A kit for detecting smad4 mutation of lung adenocarcinoma

Abstract

The application discloses a kit for detecting lung adenocarcinoma SMAD4 mutation. The application selects 13 genes TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN and HKDC1 through Lasso regression finally by using a limma package to screen differential genes closely related to SMAD4, and constructs a scoring model composed of the above genes through logistic regression; a receiver operating characteristic curve is drawn to obtain a cutoff value, which can be used for detecting lung adenocarcinoma SMAD4 mutation. Experiment verifies that the method has the characteristics of high sensitivity and specificity, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to a kit for detecting SMAD4 mutations in lung adenocarcinoma, belonging to the field of molecular diagnostic technology. Background Technology

[0002] Lung cancer is one of the malignant tumors with the highest incidence and mortality rates worldwide, with an average five-year survival rate of less than 15% [1,2]. According to data from the National Cancer Center, in 2015, there were 788,000 new cases of lung cancer and 631,000 deaths in my country, both ranking first among malignant tumors [3]. Lung adenocarcinoma (LUAD) is currently the main subtype of lung cancer, accounting for nearly 60% of new cases, and is characterized by low survival rate [4]. Non-small cell lung cancer (NSCLC) is the most common histological type [5], accounting for 85% of all lung cancers. More than 40% of patients are diagnosed with locally advanced or overt metastases, thus losing the opportunity to undergo surgery.

[0003] Mothers Against Decapentaplegic Homolog 4 (SMAD4) is an intracellular signaling molecule in the TGF-β pathway, and its mutations are commonly found in gastrointestinal tumors, such as pancreatic cancer. Inactivation or low expression of SMAD4 affects TGF-β signal transduction and participates in tumorigenesis [6]. It has been reported that SMAD4 expression is significantly reduced in all stages of NSCLC patients [7]. Reduced SMAD4 expression is more common in patients with poor disease-free survival and those resistant to platinum-based chemotherapy. SMAD4 mutation is an independent risk factor for survival in NSCLC patients. SMAD4 may promote NSCLC progression by regulating proliferation, adhesion, and immune responses [8]. Currently, research on the mechanism by which SMAD4 mutation promotes lung cancer metastasis and potential therapeutic targets such as miRNA-495 is a hot topic in targeted therapy [7]. Early identification of high-risk patients with SMAD4 mutation is of great significance for predicting patient prognosis, initiating targeted therapy as early as possible, and improving patient survival.

[0004] Current methods for detecting SMAD4 gene mutations in lung adenocarcinoma involve direct single-gene testing of pathological tissue or blood from patients. This approach is costly, cumbersome, and has low accuracy, and SMAD4 gene testing is not routinely included in these methods. Therefore, developing an accurate and convenient method for detecting SMAD4 mutations would contribute to achieving precision medicine for patients.

[0005] References:

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[0008] 3.Chen W,Zheng R,Baade P D,Zhang S,Zeng H,Bray F,Jemal A,Yu X Q andHe J.Cancer statistics in China,2015.CA Cancer J Clin.2016;66:115-32.

[0009] 4.Lu T,Yang X,Huang Y,Zhao M,Li M,Ma K,Yin J,Zhan C and Wang Q.Trendsin the incidence,treatment,and survival of patients with lung cancer in thelast four decades.CANCER MANAG RES.2019;11:943-53.

[0010] 5.Zappa C and Mousa S A.Non-small cell lung cancer:current treatmentand future advances.Transl Lung Cancer Res.2016;5:288-300.

[0011] 6. Zhao M, Mishra L and Deng C X. The role of TGF-beta / SMAD4 signaling in cancer. INT J BIOL SCI. 2018; 14:111-23.

[0012] 7.Tan PAK3 via miRNA regulation.NAT COMMUN.2021;12:4853.

[0013] 8. Wang Y, Xue Q, Zheng Q, Jin Y, Shen X, Yang M, Zhou X and Li Y.SMAD4mutation correlates with poor prognosis in non-small cell lung cancer. LABINVEST. 2021; 101:463-76. Summary of the Invention

[0014] The purpose of this invention is to address the shortcomings of existing methods for detecting SMAD4 mutations, such as high cost, cumbersome procedures, and low detection accuracy. This invention proposes a scoring model and kit based on the expression levels of a set of gene combinations for detecting SMAD4 mutations in lung adenocarcinoma, thereby improving detection sensitivity and specificity.

