CN118086512A - Method for detecting ALK fusion gene by digital PCR method and kit thereof - Google Patents
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Abstract
The invention provides a method for detecting an ALK fusion gene by a digital PCR method and a kit thereof. On one hand, the method overcomes the situation that only using TKD/IC detection parameters can misjudge the ALK RNA expression increase caused by ALK gene amplification as false positive; on the other hand, the problem that the interpretation cannot be realized when only TKD/NO detection parameters are used because NO expression is 0 is solved.
Description
Technical Field
The invention relates to the field of digital PCR detection, in particular to a method for determining fusion gene digital PCR detection parameters.
Background
Currently, ALK fusion is widely recognized in a variety of cancers and becomes an important target for personalized therapy. Traditional detection methods, such as Immunohistochemistry (IHC), fluorescence In Situ Hybridization (FISH), quantitative polymerase chain reaction (qPCR) and the like, detect ALK fusion genes, and have the problems of missed detection, insufficient sensitivity and the like although having certain advantages.
There has been reported a method (Liu Y et al, thoracic Cancer, 2020) in which the 3-and 5-terminal expression imbalance of the ALK gene is detected by digital PCR to detect the ALK fusion gene, but in this method, only the 3-terminal/reference gene (IC) is used as a detection parameter, and it has been reported that about 10% of ALK gene amplification occurs in lung cancer patients, and that the ALK RNA expression level is increased due to the partial ALK gene amplification, and that there is a risk of false positive interpretation of the sample by using the ALK 3-terminal/reference gene (IC) as a detection parameter. Therefore, it is important to find a more rapid, accurate and reliable detection method.
Disclosure of Invention
In order to solve the above problems, the present invention proposes a method for using TKD/IC, NO/IC and TKD/NO as detection parameters (wherein TKD represents tyrosine kinase domain, NO represents non-tyrosine kinase domain, and IC represents internal reference gene) in combination to overcome the risk of false positive interpretation and accurately detect ALK fusion gene.
In one embodiment, a method of detecting an ALK fusion gene by a digital PCR method, the method comprising:
Step 1: detecting RNA expression amounts of a TKD region and a NO region of each negative and positive tissue sample by collecting negative and positive tissue samples meeting the statistical analysis requirement number, and calculating a ratio TKD/IC of the RNA expression amount of the TKD region to the RNA expression amount of the internal reference gene, a ratio NO/IC of the RNA expression amount of the NO region to the RNA expression amount of the internal reference gene and a ratio TKD/NO of the RNA expression amount of the TKD region to the RNA expression amount of the NO; determining a TKD/IC threshold value, a NO/IC threshold value and a TKD/NO threshold value respectively, wherein the ratio of the RNA copy number of the internal reference gene to the RNA copy number of the TKD region of ALK fusion positive is 0.01 times to 100 times;
step 2, detecting the RNA copy number of an ALK gene TKD region of a sample to be detected by a digital PCR method, detecting the RNA copy number of an ALK gene NO region of the sample to be detected by a digital PCR method, detecting the RNA copy number of an internal reference gene by a digital PCR method, and calculating TKD/IC, NO/IC and TKD/NO of the sample to be detected;
Step 3: and (3) determining whether the ALK gene in the sample to be detected is an ALK fusion gene according to the TKD/IC, the NO/IC and the threshold value of the TKD/NO determined in the step (1) and the TKD/IC, the NO/IC and the TKD/NO of the sample to be detected determined in the step (2).
In one embodiment, when the TKD/IC of the sample to be tested is smaller than the threshold value, the sample to be tested is judged to be an ALK gene fusion negative sample; when the TKD/IC of the sample to be detected is not smaller than the threshold value and the NO/IC is smaller than the threshold value, judging that the sample to be detected is an ALK gene fusion positive sample; when the ALK TKD/IC of the sample to be detected is not smaller than the threshold value, the NO/IC is not smaller than the threshold value, and the TKD/NO is not smaller than the threshold value, the sample to be detected is judged to be an ALK gene fusion positive sample; when the ALK TKD/IC of the sample to be detected is not smaller than the threshold value, the NO/IC is not smaller than the threshold value, and the TKD/NO is smaller than the threshold value, the sample to be detected is judged to be an ALK gene fusion negative sample.
In one embodiment, the threshold value of TKD/IC is determined from the negative sample of the fusion gene to be tested according to the confidence interval; alternatively, the threshold for TKD/IC is determined using a subject's working profile method that fuses positive and negative samples.
