ALK fusion gene
The forms of ALK mutation include overexpression, fusion with other genes, point mutation and so on.
? ALK is a common driver gene of non-small cell lung cancer (NSCLC), and the mutation positive rate of NSCLC in China is 5.3%.
The mutation of ALK fusion gene is mainly found in lung adenocarcinoma, and the mutation probability of ALK fusion gene in patients with lung squamous cell carcinoma is very low.
Studies have shown that the ALK positive rate of Asian patients with wild-type adenocarcinoma of EGFR and KRAS is as high as 30%-42%, so it is more necessary to detect ALK gene if EGFR and KRAS are found to be wild-type.
The following figure shows the rearrangement form of ALK in non-small cell lung cancer.
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Immunohistochemistry (IHC)
Principle: It was detected by antigen-antibody binding reaction and signal cascade amplification. The results of Roche Ventana IHC kit and FISH can reach 94%~ 100% consistency.
The standard of explanation is that clusters of yellowish brown stained granules appear in the cytoplasm of tumor cells, while membrane staining is negative. Both EU and China have approved Ventana IHC to detect ALK rearrangement.
Disadvantages: Due to tumor heterogeneity, the expression intensity of target protein is uneven. Because of the possibility of protein or RNA degradation, it is not recommended to detect the ventana IHC when the FFPE slices are stored for more than 3 months.
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fluorescence in situ hybridization
Principle: The probes were separated by fluorescence in situ hybridization, and red and green probes were designed to label both ends of ALK gene respectively. Once ALK gene breaks, red and green signals will be separated, while those without break will show yellow fluorescence signal.
The standard of interpretation is to count 50 tumor cells in a single visual field. When separation signals are displayed, > 50% cells are interpreted as positive, for example.
Disadvantages: it is impossible to judge the fusion type of ALK, and it must be done by an experienced pathologist. The critical value of 15% is controversial, which may lead to false negative due to tumor heterogeneity and sampling deviation. In other words, patients with ALK mutation were missed.
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Reverse transcription polymerase chain reaction
Principle: Using pre-designed primers, it is simple and easy to detect fusion mutation from reverse transcription RNA in samples, and the fusion type of ALK can be determined.
Disadvantages: we need high-quality ALK RNA samples, most of which are paraffin samples in clinic. RNA degradation is serious, which affects the detection rate and leads to false negative results. RT-PCR detection is not recommended for samples over 2 years, and fresh tumor tissue samples are recommended. PCR primers can only detect known fusion genotypes, but not all fusion genotypes.
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Second generation sequencing (NGS)
At present, it has been proved that it is feasible to detect gene amplification and gene rearrangement by second-generation sequencing technology, and second-generation sequencing is helpful to find new fusion mutation forms of ALK. And only a few samples are needed to detect many gene mutations.
Disadvantages: the price is high, and the interpretation standard and subsequent verification need to be solved urgently.
The three methods commonly used in clinic are FISH, Ventana IHC and RT-PCR, and FISH has the lowest sensitivity. Therefore, if it is a wax block made of pleural effusion or a cytological specimen obtained by fine needle aspiration, it is not recommended to use FISH to avoid false negative. In addition, circulating tumor DNA(ctDNA) and circulating tumor cells (CTC) are also being developed through blood sampling. In a word, when the ALK test results are ambiguous, it must be verified by another test method, and none of them has the sensitivity and specificity of 100%.
References:
DOI: 10.20892/ISSN . 2095-394 1.20 17.00 17
, invasion and deletion.