[0015] To achieve the above objectives, the present invention provides an application of a detection reagent in the preparation of a kit for detecting SMAD4 mutations in lung adenocarcinoma. The detection reagent comprises reagents for detecting the expression levels of the following genes: TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN, and HKDC1. This detection reagent is the sole key component of the kit for detecting SMAD4 mutations in lung adenocarcinoma.

[0016] Preferably, the kit also includes an instruction manual, which describes the following scoring model:

[0017] Score=2.4750×TRIM48+1.6121×HS3ST1+1.7751×GPA33+1.1035×STK39+0.3149×GPR110+0.6898×IPCEF1+2.1786× ALPPL2+1.3422×KCNQ3+1.9323×ADAM28+1.3519×CDC42EP1+1.1779×ZBTB18+3.5127×MYCN+1.7359×HKDC1-8.9545;

[0018] In the scoring model, each gene represents a gene expression variable. When the expression level of a detected gene is higher than the gene threshold, it is recorded as 1; when the expression level is lower than the gene threshold, it is recorded as 0.

[0019] The gene thresholds for TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN, and HKDC1 are 4.572089, 2.308389, 3.990620, 4.061866, 3.520856, 1.819687, 1.653209, 2.565847, 2.212941, 6.471321, 4.348365, 4.048287, and 3.617944, respectively.

[0020] Preferably, the cutoff value for the Score in the scoring model is -3.686. When the Score of a detected sample is higher than the cutoff value, the sample has a SMAD4 mutation; when the Score of a detected sample is lower than the cutoff value, the sample does not have a SMAD4 mutation.

[0021] Preferably, the test sample is a fresh tissue tumor sample.

[0022] The present invention also provides a kit for detecting SMAD4 mutations in lung adenocarcinoma, comprising at least reagents for specifically detecting the expression levels of the following genes: TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN, and HKDC1.

[0023] The present invention also provides a method for detecting SMAD4 mutations in lung adenocarcinoma for non-disease diagnosis and treatment purposes, comprising the following steps:

[0024] Step 1: Detect the expression levels of the following genes in the sample: TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN, and HKDC1.

[0025] Step 2: Calculate the Score value according to the scoring model: Score = 2.4750×TRIM48 + 1.6121×HS3ST1 + 1.7751×GPA33 + 1.1035×STK39 + 0.3149×GPR110 + 0.6898×IPCEF1 + 2.1786×ALPPL2 + 1.3422×KCNQ3 + 1.9323×ADAM28 + 1.3519×CDC42EP1 + 1.1779×ZBTB18 + 3.5127×MYCN + 1.7359×HKDC1 - 8.9545;

[0026] In the scoring model, each gene represents a gene expression variable. When the expression level of a detected gene is higher than the gene threshold, it is recorded as 1; when the expression level is lower than the gene threshold, it is recorded as 0.

[0027] The gene thresholds for TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN, and HKDC1 are 4.572089, 2.308389, 3.990620, 4.061866, 3.520856, 1.819687, 1.653209, 2.565847, 2.212941, 6.471321, 4.348365, 4.048287, and 3.617944, respectively.

[0028] Step 3: Judgment: The cutoff value for the score is -3.686. When the score of the tested sample is higher than this cutoff value, the sample has the SMAD4 mutation; when the score of the tested sample is lower than this cutoff value, the sample does not have the SMAD4 mutation.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] This invention is the first to propose a scoring model and kit based on the expression levels of a set of gene combinations. This model or kit can be used to assess SMAD4 mutations in lung adenocarcinoma. Compared with commonly used detection methods, it has the advantages of convenient operation, high accuracy, good sensitivity and specificity, and has good application prospects in clinical practice. Attached Figure Description

[0031] Figure 1 This is the receiver operating characteristic (ROC) curve for the scoring model. Detailed Implementation

[0032] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0033] Example

[0034] (I) Construction of a scoring model for predicting SMAD4 mutations

[0035] First, gene expression data from 563 lung adenocarcinoma samples were obtained from the TCGA database and divided into SMAD4-mutant, wild-type, and normal tissue groups. Differentially expressed genes among the three groups were screened using the limma package in R, identifying 43 genes most strongly associated with SMAD4 mutation. Lasso regression was then used to select 13 genes: TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN, and HKDC1. The threshold for each gene was determined using ROC curves, as shown in Table 1. A scoring model was constructed using Logistic regression.

