Clinical Evaluation of Genetron D842V PCR Kit in GIST Patients
Conditions: GIST, Gastrointestinal Stromal Tumors
Sponsor: Genetron Health

 
 
 
Clinical Evaluation of Genetron D842V PCR Kit  
in GIST Patients 
 
 
 
Study Document 
 
 
 
 
 
 
 
 
NCT number:NCT05400018 
Date: 05/30/2022 
 
 
 

 
Content 
1 Background ................................................................................................................. 3 
2 Study Purpose ............................................................................................................. 4 
3 Study Design ............................................................................................................... 4 
3.1 Overall Study Design ........................................................................................... 4 
3.2 Sample Screening ................................................................................................. 5 
3.2.1 Basis to Determine Sample and Sample Size ................................................... 5 
3.2.2 Inclusion, exclusion and elimination criteria .................................................... 6 
3.2.2.1 Inclusion criteria ......................................................................................... 6 
3.2.2.2 Exclusion criteria ........................................................................................ 6 
3.2.2.3 Elimination criteria ..................................................................................... 6 
3.2.3 Sample collection, storage and transportation................................................... 6 
3.2.4 Sample numbering............................................................................................. 7 
3.3 Basis to Determine Comparison Method ............................................................. 7 
3.4 Study Duration and Reasons for Determination .................................................. 8 
4 Statistical Method ....................................................................................................... 8 
4.1 Positive, Negative and Total Coincidence Rates Between Test Reagent and 
Comparison Method ................................................................................................... 8 
4.2 Kappa test ............................................................................................................. 9 
5 Quality Control Method .............................................................................................. 9 
6 Provisions for Protocol Amendment ......................................................................... 10 
7 Ethical issues and explanations involved in the clinical trial ................................... 10 
8 Data Processing and Record Storage ........................................................................ 11 
9 Responsibilities of Involved Parties.......................................................................... 11 
10 Study Technology Roadmap ................................................................................... 12 
 
 
 

 
1 Background 
Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor in 
gastrointestinal tract, biologically manifested from benign to malignant, usually 
expressed as CD117 and DOG1 positive in immunohistochemical testing, showing 
differentiated Cajal cells. Most cases have c-kit or platelet derived growth factor 
receptor alpha (PDGFRA) gene activation mutation, and a few cases involve other 
molecular changes, including SDHX, BRAF, NF1, K/N-RAS and PIK3CA gene 
mutations. These mutations force the protein kinase into an increasingly active state. 
Since other available treatments mainly bind to inactive protein conformations, 
certain primary and secondary mutations usually cause therapeutic resistance and 
disease progression. In unresectable or metastatic GIST, the clinical benefits of 
current treatments may vary depending on the type of mutation. Mutation detection 
plays an important role in tailoring treatments for potential disease drivers and is 
recommended in expert guidelines. 
The full name of the protein encoded by PDGFRA gene is platelet derived growth 
factor receptor alpha α. As a cell surface receptor tyrosine kinase, PDGFRA can be 
activated after binding with its corresponding ligand PDGF, and then activate the 
phosphorylation pathways of phosphatidylinositol, cAMP and various proteins to 
regulate cell division and proliferation. Abnormal gene activation may cause 
tumorigenesis and promote tumor angiogenesis. PDGFRA mutation is closely related 
to GIST. Currently, up to 85% of patients with GIST have PDGFRA or KIT gene 
mutations. These mutations are associated with the production of abnormal KIT and 
PDGFRA proteins, which drive cancer. The two proteins can usually be shut down by 
imatinib (Glivec) and similar drugs, so as to block the activity of proteins. However, 
the PDGFRA Exon 18 mutant is very special. It changes the shape of PDGFRA 
protein, thus preventing the protein from binding to drugs. Meanwhile, clinical studies 
found that Avapritinib was able to bind to all mutated PDGFRA proteins tested and 
inhibit their activity in cancer cells. Since the most common mutant of PDGFRA is 
D842V mutant on Exon 18, the qualitative detection of D842V mutant on PDGFRA 

 
in patients with GIST can be used to assist the concomitant diagnosis of Avapritinib in 
clinical drug treatment. 
Genetron D842V PCR Kit is used for qualitative detection of D842V mutation in 
Exon 18 of PDGFRA gene in formalin-fixed paraffin-embedded (FFPE) tissue 
samples of patients with gastrointestinal stromal tumors (GIST), and for concomitant 
diagnosis of Avapritinib tablets in drug therapy. The result of this kit is for the related 
sites only, used as a reference for clinicians to choose proper tumor-targeted drugs for 
patients with GIST, and cannot be used as the only basis for individualized treatment 
of patients. Clinicians should thoroughly evaluate the detection results based on 
factors such as patients' condition, drug indications, treatment response and other 
laboratory indicators. 
2 Study Purpose 
The primary purpose of this study is to validate and evaluate the safety and 
effectiveness of Genetron D842V PCR Kit by comparing the results of this kit to 
those of the simultaneous detection with Sanger sequencing method, calculating the 
coincidence rate and consistency between the test reagent and the comparison method. 
3 Study Design 
3.1 Overall Study Design 
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. When the comparison results 
are inconsistent with the detection results of Sanger sequencing method, the detection 
results of sanger will be acceptable. Results will be interpreted independently 
according to the critical value or interpretation requirements provided by each method, 
and relevant statistics will be carried out to evaluate the performance of the 
investigation kits in clinical application. 

