September 23, 2023

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How to choose genetic testing methods for lung cancer?

How to choose genetic testing methods for lung cancer?

How to choose genetic testing methods for lung cancer? With so many genetic testing methods for lung cancer, which is better? How to choose?

In the past ten years, the treatment of advanced non-small cell lung cancer (NSCLC), especially targeted therapy, has made great progress, which can significantly improve the objective remission rate of patient treatment and prolong progression-free survival (PFS), and significantly improve Quality of Life. Molecular typing is the prerequisite for the implementation of targeted therapy for NSCLC .

It is of great clinical significance to select accurate, fast, and appropriate detection methods and comprehensively screen out the target population for targeted drugs. At the same time, with the continuous discovery of rare gene mutations and the improvement of the mechanism of acquired drug resistance of targeted drugs, clinics have put forward more demands for the connotation of genetic testing.

There are various methods of molecular pathology detection, including Sanger sequencing, real-time quantitative PCR (qRT-PCR), fluorescence in situ hybridization (FISH), next-generation sequencing (NGS) and immunohistochemistry (IHC), etc.

The various detection methods have different The advantages and disadvantages, the appropriate testing method should be selected according to the testing content and clinical needs.

Single-gene testing is a widely approved testing method at this stage, which is fast and easy to carry out; multi-gene testing can provide patients with multiple genetic mutation information at one time.

At this stage, the multiplex PCR detection and the small panel second-generation sequencing kits have been approved by the National Medical Products Administration (NMPA), which can detect multiple gene mutations at the same time; the second-generation sequencing technology can perform more genes at the DNA and RNA level , Multi-site detection is the development direction of molecular pathology in the future.

NSCLC gene mutation detection mainly includes targeted therapy and immunotherapy-related molecular pathology detection . 

Immunotherapy-related molecular pathology tests include PD-L1 protein expression and TMB . Other biomarkers such as high microsatellite instability (MSI-H) are rare in NSCLC.

How to standardize the genetic testing of NSCLC patients, so that lung cancer patients can get the best stratified treatment plan, has always been the direction of clinicians’ efforts . 

Applicable population for genetic testing

1. Pulmonary invasive adenocarcinoma (or NSCLC containing adenocarcinoma) that is intended to receive targeted therapy.

2. Advanced NSCLC patients with non-adenocarcinoma confirmed by biopsy histopathology can be recommended for target molecular gene testing. In addition to lung adenocarcinoma, target molecular gene mutations exist in other NSCLC patients (such as lung adenosquamous carcinoma, non-specific NSCLC, large cell lung cancer, and sarcomatoid carcinoma, etc.), and patients can benefit from targeted drug therapy .

3. For all EGFR and ALK gene mutation-negative advanced NSCLC patients, if they plan to undergo PD-1/PD-L1 antibody drug immunotherapy, PD-L1 expression detection is recommended. For NSCLC patients who intend to undergo anti-PD-1/PD-L1 immunotherapy, TMB testing can be selected.

Specimen type specification for genetic testing

1. Paraffin specimens of tumor tissues are preferentially used for test specimens :

mainly include specimens obtained by surgery, fiberoptic bronchoscopy biopsy, CT-guided lung puncture, thoracoscopy, lymph node biopsy and other methods. The proportion of tumor cells must be evaluated before the test, and the test can be performed only after the test requirements are met. For surgical specimens, preference is given to selecting specimens with a higher proportion of tumor cells for genetic testing . If the proportion of tumor cells is low, enrichment can be used to ensure the accuracy and reliability of the test results.

2. Cytology specimens :

including pleural effusion, percutaneous lung biopsy, intrabronchial ultrasound guided fine needle aspiration biopsy (EBUS-FNA), sputum, alveolar lavage fluid, etc., which need to be made into paraffin-embedded specimens for tumor cells Proportion evaluation, testing can be carried out after meeting the testing requirements.

3. For a small number of patients with advanced lung cancer who cannot obtain tissue or cytology specimens, blood testing (liquid biopsy) is recommended .

There is circulating free tumor DNA (ctDNA) in the blood of patients with advanced NSCLC, and the detection rate of ctDNA in their plasma is higher.

4. For some NSCLC patients with advanced meningeal metastasis:

the cerebrospinal fluid can enrich the ctDNA of intracranial tumors , and the cerebrospinal fluid can be obtained by lumbar puncture for related gene detection.

Different mutant gene testing specifications


EGFR gene mutations include gene mutations (point mutations, insertion/deletion mutations), which mainly occur in exons 18-21 of the EGFR tyrosine kinase region, and acquired drug resistance mutations (including first and second generation TKIs). The resistance mutation T790M and the third-generation TKI resistance mutation C797S, etc.).

