Patent Application
20190078167
The invention relates to the technical field of molecular biology, in particular to a genetic marker used for identifying benign and malignant pulmonary micro-nodules and the application thereof.
Lung carcinoma is the primary cause for cancer induced death in urban population in China. According to the data of the statistical yearbook by the Ministry of Health in 2011, the mortality rate induced by lung carcinoma in China was 46.46 persons/100,000 persons in 2010, ranking first in the mortality rate induced by malignant tumors, almost equivalent to the total mortality rate induced by liver cancer, gastric cancer and colorectal cancer. The prognosis of lung carcinoma is closely related to the clinical stage of definite diagnosis, wherein, the treatment of ultra-early Stage 0 in situ lung carcinoma has the best therapeutic effect, and the postoperative 5-year survival rate of the patents is as high as 90%; the postoperative 5-year survival rate of patents with Stage Ia lung carcinoma is 61%, while the overall 5-year survival rate for patients at Stages II-IV decreased from 34% to 5% and below. Therefore, the key to improve the cure rate and reduce the mortality of lung carcinoma is early detection, and especially the ultra-early detection of Stage 0 peripheral in situ lung carcinoma is of great value for improving the cure rate of lung carcinoma.
At present, it is a main method for the early screening of lung carcinoma to examine high-risk populations by using low-dose spiral CT (LDCT) and other high-resolution imaging methods. In the large-scale early screening practice of lung carcinoma, a large number of patients with micro-nodules (with the nodule size less than 10 mm) were found, these micro-nodules may be the benign lesions of lung cancer such as inflammation, etc., or may be micronodular lung carcinoma (accounting for about 30%˜40%) containing malignant carcinoma cells, of which most of the micronodular lung carcinomas belong to ultra-early peripheral in situ lung carcinoma (tumor TNM Stage 0) and partially Stage Ia lung carcinoma, and good therapeutic results can be achieved in case of early diagnosis. However, it is very difficult to judge benign and malignant pulmonary micro-nodules clinically, it is mainly because that the sizes of micro-nodules are less than 10 mm, it is difficult to perform biopsy and pathological examination by fine needle puncture, and even if PET-CT examination is performed, the obtained results also have very limited diagnostic value. The detection of other lung carcinoma related serum tumor markers, such as carcinoembryonic antigen (CEA), neuron-specific enolase (NSE), squamous cell carcinoma antigen (SCC-Ag), cytokeratin 19-fragment, etc., has certain reference value for the auxiliary diagnosis of middle-stage and advanced lung carcinoma, but it has little value for the diagnosis of ultra-early peripheral in situ lung carcinoma (Stage 0) and Stage Ia lung carcinoma. In addition, conventional tumor serum protein markers usually have low detection sensitivity; for example, CEA and NSE have a sensitivity (positive detection rate) of only about 30% for middle-stage and advanced lung carcinoma, besides, the serum protein markers have poor specificity for lung carcinoma detection, pneumonia and other benign lesions can also cause abnormal concentrations of protein markers, leading to false positive results of detection. Therefore, in order to accurately identify the benign and malignant micro-nodules found in CT imaging and to discover ultra-early micronodular lung carcinoma as early as possible, it is urgent to develop a detection technique and product that can accurately identify benign and malignant micronodules.
Blood is the largest organ of human body, and blood cell is one of the few cell types that can communicate with almost all tissue cells. If the tissues and organs in vivo have some injuries, inflammation, tumor or other malignant diseases, a series of specific changes will occur in the microenvironment around the diseased tissue cells. When blood flows through various tissues and organs, the microenvironment of the diseased tissue cells exchanges information with blood cells, whereas the blood cells will respond directly or indirectly to these changes have the corresponding gene expression changes, and participate in the information transmission and exchange of the immune system and other systems of the whole body. Such gene expression changes of blood cells are much earlier than the obvious physical signs of the body, containing the distinctive gene expression changes of some diseases. Therefore, it is possible to sensitively capture the early molecular information of tumor or malignant diseases in vivo and screen out the distinctive gene expression signals (markers) of the disease by closely monitoring the expression profile of blood cell genes, providing reliable basis for early detection and monitoring of diseases. Moreover, as a simple and noninvasive examination method, the detection of peripheral blood gene expression is easy for the subject to accept with high compliance of examination, and it has great application value for the early screening/diagnosis of malignant tumors.
