Expert Consensus on Mutation Detection of Epidermal Growth Factor Receptor Gene in Chinese Patients

Issuing time:2018-10-23 00:00

一、Epidermal growth factor receptor, EGFR Clinical significance of gene mutation detection


EGFR is a transmembrane receptor tyrosine kinase, the activation of this region is phosphorylated on cancer cell proliferation, growth-related signal transmission is of great significance.Therefore, EGFR as a molecular target of cancer therapy has been widely concerned, and gradually developed gefitinib,eGFR tyrosine kinase inhibitors such as erlotinib and icotinib Kinase And anti-EGFR antibodies.


A large number of studies have shown that EGFR mutation status is the most important predictor of EGFR-TKI efficacy.The incidence of EGFR mutations was higher in women, non-smokers, adenocarcinomas, and Asian populations. The incidence of EGFR mutations is higher in adenocarcinomas, but higher in undifferentiated adenocarcinomas, adenosquamous carcinomas, and small cell carcinomas, especially in combination with adenocarcinomasmutations in the EGFR gene are also often detected.


Detection of EGFR mutation status in patients with non-small cell lung cancer is of clinical significance and is a prerequisite for determining whether patients can be treated with EGFR-TKI.Piece. In view of this, Japan, the EU's authoritative academic institutions have organized experts to develop their own expert consensus on EGFR testing. In 2011, in order to standardize the detection of EGFR gene mutation in China and to ensure the accuracy of test results, EGFR gene mutation detection in Chinese patients with non-small cell lung cancer the group developed a Chinese non-small cell lung cancer patients EGFR gene mutation detection expert consensus.


In clinical practice, however, about 10%~ fifteen percent of patients with advanced non-small cell lung cancer do not have a molecular test because the tumor tissue is insufficient or cannot be obtained.Some prospective studies showed that compared with tumor tissue, the detection of EGFR mutation in blood circulating tumor DNA ( ctDNA ) was also highly specific, but the sensitivity needs to be improved. The objective remission rate of ctdnaegfr mutation positive patients treated with gefitinib was significantly higher than that of standard chemotherapy.Progression-free survival was significantly prolonged.Therefore, ctDNA detection is a suitable alternative for mutation analysis when tumor tissue is difficult to obtain.Based on this, we have updated the consensus for the 2011 edition, which is the updated 2016 edition.




二、EGFRGeneral principles of gene mutation detection

When carrying out EGFR gene mutation detection, clinicians should cooperate with pathologists to improve the awareness of clinicians' routine detection of EGFR gene mutation, optimize the detection process, shorten the detection cycle.This is beneficial for patients with egfr-sensitive mutations to benefit as much as possible from EGFR-TKI treatment.


1. Which patients should be tested for EGFR mutations?

The mutation rate of EGFR gene was approximately 30 % in unselected Chinese patients with NSCLC. To enable patients to benefit as much as possible from the most effective treatment, all patients with non-small cell lung cancer and other types of lung cancer with adenocarcinoma components are eligible for as long as conditions permit should try to detect mutations in the EGFR gene.However, based on known epidemiological data, the mutation rates vary widely among populations.In terms of pathological type, the mutation rate of adenocarcinoma patients is much higher than that of non-adenocarcinoma patients, and the mutation rate of Asian adenocarcinoma patients can be as high as about 50%.Non-smoking, female, non-mucinous adenocarcinoma patients had a higher mutation rate. In terms of smoking history, the mutation rate of non-smoking patients was also higher than that of smoking patients, even the mutation rate of cancer patients with smoking history reached 30%.EGFR mutations should be routinely detected in patients with lung adenocarcinoma.


2. Basic requirements for conducting EGFR mutation testing laboratories:

Laboratories that carry out EGFR gene testing must establish PCR standard laboratories, which are certified by relevant institutions and meet the quality standards of China's health management institutions and the international community.Avoid contamination in the detection process and ensure the accuracy of the test results.The staff should be trained and skilled in PCR.The laboratory must participate in quality control programs such as EMQN (International) and the National Center for quality control and evaluation of pathology of the national health and Family Planning Commission (National) room quality assessment.


3. Time required for EGFR mutation detection:

For non-small cell lung cancer treatment strategies, the time required for EGFR mutation detection is a key factor, and clinical competent physicians need to obtain a test node as soon as possible the result is to determine the patient's follow-up treatment plan. It is recommended that the total process from the receipt of the specimen and the application to the report of the test results should not exceed 7 working days.



三、EGFRMutation Detection Specimen and Its Management

(一)Types of specimens

Surgically resected tumors, biopsy tissue and cytological specimens can be used to detect EGFR gene mutation. The clinical methods include surgery, biopsy under fiberbronchoscope, percutaneous lung biopsy, pleural effusion, thoracoscope, lymph node biopsy, intrabronchial ultrasound-guided fine needle biopsy ( EBUS FNA), and so on. Patients with advanced lung adenocarcinoma who could not get enough tumor tissue and cytological samples could be detected by EGFR mutation in blood samples. Both primary and metastatic foci were suitable for detection. Repeated biopsies are recommended for patients with advanced metastatic lung cancer or drug-resistant patients to monitor drug-resistant mutations in a timely manner.