[0036] Score=2.4750×TRIM48+1.6121×HS3ST1+1.7751×GPA33+1.1035×STK39+0.3149×GPR110+0.6898×IPCEF1+2.1786× ALPPL2+1.3422×KCNQ3+1.9323×ADAM28+1.3519×CDC42EP1+1.1779×ZBTB18+3.5127×MYCN+1.7359×HKDC1-8.9545

[0037] Finally, based on the gene thresholds shown in Table 1 and the scoring model described above, a score is calculated for each sample, and then... Figure 1 The ROC curve shown has a cutoff value of -3.686. The TCGA samples are divided into two groups: those with scores higher than the cutoff value are assigned to the SMAD4 group, and those with scores lower than the cutoff value are assigned to the non-SMAD4 mutation group. The prediction results of this prediction model in lung adenocarcinoma in the test set are shown in Table 2.

[0038] Table 1 Gene Thresholds

[0039] Gene threshold Gene threshold TRIM48 4.572089 KCNQ3 2.565847 HS3ST1 2.308389 ADAM28 2.212941 GPA33 3.990620 CDC42EP1 6.471321 STK39 4.061866 ZBTB18 4.348365 GPR110 3.520856 MYCN 4.048287 IPCEF1 1.819687 HKDC1 3.617944 ALPPL2 1.653209

[0040] Table 2. Sensitivity and specificity of gene sets in predicting lung adenocarcinoma in the test set.

[0041] Clinical testing showed SMAD4 mutation positive. Clinical testing for SMAD4 mutations - Predict SMAD4 mutation+ 19 30 Predicting SMAD4 mutations - 1 513

[0042] Based on the results in Table 2, the sensitivity predicted by the detection model is 0.95 and the specificity is 0.94.

[0043] (II) Effect Verification

[0044] Gene sequencing was performed on 47 lung adenocarcinoma samples collected from the Department of Thoracic Surgery at Zhongshan Hospital affiliated with Fudan University. The gene expression data were divided into two groups according to the scoring model. Those with a value higher than the cutoff value of -3.686 were classified as the SMAD4 group, and those with a value lower than the cutoff value of -3.686 were classified as the non-SMAD4 mutation group. The prediction results of this prediction model in the validation set of lung adenocarcinoma are shown in Table 3.

[0045] Table 3. Sensitivity and specificity of the gene set in predicting lung adenocarcinoma in the validation set.

[0046] Clinical testing showed SMAD4 mutation positive. Clinical testing for SMAD4 mutations - Predict SMAD4 mutation+ 18 4 Predicting SMAD4 mutations - 2 23

[0047] Based on the results in Table 3, the sensitivity of the prediction model is 0.90 and the specificity is 0.85.

[0048] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several 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.

Claims

1. Use of a detection reagent in the preparation of a kit for detecting a SMAD4 mutation of lung adenocarcinoma, characterized in that, The detection reagent is composed of reagents for detecting the expression amounts of TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN and HKDC1; and the detection reagent realizes the detection of lung adenocarcinoma SMAD4 mutation as the only key component of a kit.

2. Use according to claim 1, characterized in that, The detection sample is a fresh tissue tumor sample.

3. A kit for detecting a SMAD4 mutation in lung adenocarcinoma, characterized by, The kit at least comprises reagents for specifically detecting the expression amounts of TRIM48, HS3ST1, GPA33, STK39, GPR110, IPCEF1, ALPPL2, KCNQ3, ADAM28, CDC42EP1, ZBTB18, MYCN and HKDC1.

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