gene mutation as the preferred molecular marker for molecular pathological typing (grade I 
evidence). 
At present, the gold standard method for clinical detection of IDH gene mutation is nucleic acid 
sequencing. However, this method is time-consuming and laborious to determine the detection 
results, and the operation process is complex, so it is difficult to widely apply this technique in 
clinical practice. 
2 Research Purpose 
By using this product to detect the IDH1 R132H gene mutation in the cancer cells of patients with 
glioma, compare it with the results of nucleic acid sequence determination, and combine the results 
of clinicopathological typing to confirm and evaluate the safety and effectiveness of this product. 
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 IDH1 gene R132H mutation in different subtypes 
was counted to evaluate the clinical application performance of this product 

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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 
IDH1 
R132H 
There was no obvious amplification of FAM 
channels  
Or the Ct value is not displayed 
Ct value < 
25 
△ 
Ct > 8 
Ct 
value < 
25 
△Ct 
≤ 8 
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. 
(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. 

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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 IDH1 gene 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 
study, while excluding research-related influencing factors, has no adverse effect on the health of 
the subjects. 

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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 
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. 

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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.