(二)Management of tissue/cell specimens

No matter which sample (except blood sample) is used for the test, the following quality standards must be achieved:


1. Fixation of specimens:

Tissue specimens were recommended to be fixed with 4 % neutral buffered formaldehyde solution for biopsy and surgical removal, avoiding the use of fixatives containing heavy metal ions, the volume of the fixed sample was 10 times.The time from in vitro to fixation should not exceed 30 min.Biopsy specimens are usually fixed~ 12h, resected specimen should be fixed 6~ 48 hours.The cytological specimens (sputum and pleural effusion) should be fixed with 95% ethanol for at least 15 minutes.When it is necessary to make a wax block of exfoliated cells, it can be fixed with 95% ethanol for 2 hours.


2. Tissue section:

No matter which type of specimen is used, sufficient tumor cells should be included and non-tumor tissues and cells should be excluded as far as possible.It is recommended that the number of tumor cells is more than 200, and the proportion of tumor cells is 50 %. The application of the method with high sensitivity can be reduced as appropriate. Samples of substandard tumor cell counts should be retaken, and tests may be continued in special cases (if no other specimens are available).Note in the report (see "quality control of DNA").General cut thickness 5~ 10 m sections were sectioned to meet the requirement of tumor cells, and some measures should be taken to avoid cross contamination among different cases. The handling and quality control of the specimens should be performed by an experienced pathologist, and all specimens should be tested in as short a time as possible.

(三)Collection and handling of blood samples

During the process of blood collection, transportation and storage, the lysis of leukocytes and the degradation of free DNA are strictly prevented. In order to obtain the best plasma sample for follow-up EGFR gene mutation analysis, it is recommended to collect 10mL whole blood and use routine EDTA anticoagulation to collect blood vessel ( no heparin anticoagulant tube is used). Blood vessels ( including free DNA protectors and anti-cell lysis protectors) were collected at room temperature ( 6~ 30℃) for 2 hours or at room temperature for 5~ 7 days, Full cryogenic centrifugation was performed twice. The first stage was centrifuged at a low speed for 10min ( 800~ 2000g). The second stage was centrifuged at a high speed ( or low speed) for 10min ( 2000~ 16000g). Two centrifugations were beneficial to the removal of all cell fragments from the plasma, and then the cell-free plasma was isolated and stored at -70℃until DNA was extracted or directly extracted.


四、EGFR Common Methods for Gene Mutation Detection


1. The best method of DNA extraction:

DNA must be extracted using an extraction kit approved by the China food and Drug Administration (CFDA).Simple adsorption column extraction is recommended when tissue samples are small.When there are more tissues, we can use DNA extraction method for formaldehyde-fixed paraffin-embedded tissue. The ctDNA extraction from blood samples is recommended for use with the CFDA-approved ctDNA extraction kit.


2. DNA quality control:

The concentration and quality of DNA template directly affect the results of detection.In order to improve the detection success rate, it is suggested that the extracted DNA template should be tested to ensure that it meets certain quality standards.DNA samples do not meet the quality testing standards, in principle, do not continue to carry out gene mutation detection, special circumstances ( such as patients without other available specimens ) can continue to open exhibition inspection.If the standard EGFR mutation test is positive, the results can be reported to clinicians;if the EGFR mutation test is negative, it should be reported as "not detected" and note that the mutation cannot be ruled out due to the substandard quality of the samplesex, it is recommended to re-sample the test.


3. EGFR gene mutation detection technology :

There are many methods to detect EGFR mutations, including direct sequencing, real-time (real-time) Time) PCR-based methods such as mutation amplification arrest System, fragment length analysis, denaturing high performance liquid chromatography, etc.These methods have their own advantages and disadvantages. Regardless of the test method used, the following sensitive and resistant mutations should be included.Current guidelines recommend egfr-sensitive mutations including exon 19 deletion mutations (19del) and exon 21 point mutations (L858R),and double mutations (19del / L858R, T790M / 19del, T790M / L858R) and some rare mutations (g719x, l861q,s768i);there should also be two drug-resistant mutations, exon 20 insertion mutation (20ins) and exon 20 point mutation (T790M). Nearly 90% of EGFR mutations are 19exon deletion mutation (19del) and exon 21 point mutation (L858R).The number of tumor cells and the amount of ctDNA in the blood samples of cytology specimens are generally less, and the detection method with high sensitivity is needed, and the blood samples are limited to conditional the lab tests. In the future, more sensitive detection methods, such as micro-drop digital PCR (DDPCR) and second-generation sequencing, are expected to be mature, validated by clinical treatment and approved by CFDA.After certification, it will bring a broader application space for the use of ctDNA specimens.


4. Repeated validation of test results:

If a new mutation is found, the test should be repeated. When the sequencing results are poor (using sequencing), or the cycle threshold is close to the defined lower limit (using ARMS), or other quality criteria are not met,tests should also be repeated.If the test fails, the following steps can be taken: extracting DNA from new tissue sections;resequencing or ARMS detection; repeat the test with different samples; discuss other measures with the pathologist.In repeat testing, it is best to start again with tissue samples, and complex samples can be discussed with the competent clinician.


