Genetic composition, kit and detection method for detecting lung adenocarcinoma KRAS mutation

Abstract

This invention relates to gene compositions, kits, and methods for detecting KRAS mutations in lung adenocarcinoma, belonging to the field of molecular diagnostic technology. This invention is the first to propose a combination of eighteen miRNA gene compositions for evaluating KRAS mutations in lung adenocarcinoma, which, compared to currently used methods, offers advantages such as ease of operation, quantitative analysis, high accuracy, and good sensitivity and specificity.

Description

Technical Field

[0001] This invention belongs to the field of molecular diagnostic technology, specifically relating to gene compositions, kits, and methods for detecting KRAS mutations in lung adenocarcinoma. Background Technology

[0002] Lung cancer is the most common cancer worldwide, and its mortality rate ranks first and second among men and women, respectively, with an average five-year survival rate of less than 15%. Among them, lung adenocarcinoma is currently the main subtype of lung cancer, accounting for nearly 60% of new cases, and is characterized by low survival rates.

[0003] Kirsten rat sarcoma viral oncogene (KRAS) mutations are among the most common mutations in lung adenocarcinoma, accounting for 32% of lung cancers [1], and play a crucial role in tumor development [1]. Point mutations are the most common type of KRAS mutation, with the most common types being KRAS-G12D mutations (41%), KRAS-G12V mutations (28%), and KRAS-G12C mutations (14%). In NSCLC, G12C is the most common type [3], which is closely associated with poor prognosis [4].

[0004] Significant progress has been made in the development of AMG510[5], a drug targeting KRAS mutations, and Sotorasib was launched in 2021, filling the gap in KRAS mutation-targeted drugs. Given the high risk of KRAS-mutant lung adenocarcinoma, early and accurate identification of KRAS-mutant patients is of great significance for improving the effectiveness of targeted therapy and reducing the social burden. Currently, the methods for directly detecting KRAS mutations in clinical practice are costly, complicated, and have low accuracy. There may still be detection methods with higher sensitivity, specificity, and application value that can be applied to the formulation of individualized targeted therapy plans for lung adenocarcinoma patients.

[0005] Detecting the expression levels of multiple miRNAs in patients as biomarkers to determine their KRAS mutation status will provide more accurate assurance for KRAS mutation detection and help achieve precision medicine for patients. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a gene composition, kit, and method for detecting KRAS mutations in lung adenocarcinoma.

[0007] To achieve the goal of solving the above problems, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a gene composition for detecting KRAS mutations in lung adenocarcinoma, comprising the following miRNA gene compositions: hsa-miR-141-3p, hsa-miR-1307-5p, has-miR-331-3p, hsa-miR-9-5p, hsa-miR-127-3p, hsa-miR-181c-5p, hsa-miR-125b-5p, hsa-mi R-21-5p, hsa-miR-146b-5p, hsa-miR-197-3p, hsa-miR-146b-3p, hsa-miR-185-5p, hsa-miR-4 24-5p, hsa-miR-375-3p, hsa-miR-99a-5p, hsa-miR-21-3p, hsa-miR-374a-3p and has-let-7c-5p.

[0009] Secondly, the present invention also provides the use of the gene composition described above in the preparation of a drug for detecting KRAS mutations in lung adenocarcinoma.

[0010] Preferably, the drug includes a reagent for detecting the expression level of the gene composition, and uses it as the sole active ingredient.

[0011] Thirdly, the present invention also provides a kit for detecting KRAS mutations in lung adenocarcinoma, comprising a reagent for detecting the expression level of the gene composition as described above, with said reagent as the sole active ingredient.

[0012] Preferably, the specification also includes a description of the threshold and score formula for the miRNA gene, wherein the threshold of the miRNA gene composition is as follows:

[0013]

[0014]

[0015] The Score scoring formula is as follows:

[0016] Score=0.4360*hsa-miR-141-3p+0.4551*hsa-miR-1307-5p+0.2065*has-miR-331-3p+0.8180*hsa-miR-9-5p+0.3668*hs a-miR-127-3p+0.7692*hsa-miR-181c-5p+0.7516*hsa-miR-125b-5p+1.1773*hsa-miR-21-5p+0.5870*hsa-miR-146b-5p+ 0.8374*hsa-miR-197-3p+0.4296*hsa-miR-146b-3p+7.4558*hsa-miR-185-5p+9.7320*hsa-miR-424-5p+0.6608*hsa-miR -375-3p+8.1049*hsa-miR-99a-5p+0.7248*hsa-miR-21-3p+0.1435*hsa-miR-374a-3p+0.5374*has-let-7c-5p-21.6752.