Commonly used detection methods and site requirements for EGFR gene mutations: There are currently many methods for EGFR gene mutation detection. Commonly used clinical detection methods include Sanger sequencing, qRT-PCR and next-generation sequencing . Regardless of the detection method used, it should include the deletion of exon 19 (19del) and exon 21 point mutation (L858R), the most important mutation site of EGFR, and the exon 18 point mutation (G719X). Exon 20 insertion mutation (20ins) and point mutation (T790M, S768I), exon 21 point mutation (L861Q), etc.

Advantages and disadvantages of different detection methods

Sanger sequencing method

It is a method that can directly detect known and unknown mutations, and is an early and widely used EGFR gene mutation detection method. However, the sensitivity of Sanger sequencing method to detect EGFR gene mutations is low, the operation steps are complicated, and it is prone to contamination, which can no longer fully meet the needs of clinical EGFR gene mutation detection.


Based on the qRT-PCR method, primers and probes need to be designed according to the known mutation types of the EGFR gene, which cannot detect all possible mutations, but the sensitivity is relatively high, the operation is simple, and there is no need to manipulate the PCR product, to a large extent It avoids the contamination of amplified products and is easy to carry out clinically. It is currently one of the most commonly used detection techniques for EGFR gene mutation detection .

Next-generation sequencing

Next-generation sequencing is a high-throughput sequencing technology that can sequence multiple genes and multiple sites at the same time. Compared with traditional sequencing technology, second-generation sequencing can detect a large number of target genes at one time, can analyze the abundance of gene variation, and is relatively low cost. However, the second-generation sequencing has many operating steps and complex procedures, and problems in any link will affect the accuracy of the detection results. The interpretation of the results depends on the accurate analysis of biological information. Next-generation sequencing detects EGFR gene mutations, and the detection sites are more comprehensive, and rare mutation sites can be found, providing a basis for the entire process management of patients with advanced NSCLC .

Resistance mechanism detection

At present, the resistance mechanism of EGFR TKI is basically clear, most of which are acquired mutations of EGFR T790M, accounting for about 50%. Other mechanisms include MET gene amplification, KRAS mutation, BRAF mutation and so on. Therefore, for first- and second-generation EGFR-TKI-resistant patients, T790M testing (qRT-PCR or second-generation sequencing) is preferred. It can also be tested with other resistance mechanisms at the same time or after T790M is negative, high-throughput technology (second-generation sequencing) can be used to detect other resistance mechanisms. For third-generation TKI-resistant patients, second-generation sequencing is recommended to detect the mechanism of resistance.


ALK gene mutation types include gene rearrangement/fusion, and acquired resistance mutations (mainly point mutations). Acquired mutations in the kinase region of ALK are one of the main mechanisms for ALK inhibitors to treat drug resistance, and have certain guiding significance for guiding the follow-up clinical treatment of drug-resistant patients.

Common detection methods and main features of ALK gene mutations: ALK gene rearrangement leads to the expression of ALK fusion genes, which can be detected at multiple molecular levels, including FISH to detect ALK gene rearrangements at the DNA level; qRT-PCR to detect ALK fusion mRNA ; IHC detects ALK fusion protein expression, and next-generation sequencing detects rearrangements at the DNA level or fusion sequences at the mRNA level.

Advantages and disadvantages of different detection methods

ALK Ventana D5F3 IHC test

It is the fastest and most economical method at present, and the interpretation standard of binary results is simple and easy to implement . This interpretation standard is only applicable to lung adenocarcinoma. This test should be used with caution when used in squamous cell carcinoma, neuroendocrine carcinoma and other types of lung cancer specimens. Suspected positive specimens need to be verified by other methods. In clinical practice, we must be alert to the pitfalls in the interpretation of IHC results to avoid non-specific staining.

FISH detection

It is the “gold standard” for detecting ALK rearrangement. The interpretation of the test results is intuitive, and the requirements for sample quality are low, but the cost is high and the economic benefit ratio is poor. And in FISH interpretation, it is necessary to be extra cautious in judging the separation signal at the critical value, atypical separation signal, etc., it is recommended to use other technology platforms to review the detection.


The ALK fusion gene detection based on the qRT-PCR method has high sensitivity and specificity, but because qRT-PCR can only detect the known type of ALK fusion gene, there are false negatives . In addition, since qRT-PCR is based on mRNA amplification technology, the strictest technical standards should be formulated for internal and external quality control in the laboratory to prevent contamination.

