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2021

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Accurate detection is essential for accurate medication use, starting from MET for lung cancer


Lung cancer is a malignant tumor that poses a serious threat to human health in the world today, and advances in molecular biology have greatly changed the treatment pattern of advanced NSCLC. The targeted treatment of lung cancer cannot be separated from molecular detection of lung cancer, which involves various aspects, and any factor may affect the accuracy of molecular detection results. In order to standardize the molecular detection of lung cancer, AZ collaborated with relevant domestic experts to comprehensively elaborate on the sample requirements, testing platform, testing treatment and standards, and testing concepts for molecular detection of lung cancer.

Accurate detection is essential for accurate medication use - Starting from MET for rare mutated genes in lung cancer

Overview of Rare Gene Mutations in 01 Non Small Cell Lung Cancer

The number of lung cancer patients in China has increased year by year, with the incidence rate and mortality ranking first among all malignant tumors, of which non small cell lung cancer (NSCLC) accounts for about 80%. However, with the continuous progress of molecular pathological diagnosis technology and targeted drug research and development, targeted therapy for NSCLC has also made significant progress. The objective response rate and progression free survival time of NSCLC patients after targeted therapy have been significantly prolonged, and their quality of life has also steadily improved. The use of genetic testing methods to diagnose and differentiate drug sensitive mutations and screen patients suitable for targeted drugs has positive significance for the clinical diagnosis and treatment of NSCLC. For example, the mutation rate of epidermal growth factor receptor (EGFR) in NSCLC patients is close to 40%, and good results have been achieved in the relationship between the type of mutation and prognosis and targeted treatment [1]. But with the promotion and application of molecular pathological diagnosis, including second-generation sequencing (NGS) and other technologies, more rare mutations in NSCLC also affect patient prognosis. They include ROS1 (mutation rate 2% - 3%), MET (2% - 4%), HER2 (2% - 4%), BRAF (1% - 2%), RET (1% - 4%), as well as rare mutant genes NTRK (<1%), NRG1/2 (<1%), FGFR2 (<1%), etc. [1]. However, the significance of the above gene mutations in NSCLC still needs to be further clarified, and their targeted therapeutic drugs are rarely marketed in China, and their effects also need to be confirmed. Nevertheless, based on the large number of NSCLC patients in China and the epidemiological status of incidence rate increasing year by year, it is still of great significance to study the significance of the above rare gene changes on the prognosis of NSCLC patients and the effect of targeted treatment for the detection of NSCLC patients with positive mutations of the above rare genes.

02 MET gene and non-small cell lung cancer

The protein c-Met encoded by the mesenchymal epithelial transition factor MET gene, as a tyrosine kinase receptor for liver cell growth factor HGF, is abnormally activated in various tumors, promoting tumor cell survival, proliferation, and invasion, including NSCLC. Although MET mutations are relatively rare in NSCLC compared to common mutations such as EGFR, recent studies have confirmed that the most common mutation in exon 14 of the MET gene in NSCLC is 1.7% -4.3% [2-3]. However, it is worth noting that MET gene abnormalities are independent drivers of cancer and independent predictors of poor prognosis. In addition, secondary MET amplification is one of the main mechanisms of tyrosine kinase inhibitor (TKI) resistance targeting EGFR, accounting for approximately 5% -20% of the EGFR TKI resistance mechanism. Therefore, MET gene abnormalities, including overexpression, amplification, exon 14 skip mutation, and relatively rare MET rearrangements, play an important role in the occurrence, development, drug resistance, and prognosis of NSCLC. At present, MET inhibitors have been approved domestically and internationally for the treatment of NSCLC patients with MET14 exon skip mutations. Phase I and II clinical studies are also underway on the combined use of MET inhibitors, EGFR-TKI, and other drugs for the treatment of NSCLC patients. Therefore, the detection of MET gene abnormalities is of great significance for targeted therapy based on MET mutations in NSCLC patients.