[0017] Preferably, when the score of the test sample is higher than the cutoff value for lung adenocarcinoma, it is determined that the test sample has a KRAS mutation; when the score of the test sample is not higher than the cutoff value for lung adenocarcinoma, it is determined that the test sample does not have a KRAS mutation; wherein, the cutoff value for lung adenocarcinoma is -0.62.

[0018] Preferably, the test sample is a fresh lung adenocarcinoma tissue sample.

[0019] Fourthly, the present invention also provides a method for detecting KRAS mutations in lung adenocarcinoma, comprising the following steps:

[0020] Step (1): Collect test samples and detect the expression levels of the following miRNA gene combinations: hsa-miR-141-3p, hsa-miR-1307-5p, has-miR-331-3p, hsa-miR-9-5p, hsa-miR-127-3p, hsa-miR-181c-5p, hsa-miR-125b-5p, hsa-miR-21-5p , hsa-miR-146b-5p, hsa-miR-197-3p, hsa-miR-146b-3p, hsa-miR-185-5p, hsa-miR-424-5 p, hsa-miR-375-3p, hsa-miR-99a-5p, hsa-miR-21-3p, hsa-miR-374a-3p and has-let-7c-5p;

[0021] Step (2): Convert the expression level of the above miRNA gene composition into a 0-1 variable according to the threshold. That is, when the expression level of the gene composition is higher than its threshold, it is recorded as 1; when the expression level of the gene composition is not higher than its threshold, it is recorded as 0.

[0022] Step (3): Calculate the Score score of the test sample based on the 0-1 variables of the gene composition, and compare it with the cutoff value for lung adenocarcinoma: when the Score score is higher than the cutoff value for lung adenocarcinoma, it indicates that the test sample has a KRAS mutation; when the Score score is not higher than the cutoff value for lung adenocarcinoma, it indicates that the test sample does not have a KRAS mutation; wherein the threshold of the miRNA gene composition is as follows:

[0023]

[0024]

[0025] The Score scoring formula is as follows:

[0026] Score=0.4360*hsa-miR-141-3p+0.4551*hsa-miR-1307-5p+0.2065*has-miR-331-3p+0.8180*hsa-miR-9-5p+0.3668*hs a-miR-127-3p+0.7692*hsa-miR-181c-5p+0.7516*hsa-miR-125b-5p+1.1773*hsa-miR-21-5p+0.5870*hsa-miR-146b-5p+ 0.8374*hsa-miR-197-3p+0.4296*hsa-miR-146b-3p+7.4558*hsa-miR-185-5p+9.7320*hsa-miR-424-5p+0.6608*hsa-miR -375-3p+8.1049*hsa-miR-99a-5p+0.7248*hsa-miR-21-3p+0.1435*hsa-miR-374a-3p+0.5374*has-let-7c-5p-21.6752;

[0027] The cutoff value for the lung adenocarcinoma was -0.62.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention is the first to propose a combination of eighteen miRNA gene sequences that can be used to evaluate KRAS mutations in lung adenocarcinoma. Compared with currently used methods, this method has the advantages of being easy to operate, capable of quantitative analysis, highly accurate, sensitive and specific. Attached Figure Description

[0030] Figure 1 This demonstrates the sensitivity and specificity of the miRNA set of this invention in predicting lung adenocarcinoma in the test set. Detailed Implementation

[0031] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0032] Example 1: Screening and efficacy verification of miRNA sets

[0033] (I) Construction of a prognostic scoring model for lung adenocarcinoma

[0034] 1. Method

[0035] First, miRNA expression data from 453 lung adenocarcinoma samples were obtained from the TCGA database, divided into KRAS-mutant, wild-type, and normal tissue groups. Differentially expressed miRNAs among the three groups were screened using the R language's limma package, identifying the 39 miRNAs most strongly associated with KRAS mutations. Lasso regression was then used to select 18 miRNA gene combinations: hsa-miR-141-3p, hsa-miR-1307-5p, has-miR-331-3p, hsa-miR-9-5p, hsa-miR-127-3p, hsa-miR-181c-5p, hsa-miR-125b-5p, hsa-miR-21-5p, and hsa-miR-146b. -5p, hsa-miR-197-3p, hsa-miR-146b-3p, hsa-miR-185-5p, hsa-miR-424-5p, hsa-miR-375-3p, hsa-miR-99a-5p, hsa-miR-21-3p, hsa-miR-374a-3p, and has-let-7c-5p; the threshold of each miRNA was determined by ROC curves, as shown in Table 1.