In one embodiment, the threshold value of NO/IC is determined from the negative sample of the fusion gene to be tested based on the confidence interval.
In one embodiment, the threshold value of TKD/NO is determined from the negative sample of the fusion gene to be tested according to the confidence interval; alternatively, the TKD/NO threshold is determined using a subject's working profile method that fuses positive and negative samples.
In one embodiment, the reference gene is selected from the group consisting of ABL1 gene, GUSB gene, TFRC gene, and TBP gene.
In one embodiment, a digital PCR kit for use in the above method is provided.
In one embodiment, the kit includes an RNA copy number primer and probe for detecting the TKD region of the ALK gene by a digital PCR method, an RNA copy number primer and probe for detecting the NO region of the ALK gene by a digital PCR method, and an RNA copy number primer and probe for detecting the internal reference gene by a digital PCR method.
In one embodiment, the kit comprises the following primers and probes:
。
In the ALK fusion gene interpretation process of the invention, the TKD/IC detection parameters are firstly used. When the parameter is below its threshold, the sample is judged to be ALK fusion negative, indicating that TKD RNA expression is low and ALK fusion does not occur. And when TKD/IC is not smaller than its threshold value, this indicates that the TKD RNA expression level is increased. However, the present invention further contemplates NO/IC, considering that ALK gene amplification also leads to an overall increase in RNA expression. If NO/IC is below its threshold, the sample is judged positive for ALK fusion, indicating high TKD RNA expression, while the lower NO expression is due to increased TKD expression due to ALK fusion. However, if NO/IC is not less than its threshold, it is indicated that the expression levels of both TKD and NO are increased, which may be due to gene amplification. In this case, the present invention further uses TKD/NO for interpretation. If TKD/NO is not smaller than the threshold value, the sample is judged to be positive to ALK fusion; otherwise, it is determined that ALK fusion is negative.
The method has accurate detection, and overcomes the situation that the ALK RNA expression quantity increase caused by ALK gene amplification is misjudged as false positive by only using TKD/IC detection parameters; on the other hand, the problem that the interpretation cannot be realized when only TKD/NO detection parameters are used because NO expression is 0 is solved.
Detailed Description
In order that those skilled in the art will better understand the present application, the following description will proceed with reference being made to illustrative embodiments, only to the extent that they are described in connection with some, but not all embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, shall fall within the scope of the application.
Example one detection of ALK fusion Gene
Step 1: detecting RNA expression amounts of a TKD region and a NO region of each negative and positive tissue sample by collecting negative and positive tissue samples meeting the statistical analysis requirement number, and calculating a ratio TKD/IC of the RNA expression amount of the TKD region to the RNA expression amount of the internal reference gene, a ratio NO/IC of the RNA expression amount of the NO region to the RNA expression amount of the internal reference gene and a ratio TKD/NO of the RNA expression amount of the TKD region to the RNA expression amount of the NO; and determining a TKD/IC threshold value, a NO/IC threshold value and a TKD/NO threshold value respectively, wherein the ratio of the RNA copy number of the internal reference gene to the RNA copy number of the TKD region of the ALK fusion positive is 0.01 times to 100 times.
(1) Detection zone selection and primer probe design
When collecting the tissue samples negative and positive for the fusion gene in an amount satisfying the requirement of statistical analysis, the number of each sample is usually not less than 10, preferably not less than 20, preferably not less than 30. Designing detection primers and probes for different regions of the ALK gene, wherein the TKD region (tyrosine kinase region) comprises exons 20-29, with exons 22-23 being selected as the detection region of the TKD region; the NO region (region other than the TKD region) includes exons 1 to 19, of which exons 17 to 18 are selected as detection regions called NO regions.
And selecting a proper internal reference gene according to the RNA expression quantity of the TKD region of the ALK fusion positive sample, so that the relative expression level of the internal reference gene and the TKD of the fusion positive sample is in a certain range, and the ratio of the internal reference gene to the TKD is 0.01-100 times, thereby providing a reliable detection result. The detection region of the reference gene should be selected to span at least two adjacent exons of the intron to exclude amplification of the DNA. Candidate internal reference genes include ABL1, GUSB, TFRC, TBP, etc., and ABL1 is used as an internal reference in this example, and primer probe sequences are shown in the following table.
TABLE 1 primer probe sequences used in the present invention
(2) RNA extraction, detection system preparation and digital PCR reaction
Lung cancer tissue RNA (ALK copy number determined by FISH) was extracted using commercial extraction reagents, the extracted RNA was quantified and then formulated for RT-dPCR detection as follows table 2 system.