Next-generation sequencing

The second-generation sequencing can detect ALK gene mutations in the blood, but the appearance of false negative results should be noted, and other platforms or detection methods should be selected for retests if necessary.

3 、 ROS1

ROS1 gene variants include ROS1 gene rearrangement. At present, more than ten kinds of ROS1 gene fusion partners have been discovered, including CD74, SLC34A2, CCDC6, TPM3, EZR, etc. The break sites are mainly located in exons 32 to 36 of the ROS1 gene.

ROS1 gene detection method and main features:

  • ROS1 gene rearrangement/fusion expression detection methods are similar to ALK, but IHC antibodies have poor specificity. Currently, they cannot be used directly to detect ROS1 gene rearrangement/fusion expression, and can only be used for the preliminary screening of ROS1 gene fusion.
  • ROS1 fusion gene detection based on qRT-PCR method has high sensitivity and specificity, and can be combined with ALK.
  • The FISH test is the “gold standard” for detecting ROS1 rearrangement .
  • The detection of ROS1 gene mutations by next- generation sequencing can also detect rearrangement sequences at the DNA level, as well as fusion sequences at the mRNA level. However, due to the particularity of the ROS1 gene sequence, the detection sensitivity of second-generation sequencing based on the DNA level is affected by the library probe design. And the ability of bioinformatics analysis has a greater impact, so you should pay attention to false negatives.


In the clinical practice of NSCLC, based on the existing evidence-based medical evidence, the current focus is on the detection of MET exon 14 skipping mutations and MET amplification. MET amplification includes primary amplification and secondary amplification , and its secondary amplification is more common in patients with positive driver genes after TKI treatment progresses, and it is one of the important mechanisms of EGFR-TKI treatment of drug resistance.

Common detection methods and main characteristics of MET gene mutations

  • MET exon 14 skipping mutation detection, including next- generation sequencing or qRT-PCR direct detection of mRNA missing MET exon 14, or next-generation sequencing at the DNA level may lead to MET exon 14 splicing Genetic variation.
  • RNA-based testing should focus on mRNA quality in clinical practice, and quality control should be done before testing. If it is found that mRNA has been degraded, it is recommended to re-test. Insufficient coverage of MET gene probes may lead to missed detections in DNA-based next-generation sequencing. It is recommended that next-generation sequencing detection should cover as far as possible the exon 14th, such as the 13th or 14th intron, which may have splicing mutations. area.
  • In situ hybridization detection is the standard method for detecting MET amplification. At present, different FISH interpretation standards [Cappuzzo standards and UCCC (University of Colorado Cancer Center) standards] are used in clinical research. In clinical practice, it is recommended to use UCCC standards that can distinguish between site-specific amplification and polyploidy as much as possible.

Compared with FISH, next-generation sequencing can be used for MET amplification detection, but the correspondence with FISH detection is more complicated, and may miss MET polysomes, but next-generation sequencing can detect MET mutations and fusions and other mutations at the same time, and can achieve Multi-gene co-examination is more widely used in clinical practice.

5. Other target molecules

In addition to the aforementioned target molecules, HER2 gene mutation or amplification, BRAF mutation, KRAS mutation, RET gene rearrangement, NTRK family gene rearrangement, etc., are all important driver gene mutations in NSCLC and are potential therapeutic targets. Their detection methods can refer to the detection methods and detection strategies of gene point mutations and gene rearrangement types. For the selection and optimization of testing protocols, more accumulation of clinical practice is still needed.

HER2 is a member of the ERBB family of tyrosine kinase receptors. The mutation or amplification of HER2 gene is one of the driving genes of NSCLC. The most common genetic mutation is the insertion mutation in exon 20 of HER2. The mutation rate in NSCLC is 2%~4% .

BRAF mutation can cause abnormal activation of the MAPK/ERK signaling pathway. The common site of BRAF mutation is V600, and the mutation frequency in NSCLC is 1% to 2%.

The mutation rate of K RAS gene in Asian population is 8%~10% . The mutation sites are located in exons 2 and 3, which are prone to occur in smoking lung adenocarcinoma patients, and are prone to be accompanied by other gene mutations, such as LKB1, p53, CDKN2A /B, etc., KRAS mutations are related to poor prognosis and drug resistance.

The mutation frequency of RET fusion genes in NSCLC ranges from 1% to 4% , and the most common RET gene fusion partners are KIF5B (70% to 90%) and CCDC6 (10% to 25%). NTRK fusion genes are rare in NSCLC, with an incidence of less than 0.1%.

How to choose genetic testing methods for lung cancer?

(source:internet, reference only)

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