[0036] Table 1

[0037]

[0038]

[0039] Constructing a scoring model using Logistic regression:

[0040] Score=0.4360*hsa-miR-141-3p+0.4551*hsa-miR-1307-5p+0.2065*has-miR-331-3p+0.8180*hsa-miR-9-5p+0.3668*hs a-miR-127-3p+0.7692*hsa-miR-181c-5p+0.7516*hsa-miR-125b-5p+1.1773*hsa-miR-21-5p+0.5870*hsa-miR-146b-5p+ 0.8374*hsa-miR-197-3p+0.4296*hsa-miR-146b-3p+7.4558*hsa-miR-185-5p+9.7320*hsa-miR-424-5p+0.6608*hsa-miR -375-3p+8.1049*hsa-miR-99a-5p+0.7248*hsa-miR-21-3p+0.1435*hsa-miR-374a-3p+0.5374*has-let-7c-5p-21.6752.

[0041] Finally, based on the miRNA threshold and scoring model, we calculated the score for each sample and obtained the cutoff value for lung adenocarcinoma through ROC curve. The cutoff value was -0.62. The TCGA samples were divided into two groups: those with scores higher than the cutoff value were assigned to the KRAS group, and those with scores lower than the cutoff value were assigned to the non-KRAS mutation group.

[0042] (II) Effect Verification

[0043] We performed miRNA sequencing on 47 lung adenocarcinoma samples collected from the Department of Thoracic Surgery at Zhongshan Hospital affiliated with Fudan University. The miRNA expression data were divided into two groups according to a scoring model: those with a value higher than the cutoff value of -0.62 were classified as the KRAS group, and those with a value lower than the cutoff value of -0.62 were classified as the non-KRAS mutation group. The sensitivity and specificity tests are listed in Tables 2 and 3, respectively.

[0044] Table 2

[0045] Clinical testing for KRAS mutation+ Clinical testing for KRAS mutations - Predicting KRAS mutations+ 109 35 Predicting KRAS mutations - 12 297

[0046] Table 3

[0047]

[0048]

[0049] As shown in Tables 2 and 3, the sensitivity was 0.90 and the specificity was 0.89. The combination of eighteen miRNA genes has been verified through clinical trials to be able to evaluate KRAS mutations in lung adenocarcinoma. It has the advantages of convenient operation, quantitative analysis, high accuracy, and good sensitivity and specificity.

[0050] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A gene composition for detecting KRAS mutations in lung adenocarcinoma, characterized in that, Including the following miRNA genes: hsa-miR-141-3p, hsa-miR-1307-5p, has-miR-331-3p, hsa-miR-9-5p , hsa-miR-127-3p, hsa-miR-181c-5p, hsa-miR-125b-5p, hsa-miR-21-5p, hsa-miR-1 46b-5p, hsa-miR-197-3p, hsa-miR-146b-3p, hsa-miR-185-5p, hsa-miR-424-5p, hsa -miR-375-3p, hsa-miR-99a-5p, hsa-miR-21-3p, hsa-miR-374a-3p and has-let-7c-5p.

2. The use of the gene composition as described in claim 1 in the preparation of a medicament for detecting KRAS mutations in lung adenocarcinoma.

3. The application according to claim 2, characterized in that, The drug includes a reagent for detecting the expression level of the gene composition, and uses it as the sole active ingredient.

4. A kit for detecting KRAS mutations in lung adenocarcinoma, characterized in that, Includes a reagent for detecting the expression level of the gene composition as described in claim 1, with said reagent as the sole active ingredient.

5. A kit for detecting KRAS mutations in lung adenocarcinoma according to claim 4, characterized in that, It also includes a specification sheet describing the threshold and score formula for the miRNA gene composition, wherein the threshold for the miRNA gene composition is as follows: The Score scoring formula is as follows: Score=0.4360 hsa-miR-141-3p+0.4551 hsa-miR-1307-5p+0.2065 has-miR-331-3p+0.8180 hsa-miR-9-5p+0.3668 hsa-miR-127-3p+0.7692 hsa-miR-181c-5p+0.7516 hsa-miR-125b-5p+1.1773 hsa-miR-21-5p+0.5870 hsa-miR-146b-5p+0.8374 hsa-miR-197-3p+0.4296 hsa-miR-146b-3p+7.4558 hsa-miR-185-5p+9.7320 hsa-miR-424-5p+0.6608 hsa-miR-375-3p+8.1049 hsa-miR-99a-5p+0.7248 hsa-miR-21-3p+0.1435 hsa-miR-374a-3p+0.5374 has-let-7c-5p-21.6752。 6. A kit for detecting KRAS mutations in lung adenocarcinoma according to claim 5, characterized in that, When the Score of the tested sample is higher than the cutoff value for lung adenocarcinoma, the tested sample is determined to have a KRAS mutation; when the Score of the tested sample is not higher than the cutoff value for lung adenocarcinoma, the tested sample is determined to not have a KRAS mutation; wherein, the cutoff value for lung adenocarcinoma is -0.

62.

7. A kit for detecting KRAS mutations in lung adenocarcinoma according to claim 6, characterized in that, The test sample was a fresh lung adenocarcinoma tissue sample.

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