TABLE 2 preparation of detection systems
。
The digital PCR analysis system of New Yi manufacturing technology (Beijing) was used for micro-droplet generation, RT-PCR amplification and micro-droplet detection, and the procedure of RT-PCR is shown below, and specific operation steps can be seen in the related specification.
The results obtained after the detection are shown in the following Table 3. The results show that when ALK is not amplified (the copy number is 2), the relative expression amounts of TKD and NO (TKD/IC and NO/IC) of the ALK fusion negative sample are low, and the relative expression amount of TKD of the ALK positive sample is remarkably increased. When the ALK gene is amplified (the copy number is greater than or equal to 3), the relative expression amounts TKD/IC and NO/IC of TKD and NO in the ALK fusion negative sample are also higher, but the TKD/IC of the ALK positive sample is remarkably increased. See tables 3 and 4 for known ALK amplification and ALK fusion negative positive tissue sample detection results.
TABLE 3 Table 3
TABLE 4 Table 4
We step-wise determine the thresholds for TKD/IC, NO/IC, and TKD/NO:
(1) Under ALK no-amplification (copy number n=2), we calculated the threshold for TKD/IC from the results of ALK fusion negative samples, with an upper limit of 2.61% according to the 95% confidence interval. Meanwhile, the TKD/IC threshold value calculated by using statistical software GRAPHPAD PRISM 8.0.2 is 5.39% by using a subject working characteristic curve (ROC curve) method of fusion positive and negative samples. To ensure higher specificity, we selected 5.39% as the threshold for TKD/IC. As the amount of detection sample increases, the threshold can be fine-tuned to further increase the sensitivity and specificity of the detection method.
(2) Similarly, we also calculated a threshold of 0.48% for the NO/IC detection parameter using the 95% confidence interval upper limit for the negative sample detection parameter under ALK NO amplification (copy number n=2) conditions.
(3) Then, under the condition of ALK genetic amplification (the copy number n is more than or equal to 3), for TKD/NO indexes, the upper limit of a 95% confidence interval of a negative sample detection parameter is adopted, and the threshold value is calculated to be 402%. Meanwhile, we calculated a TKD/NO threshold of 492% using statistical software GRAPHPAD PRISM 8.0.2 using the subject work characteristic curve (ROC curve) method of fusion positive and negative samples. We select 492% in this embodiment.
Step 2, detecting the RNA copy number of an ALK gene TKD region of a sample to be detected by a digital PCR method, detecting the RNA copy number of an ALK gene NO region of the sample to be detected by a digital PCR method, detecting the RNA copy number of an internal reference gene by a digital PCR method, and calculating TKD/IC, NO/IC and TKD/NO of the sample to be detected;
Designing detection primers and probes for different regions of the ALK gene as in step 1, wherein the TKD region (tyrosine kinase region) comprises exons 20-29, with exons 22-23 selected as the detection region of the TKD region; the NO region (region other than the TKD region) includes exons 1 to 19, of which exons 17 to 18 are selected as detection regions called NO regions.
The sample test results are shown in Table 5. The sample is tested for the RNA expression levels of TKD, NO and reference genes, and the result shows that the sample has high or low TKD expression level and basically has low NO copy number, and the NO expression level of individual samples is 0 under 200 copies, for example, the sample 69 and the IC copy number is about 10000.
TABLE 5 sample detection results
The values of TKD/IC, NO/IC and TKD/NO detection parameters of the samples in Table 5 were calculated, and the results were as shown in Table 6 below.
Table 6 calculation results of the detection parameters
The following table 7 is established as an ALK fusion interpretation standard for lung cancer tissue samples in which a fusion negative sample was determined when ALK TKD/IC was less than 5.39%. When ALK TKD/IC is not less than 5.39% and NO/IC is less than 0.48%, a fusion positive sample is determined; a fusion positive sample was determined when ALK TKD/IC was not less than 5.39% and NO/IC was not less than 0.48% and TKD/NO was not less than 492%, and a fusion negative sample was determined when ALK TKD/IC was not less than 5.39% and NO/IC was not less than 0.48% and TKD/NO was less than 492%. Because the method established in the embodiment fully considers the judgment rules under the normal and abnormal conditions of ALK copy number, the ALK copy number state of the sample does not need to be determined when the ALK fusion detection is carried out by using the judgment standards. The results are shown in Table 8. The interpretation result is completely consistent with the theoretical result, which indicates that the detection result of the method is accurate.