五、EGFRContent of gene mutation test report


The complete molecular test report should include sample information, test items and methods, test results interpretation, report signature and date.


1. Sample information :

Patient's basic information ( name, gender, age ) and patient sample information ( clinical diagnosis ), source of specimen, method of biopsy, size and quality of specimen, type of pathological diagnosis, quality control of pathology and DNA, date of acceptance).


2. Test items and methods:

Detection items and methods used, detection range and sites, sensitivity and limitations of detection methods.


3. Interpretation of test results:

Clinical interpretation of the test results, clearly pointed out that the results of genetic testing and targeted therapy association.In view of the limited sensitivity and specificity of the current blood sample testing methods, it is recommended that when the blood test results are negative, the report is "no mutation detected."he added that "based on the detection technology and specimen reasons, patients are advised to take biopsy specimens for re-testing".


4. Signature and date of report:

The EGFR gene test report should be signed and dated by the tester and the trained physician, leaving the laboratory contact information.Other relevant comments should also be recorded to better explain the test results.


Chinese non-small cell lung cancer patients epidermal growth factor receptor gene mutation detection expert group members (in alphabetical order by the name of the unit):

Department of Pathology, Peking University School of Medicine (Zhang Bo);department of Pathology; Cancer Hospital of Peking University; Beijing 100083; China;department of Pathology, Beijing Chest Hospital ( Zhang Haiqing ); department of Pathology, Beijing Hospital ( Wang Zheng ); department of Pathology; Kunming General Hospital of Chengdu Military Area Command;department of Pathology, Changhai Hospital, Second Military Medical University (Zheng Jianmin, Zhu Minghua);department of Pathology; Southwest Hospital Affiliated to the Third Military Medical University (Yan Xiaochu);department of Pathology, Xijing Hospital, Fourth Military Medical University; department of Pathology, Tumor Hospital of Fujian Province ( Chen Gang ); department of Pathology; Zhongshan Hospital; Fudan University;department of Pathology; Cancer Hospital of Fudan University;department of Pathology, School of medicine, Fudan University (Zhu Hongguang);department of Pathology; Guangdong Provincial People's Hospital;department of Pathology; people's Hospital of Guangxi Zhuang Autonomous Region;department of Pathology; the First Affiliated Hospital of Harbin Medical University (Wu He);department of Pathology; Henan Provincial People's Hospital;department of Pathology; Henan Cancer Hospital;department of Pathology; Tongji Hospital; Tongji Medical College; Huazhong University of science and technology; Wuhan 430030;department of Pathology, Second Affiliated Hospital, Bethune Medical College, Jilin University ( Gao Hongwen ); department of Pathology; Jiangsu Provincial People's Hospital;department of Pathology; Jiangsu Provincial Hospital of traditional Chinese medicine;department of Pathology; PLA General Hospital;department of Pathology, Air Force General Hospital ( Ren Li ); department of Pathology, Nanfang Hospital of Southern Medical University ( Liang Li ); department of Pathology, Nanjing General Hospital of Nanjing Military Region (RAO Qiu, Zhou Xiaojun);department of Pathology, Shanghai Chest Hospital Affiliated to Shanghai Jiao Tong University (Zhang Jie), Department of pulmonary medicine (Han Baohui);department of Pathology, Shanghai Pulmonary Hospital (Wu Chunyan), Department of respiratory medicine (Zhou caicun);department of Pathology, Xuanwu Hospital, Capital Medical University ( Teng Lianghong ); department of Pathology, West China Hospital of Sichuan University ( Buhong and Liu Weiping ), Department of Thoracic Oncology ( Lu U ), Cancer Center; department of Pathology; Cancer Hospital; Tianjin Medical University; Tianjin 300730; China;department of Pathology; the First Affiliated Hospital of Xi'an Jiaotong University (Zhang Guanjun);department of Pathology, School of medicine, Zhejiang University (Mao Zhengrong);department of Pathology; the First Affiliated Hospital; Medical College of Zhejiang University;department of Pathology, Shao Yifu hospital, Medical College of Zhejiang University (Xu songxiao);department of Pathology; Zhejiang Cancer Hospital;department of Pathology, the First Affiliated Hospital of Zhengzhou University (Jiang Guozhong, Li Wencai);department of Pathology; the First Affiliated Hospital of China Medical University;department of Pathology, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences (CAS) (Liu Tonghua, Liang Zhiyong, Zeng Xuan); Department of respiratory medicine (Wang Mengzhao);department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences, Oncology ( Shi Yuankai ); department of Pathology, Xiangya Hospital, Central South University (Zhou Jianhua);department of Pathology; China-Japan Friendship Hospital;department of Pathology; the First Affiliated Hospital of Sun Yat-sen University;department of Pathology, Cancer Hospital, Sun Yat-sen University ( Shao Jianyong ).


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