03 Problems in MET gene testing

Due to the relatively rare occurrence of MET gene abnormalities and insufficient experience in molecular pathological diagnosis, there are still many problems in the detection of MET gene abnormalities mentioned above in clinical practice. If the number of puncture samples is small, it is not possible to detect c-MET protein expression using immunohistochemistry (IHC); There is also controversy over the interpretation of positive results when detecting MET amplification using in situ hybridization (FISH) technology; The hopping mutation of exon MET14 has base pair deletion, point mutation and insertion deletion mutations with different lengths, which poses challenges to detection and subsequent bioinformatics analysis. Based on the above reasons, the clinical detection rate of MET pathway abnormalities in China is relatively low. Many advanced NSCLC patients with MET pathway abnormalities but without other common drug sensitive mutations will be treated with conventional radiotherapy and chemotherapy according to the wild-type mutation, with very limited effectiveness. With the continuous release of clinical data on MET targeted drugs, there is an urgent need for accurate, efficient, and convenient MET gene detection methods in clinical practice.

The detection strategy of 04 MET gene in non-small cell lung cancer

1) MET 14 skip mutation detection

At present, multiple MET inhibitors targeting MET 14 exon hopping mutations have been approved both domestically and internationally, such as cefotinib and camatinib. Therefore, their precise detection is of great significance for targeted drug use. The detection of MET 14 exon skip mutations is currently mainly based on NGS and RT-PCR. For NGS detection, in order to prevent missed detection, the NGS probe should try to cover areas outside the 14th exon, such as those on the 13th or 14th intron, where shear mutations may occur. The RNA-Base NGS method is superior to the DNA-Base method in terms of technology, and RT-PCR is also based on RNA level detection, with the characteristics of fast and high sensitivity. Due to the ability of NGS to simultaneously detect multiple mutations such as MET mutations and fusion, and to achieve multi gene co detection, it is more widely used in clinical practice. In addition, when tissue samples are not available in a timely manner, plasma samples can also be considered for the detection of MET exon 14 mutation as a supplement.

2) MET amplification detection

The detection methods for MET amplification mainly include FISH, NGS, and dPCR. FISH technology can be used for tissue detection of gene amplification or rearrangement, which is currently the gold standard for MET amplification detection and can distinguish between site-specific amplification and polyploidy. Taking FISH as a reference, the sensitivity and specificity of tissue NGS detection for targeted amplification can reach 88% and 98% [4]. Both NGS positive patients and FISH positive patients have a clinical benefit trend after treatment with EGFR combined with MET-TKI [5], but polyploid amplification is easy to be missed. In the future, NGS detection technology should be further optimized to improve the detection rate of NGS for polyploid. DPCR has the characteristics of high sensitivity, accurate quantification, and simple operation, which can be used for detecting MET site-specific amplification and polyploid amplification. The biggest advantage of dPCR is its ability to detect quantitative information more accurately, making it possible to become another effective method for detecting MET gene copy number. It can effectively detect MET gene amplification in both tissues and blood. Both IHC positive patients with MET overexpression and FISH positive patients have a clinical benefit trend after treatment with EGFR-TKI combined with MET-TKI [5]. Currently, multiple clinical studies have shown that c-MET overexpression patients have better feedback on the efficacy of MET inhibitors, and IHC will be more widely used in MET anomaly detection in the future. At present, there is no consensus on the threshold for clinical benefits of MET inhibitors in different drug clinical studies and detection methods, and further clarification is needed. For the detection of MET amplification, especially in EGFR TKI resistant populations, if there are sufficient tumor tissue samples, priority can be given to selecting NGS. When the NGS detection results cannot be determined, there is amplification signal but it is not typical, or it is at the critical value of patients, it is recommended to use FISH for retesting. For special patient populations, such as those with EGFR-TKI resistance and negative T790M or other drug resistance mechanisms, or when the tumor cell content in tissue samples is low, considering the potential risk of missed detection due to copy number variation in NGS testing, FISH retesting can still be considered when NGS reports negative MET amplification. Taking the organization as a reference, existing data on plasma testing for MET amplification indicate low sensitivity, and necessary explanations should be provided in the report. Plasma testing is only for clinical reference, and negative testing cannot exclude MET amplification.