TABLE 7 ALK fusion interpretation Standard
TABLE 8 sample interpretation results
。
Example 2 clinical sample testing
According to the method and the kit provided by the invention, 18 clinical lung cancer FFPE samples are detected, the detection results are shown in the following table 9, and according to the interpretation standards of the table 5, the detection results are completely consistent with the theoretical results. However, if the method in the article (Liu Y et al Thoracic Cancer, 2020) is used, only the 3-terminal/reference gene (IC) is used as the detection parameter, then 2 samples are interpreted as false positives. Thus, using only TKD/IC as the detection parameter, a false positive risk may occur. Therefore, the detection result of the method is more accurate, and the risk of false positive can be avoided.
TABLE 9 clinical sample test results
It is to be understood that this invention is not limited to the particular methodology, protocols, and materials described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
Those skilled in the art will also recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also encompassed by the appended claims.
Claims (9)
1. A method for detecting an ALK fusion gene by digital PCR, comprising:
Step 1: detecting RNA expression amounts of a TKD region and a NO region of each negative and positive tissue sample by collecting negative and positive tissue samples meeting the statistical analysis requirement number, and calculating a ratio TKD/IC of the RNA expression amount of the TKD region to the RNA expression amount of the internal reference gene, a ratio NO/IC of the RNA expression amount of the NO region to the RNA expression amount of the internal reference gene and a ratio TKD/NO of the RNA expression amount of the TKD region to the RNA expression amount of the NO; determining a TKD/IC threshold value, a NO/IC threshold value and a TKD/NO threshold value respectively, wherein the ratio of the RNA copy number of the internal reference gene to the RNA copy number of the TKD region of ALK fusion positive is 0.01 times to 100 times;
step 2, detecting the RNA copy number of an ALK gene TKD region of a sample to be detected by a digital PCR method, detecting the RNA copy number of an ALK gene NO region of the sample to be detected by a digital PCR method, detecting the RNA copy number of an internal reference gene by a digital PCR method, and calculating TKD/IC, NO/IC and TKD/NO of the sample to be detected;
Step 3: and (3) determining whether the ALK gene in the sample to be detected is an ALK fusion gene according to the TKD/IC, the NO/IC and the threshold value of the TKD/NO determined in the step (1) and the TKD/IC, the NO/IC and the TKD/NO of the sample to be detected determined in the step (2).
2. The method of claim 1, wherein the test sample is determined to be an ALK gene fusion negative sample when TKD/IC of the test sample is less than its threshold; when the TKD/IC of the sample to be detected is not smaller than the threshold value and the NO/IC is smaller than the threshold value, judging that the sample to be detected is an ALK gene fusion positive sample; when the ALK TKD/IC of the sample to be detected is not smaller than the threshold value, the NO/IC is not smaller than the threshold value, and the TKD/NO is not smaller than the threshold value, the sample to be detected is judged to be an ALK gene fusion positive sample; when the ALK TKD/IC of the sample to be detected is not smaller than the threshold value, the NO/IC is not smaller than the threshold value, and the TKD/NO is smaller than the threshold value, the sample to be detected is judged to be an ALK gene fusion negative sample.
3. The method of claim 1, wherein the threshold value of TKD/IC is determined from the fusion gene negative sample to be tested based on a confidence interval; alternatively, the threshold for TKD/IC is determined using a subject's working profile method that fuses positive and negative samples.
4. The method of claim 1, wherein the threshold value of NO/IC is determined from the negative sample of the fusion gene to be tested based on a confidence interval.
5. The method of claim 1, wherein the threshold TKD/NO is determined from the fusion gene negative sample to be tested based on a confidence interval; alternatively, the TKD/NO threshold is determined using a subject's working profile method that fuses positive and negative samples.
6. The method of claim 1, wherein the reference gene is selected from the group consisting of ABL1 gene, GUSB gene, TFRC gene and TBP gene.
7. A digital PCR kit for use in the method of any one of claims 1-6.
8. The PCR kit of claim 7, comprising a digital PCR method to detect RNA copy number primers and probes for the TKD region of the ALK gene, a digital PCR method to detect RNA copy number primers and probes for the NO region of the ALK gene, and a digital PCR method to detect RNA copy number primers and probes for the internal reference gene.
9. The PCR kit of claim 8, wherein the kit comprises the following primers and probes:
。
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