In order to solve the technical problem that it is clinically lack of a biological marker for accurately identifying benign and malignant pulmonary micro-nodules, the invention provides a peripheral blood genetic marker for identifying benign and malignant pulmonary micro-nodules, the marker can more accurately identifying malignant micro-nodules to discover ultra-early micronodular lung cancer with higher detection sensitivity and specificity, besides, it is only necessary to collect 2 ml of peripheral venous blood for detection, and owing to the simple detection process, it is especially applicable for the ultra-early screening of lung carcinoma for large-scale population by matching with CT and other imageological examinations.
In order to solve the above technical problem, the invention is realized by the following technical schemes:
On one aspect of the invention, it provides a product comprising one or more polynucleotides or their fragments, the polynucleotide exhibits differential expression in the peripheral blood of micronodular lung carcinoma patients and patients without lung carcinoma, and the polynucleotide comprises at least one of the 12 gene sequences characterized by lung carcinoma as shown in SEQ ID NO.1˜SEQ ID NO.12, namely HSP90AA1, UQCRQ, COX7A2, CAPZA2, CHMP5, NDUFB2, RPL24, CKLF, C14orf2, CD52, FGFBP2 and GLRX.
Preferably, the product comprises multiple polynucleotides or their fragments, the polynucleotides comprise 6˜12 gene sequences characterized by lung carcinoma among the gene sequences characterized by lung carcinoma as shown in SEQ ID NO.1˜SEQ ID NO.12.
The micronodular patients without lung carcinoma contain patients with benign pulmonary lesion and healthy subjects.
On another aspect of the invention, it provides a composition, comprising a primer and/or a probe used for detecting differential expression of gene in the peripheral blood of micronodular lung carcinoma patients and patients without lung carcinoma, and the gene comprises at least one of the gene sequences characterized by lung carcinoma as shown in SEQ ID NO.1˜SEQ ID NO.12.
The probe comprises: (1) polynucleotides specifically hybrid with the transcription products of gene expression, or their fragments; (2) a polypeptide binding agent specifically binding with the translation products of gene expression, such as antibody, or its fragment.
On another aspect of the invention, it also provides the application of the above product comprising one or more polynucleotides or their fragments, and it is used for preparing products for identifying, diagnosing or screening ultra-early lung carcinoma.
Preferably, the product for identifying, diagnosing or screening ultra-early lung carcinoma comprises: a product for detecting micronodular lung carcinoma using real-time quantitative PCR, RNA sequencing or gene chip.
The product for identifying, diagnosing or screening ultra-early lung carcinoma using real-time quantitative PCR comprises a primer for specifically amplifying at least one of the gene sequences characterized by lung carcinoma as shown in SEQ ID NO.1˜SEQ ID NO.12.
The product for identifying, diagnosing or screening ultra-early lung carcinoma using gene chip comprises: a probe hybrid with at least one of the gene sequences characterized by lung carcinoma as shown in SEQ ID NO.1˜SEQ ID NO.12.
On another aspect of the invention, it also provides a detection kit for identifying, diagnosing or screening ultra-early lung carcinoma, and the kit comprises a primer and/or a probe specifically pertinent to at least one of the gene sequences characterized by lung carcinoma as shown in SEQ ID NO.1˜SEQ ID NO.12. The kit also comprises a primer specifically pertinent to the internal control gene GAPDH.
Preferably, the kit also comprises a fluorescent probe specifically binding with PCR amplified fragments or the SYBR Green dye non-specifically binding with PCR amplified fragments.