05 Clinical Laboratory Self built Project

The self built project of clinical laboratory, abbreviated as LDT, refers to an in vitro diagnostic project that has internal research and development, verification and use of the laboratory, and uses biochemical, cytogenetic and molecular biological test methods to analyze biomarkers such as DNA, RNA, mitochondria, proteome and metabolomic diseases for the purpose of diagnosis; LDT can only be used in research and development laboratories; Purchased or self-made reagents can be used, but cannot be sold to other laboratories, hospitals, or doctors. According to the Expert Consensus on Basic Requirements for Laboratory Self built Molecular Diagnosis Projects [6], conducting LDT should not only meet certain laboratory qualifications and environmental requirements, but also meet regulatory requirements for personnel. The technical indicators of LDT should meet certain requirements such as accuracy, precision, analysis sensitivity, and analysis specificity. And LDT should also be clinically validated.

LDT can compensate for the shortage of IVDs testing projects that are insufficient to meet clinical needs, and is also a compliant testing project. At present, the clinical testing projects carried out in the hospital mainly refer to the "Clinical Testing Catalogue of Medical Institutions (2013)", and all testing reagents need to be purchased with approved IVDs (in vitro diagnostic reagents). However, due to the fact that the "Catalogue" projects are far from sufficient to meet the clinical testing needs, especially the individualized diagnosis and treatment testing needs of tumors, on March 3, 2016, the National Health and Family Planning Commission should promptly demonstrate and meet clinical needs for clinical testing projects that are not included in the "Catalogue (2013 Edition)" but have clear clinical significance, good specificity and sensitivity, and reasonable cost-effectiveness. On September 2, 2020, the National Drug Administration publicly stated that there are no in vitro diagnostic reagents of the same variety available in China, and medical institutions can develop and use them in their own institutions. On February 9th of this year, the latest version of the "Regulations on the Supervision and Administration of Medical Devices" was released, Among them, 53 provisions For in vitro diagnostic reagents that have not yet been launched with the same variety of products in China, eligible medical institutions can develop and use them in their own units under the guidance of licensed physicians according to their clinical needs. From then on, the identity of LDT testing has been officially legalized. Taking T790M blood testing as an example, there are no approved reagent kits for NGS and ddPCR blood high sensitivity testing. LDT can be used in medical institutions to provide more accurate individuals for clinical use Chemical diagnosis and treatment plan.

To sum up, the incidence rate of NSCLC is increasing year by year. Although MET gene abnormalities are relatively rare in NSCLC, based on the large number of NSCLC patients in China, and recent clinical and laboratory studies related to MET, it is confirmed that MET gene abnormalities are also important in NSCLC patients, and are often associated with drug resistance to targeted drugs such as EGFR. Clinical studies have confirmed that advanced MET positive NSCLC patients can significantly benefit from MET targeted drug therapy. Therefore, MET pathway mutations play an important role in the occurrence, development, drug resistance, and prognosis of NSCLC. However, due to various reasons, there are problems in the identification and targeted treatment of MET mutation positive patients. The commonly used molecular pathological detection methods in the diagnosis of MET gene abnormalities also need to be further optimized, such as the lack of reliable and unified thresholds and the establishment of low, medium, and high stratification standards based on clinical indicators. Therefore, clarifying the role of MET gene testing in NSCLC, ensuring accurate detection results, is of great clinical significance for patient screening and efficacy evaluation.

reference

1. Clinical Practice Guidelines for Molecular Pathological Testing of Non Small Cell Lung Cancer (2021 Edition). Chinese Journal of Pathology, 2021, 50 (4): 323-332

2. Matikas A, et al. Current and Future Approaches in the Management of Non-Small-Cell Lung Cancer Patients With Resistance to EGFR TKIs. Clin Lung Cancer. 2015; 16(4):252-61..

3. Drilon A, et al. Targeting MET in Lung Cancer: Will Expectations Finally Be MET?. J Thorac Oncol. 2017; 12(1):15-26.

4.Hartmaier RJ, et al. 2019 AACR Abstract 4897.

5. Ryan Hartmaier presented in 2021 AACR, CT127

6. Expert Consensus on Basic Requirements for Laboratory Self built Molecular Diagnosis Projects

Attachment: Summary Table of Targeted and Immunotherapy Drugs for Lung Cancer (October 2021)

Non small cell lung cancer