 
3.2 Sample Screening 
3.2.1 Basis to Determine Sample and Sample Size 
 
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. 
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 99%, the minimum sample size of positive control group is estimated to be 32 cases; 
if 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 
cases. 
This product is classified as Class III IVD reagent medical device, and the overall 
sample size of clinical trials should meet the requirements of current relevant laws 
and regulations. According to the Measures for the Administration of Registration of 
In-Vitro Diagnostic Reagents (CFDA [2014] No. 5) and the Technical Guidelines for 
Clinical Trials of In-Vitro Diagnostic Reagents (CFDA [2014] No. 16), the overall 
sample size of this clinical trial should not be less than 1,000. 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 positive and negative control samples 
should be statistically significant. 

 
3.2.2 Inclusion, exclusion and elimination criteria 
3.2.2.1 Inclusion criteria 
(1) The paraffin-embedded tissue samples of patients clinically diagnosed as 
gastrointestinal stromal tumors (GIST)are included, and a small number of other 
cancers or benign lesions in gastrointestinal sites are included as interference samples. 
(2) Samples should have appropriate basic clinical information, including: The 
patient's unique traceable number, age, sex, pathological diagnosis results, etc. 
(3) The samples are FFPE gastrointestinal stromal tumor tissue samples, and each 
sample can provide 5 paraffin sections or 5 paraffin rolls with a thickness of at least 5 
μm. 
(4) The content of tumor cells meets the requirements of test reagent and comparison 
method. 
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) Samples that cannot complete the whole detection process. 
(3) Patients with incomplete sample information. 
3.2.3 Sample collection, storage and transportation 
Sample collection 
The samples will be collected according to the standard pathological operating 
procedures, the paraffin-embedded pathological section samples should be tested for 
the content ratio of tumor lesion cells which should meet the requirements of test 
reagents. 
Sample storage and transportation 
The FFPE tissue samples within the retention period are transported at normal 

 
temperature, and the extracted nucleic acid is recommended to be detected 
immediately. Otherwise, DNA should be stored at -20°C±5°C for no more than 6 
months, and repeated freezing and thawing should be avoided. 
3.2.4 Sample numbering 
In principle, only one sample should be collected from one subject, and each subject 
should have a unique clinical trial number (secondary code). After sample enrollment, 
before the experimental procedures, the blinding personnel marks the secondary code 
outside the sample tube, and carefully records the comparison table between the 
primary code (the unique traceable number of the patient) and the secondary code as 
the blind codes. Blinding documents shall be kept independently by the blinding 
personnel until breaking of blindness. Blindness should not be broken under any 
circumstance during the trial. 
During experimental procedures, only the secondary code of samples is reflected. 
3.3 Basis to Determine Comparison Method 
According to "Technical Guidelines for Clinical Trials of in Vitro Diagnostic 
Reagents", for newly developed IVD reagents, appropriate subjects will be selected, 
and the investigational IVD reagents will be simultaneously compared with the "gold 
standard" for diagnosis in a blinded way in accordance with Technical Guidelines for 
Clinical Trials of In-Vitro Diagnostic Reagents. 
For products that have similar products approved for marketing, it is best to use 
investigational IVD reagents and commercially available products of the same kind as 
clinical samples for comparative study, so as to demonstrate the equivalency between 
investigational IVD reagents and commercially available products of the same kind. 
Based on this principle, for the sites with commercially available products, the 
commercially available kits will be used for the comparison method; for the sites 
without commercially available products, Sanger sequencing will be used for the 
comparison method. There is similar product on the market for the human PDGFRA gene 
D842V mutation detection kit (PCR-fluorescence probe method), and Sanger 
sequencing is used as a comparison method. 

 
3.4 Study Duration and Reasons for Determination 
This clinical study will last about 8 - 12 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 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 detection methods. 
Table 1 Result Summary of Clinical Trial Comparing Test Reagent and Comparison Method 
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). 
4.2 Kappa test 
Kappa test will be used to analyze the equivalence of test reagent and comparison 
method. Using statistical software, Kappa test will be used to compare the results of 
investigational reagents with gold standards, and k value will be calculated. When 
k>0.75, the investigational reagent and comparison method show good 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





 
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. 
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 PDGFRA 
gene D842V mutation detection kit (PCR-fluorescence probe method) in clinical 
application. The samples used in this clinical trial are the remaining pathological 
specimens detected during the treatment. These samples are only used for in vitro 
diagnosis without direct contact with patients and any harm to patients. These sample 
are only used to compare performance of the test reagent and the comparison method. 
The test results are only used for research and analysis related to this test, and will not 
guide the diagnosis and treatment of patients, therefore it will not have any adverse 
effects on human body. Informed consent can be exempted after being approved by 
the Ethics Committee. 
This clinical trial is conducted in strict accordance with the Declaration of Helsinki, 
and the validation of this clinical trial involves relevant data of patients. For example: 
The patient's unique traceable number, age, sex, pathological diagnosis results, etc. 
The patient's relevant data will be kept confidential by the hospital and Genetron 
Health (Beijing) Co., Ltd, and the implementing party and verifier of the clinical trial 
are committed not to disclose the contents related to the subject specimens. 

 
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 
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 
reagents free of charge, and provide necessary test equipment; 
（3） Provide training for study staff. 
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 
 
伦理委员会审批
签订临床试验协议
临床试验备案
临床试验启动
患者筛选
样本入组随机编码
HE染色（或提供同
一区域既往信息）
根据染色结果获取
肿瘤组织
结果对比
数据管理
统计分析
临床报告
考核试剂检测
对比方法检测
（Sanger）
 
 
Ethics Committee Approval 
Singing Clinical Trial Agreement 
Clinical trial Filing 
Clinical Trial Initiation 
Patient Screening 
Sample Enrollment and Random 
Coding 
HE Staining (or provision of previous 
information from the same area) 
Collection of Tumor Tissues Based on 
Staining Results 
Detection by Comparison Method(Sanger) 
Detection by Test Reagent 
Clinical Report 
Statistical Analysis 
Data Management 
Result Comparison