Preferably, the primer comprises any 1 pair or 2 and more pairs of combinations among the primer sequenes indicated by SEQ ID NO.13˜SEQ ID NO.36, and the probe comprises any 1 or 2 and more combinations among the probe sequenes indicated by SEQ ID NO.37˜SEQ ID NO.48; the primer and/or probe of the reference gene sequences are as indicated by SEQ ID NO.48˜SEQ ID NO.51.
On another aspect of the invention, it also provides a detection chip for identifying, diagnosing or screening ultra-early lung carcinoma, the chip comprises a probe hybrid with at least one of the gene sequences characterized by lung carcinoma as shown in SEQ ID NO.1˜SEQ ID NO.12.
By using the kit or detection chip of the invention, the expressions of the feature gene sequences of micronodular lung carcinoma indicated by SEQ ID NO.1˜SEQ ID NO.12 in the peripheral blood of the subjects can be detected, and then the risk value that the micro-nodule of the subject is lung carcinoma can be obtained by inputing the information of expression up-regulation or down-regulation into the micronodular lung carcinoma identifying model, so as to realize the early screening and diagnosis of lung carcinoma.
The genetic markers of the invention can identify benign and malignant pulmonary micro-nodules, and they have higher detection sensitivity and stronger specificity for ultra-early micronodular lung carcinoma; besides, they take peripheral blood, which is the easiest to collect in clinic, as the test sample. Owing to the noninvasive and simple sampling mode and high inspection compliance, it is especially applicable for the ultra-early screening of lung carcinoma for large-scale population to promote the early discovery of lung carcinoma and improve the cure rate of lung carcinoma, and it has a broad application prospect.
The invention is further explained in details below by combining with the the drawings and specific execution modes.
Aiming at the technical blank that at present it is difficult to identify benign and malignant pulmonary micro-nodules (with the nodular size less than 10 mm) in clinic, the invention provides a combination of genetic markers for identifying benign and malignant pulmonary micro-nodules. Taking the quantitative detection of peripheral blood gene expression profile as the basis, by quantitatively analyzing the differences in the gene expression of total RNA in peripheral blood between micronodular patients (including the patients with benign pulmonary lesions and micronodular lung cancer, with the nodular size less than or equal to 10 mm for all the patients) and healthy subjects without pulmonary lesion, the invention screens out 12 significantly different feature genes of lung cancer from the peripheral blood gene expression profile based on the logistic regression statistical method, and constructs the corresponding predictive model of micronodular lung cancer according to different gene combination products. Then the relative expressions of the genetic marker of micronodular lung cancer in peripheral blood of the subjects are quantitatively detected using fluorescent quantitative PCR, gene expression profile chip or RNA sequencing technology, and benign and malignant pulmonary micro-nodules are identified by combining with the identifying model, so as to identify whether the subject had lung carcinoma to realize the early discovery of lung carcinoma.
The screening of feature genes of lung carcinoma comprises the following steps:
1) Collect the peripheral blood samples of 40 malignant mocrinodular patients diagnosed as lung cancer by operation and pathological examination, 16 patients with benign micro-nodules (benign pulmonary lesion) and 28 healthy subjects without pulmonary lesion. The nodular sizes of all the mocrinodular patients were less than or equal to 10 mm by CT examination, and 2-3 ml of peripheral blood was collected for each sample.
2) Extract the total RNA of the above samples using PAXgene Blood RNA Kit, detect the fragment integrity (RIN) of the RNA samples using AgilentBioanalyzer 2100, and detect the purity of the RNA samples using Nano1000 micro ultraviolet spectrophotometer. All the samples must conform to the following conditions for quality control: RNA yield >2 mg, 28S/18S peak ratio >1, RIN value >7, and the 260 nm/280 nm absorbancy ratio >1.8.
3) Detect the total RNA samples in the above peripheral blood using Affymetrix Gene Profiling Array U133Plus2chip (human total gene expression profile chip), obtain the data of the peripheral blood gene expression profile of the samples, then perform normalization for the data of the peripheral blood gene expression profile using the MASS method in the AffymetrixExpression Console software, eliminate the system error possibly produced in the detection process of the expression profile chip to obtain the data of peripheral blood gene expression profile for unified comparison.
4) Remove the over-high and over-low gene expression signals in the peripheral blood gene expression profile, choose the genes with moderate expression (with the signal value ranging 100-10000) from all the samples for T test analysis, compare the differences in the gene expression in peripheral blood between the subjects with micronodular lung cancer and the subjects with benign nodules and healthy subjects, and take the genes with the statistical P value <0.05 and the gene expression varying above 1.1 times as the candidate genes for the follow-up analysis.
5) Analyze the correlation between the above candidate genes with micronodular lung cancer, rank them according to the correlation coefficient between the genes and micronodular lung cancer, select a set of gene queue (gene queue I) highly correlated with micronodular lung cancer; furthermore, perform correlation analysis between the remaining genes and the genes in queue I, and select another set of gene queue (gene queue II) highly correlated with gene queue I. Then perform pairwise coupling for the genes in gene queue I and gene queue II to form a series of candidate gene combination.
6) Evaluate the effect of each candidate gene combination in identifying micronodular lung cancer using the logistic regression statistical analysis method, calculate the receiver operator characteristic curve (ROC curve) and area under curve (AUC) of each candidate gene combination, and screen out a series of gene combination having favorable identifying capability for micronodular lung cancer.
7) Verify the screened series of gene combination using real-time fluorescent PCR method, keep the genes having consistent expression changes in the detection using quantitative PCR and gene expression profile chip as the peripheral blood feature genes of micronodular lung cancer, and mainly screen out 12 feature genes of lung cancer (of which the gene sequences are as indicated by SEQ ID NO.1˜SEQ ID NO.12), namely HSP90AA1, UQCRQ, COX7A2, CAPZA2, CHMP5, NDUFB2, RPL24, CKLF, C14orf2, CD52, FGFBP2 and GLRX; for the significance of these 12 feature genes of lung cancer, see
8) Evaluate the diagnostic effect of 6 gene combinations randomly selected from the above 12 genes on micronodular lung cancer using the logistic regression statistical analysis method, calculate the ROC and AUC of the gene combinations, and construct the micronodular lung cancer identifying model as shown below:
Where, P is the risk value for micronodular lung cancer (malignant pulmonary micro-nodules); b0˜b6 are the corresponding parameters of the logistic regression model, respectively; ΔCt1˜ΔCt6 are the differences in the value of quantitative PCR cycles Ct between the 6 genetic markers of micronodular lung cancer and the reference genes; X is the logistic regression log-likelihood ratio.
1. Methods and Steps:
1) Collection of peripheral blood samples of the samples to be detected: Collect the peripheral blood samples of the patients using BD PAXgeneRNA blood collecting vessels (QIAGEN).
2) Extraction and purification of the total RNA in the peripheral blood samples of the samples to be detected: Extract and purify the total RNA in the peripheral blood using the PAXgeneBlood RNA Kit (QIAGEN), and identify the fragment integrity and yield of the extracted total RNA using the Agilent BioAnalyzer 2100 microelectrophoresis analyzer. Detect the purity of the RNA samples using the Nano1000 micro ultraviolet spectrophotometer.
3) Reverse transcription reaction: Use the High-Capacity cDNA Reverse Transcription kit (Life Technology), take the total RNA as the template, and use Olig(dT) as the primer for reverse transcription, and perform reverse transcription to synthetize cDNA.
4) Fluorescent quantitative RT-PCR detection: According to the related sequences of the 6-gene Panel combined product (a total of 6 gene combinations of CKLF, GLRX, HSP90AA1, NDUFB2, RPL24 and UQCRQ) and the reference gene of GAPDH, design the corresponding specific primer and/or probe, or perform real-time fluorescent quantitative PCR reaction using the SYBR Green dye which is nonspecific binding with PCR amplified fragment and taking the cDNA obtained by reverse transcription as the template for amplification, use the GAPDH gene as the reference gene (the amplimer sequence of the reference gene is as indicated by SEQ ID NO.49˜SEQ ID NO.50, and the probe sequence is as indicated by SEQ ID NO.51), and obtain the relative mRNA content of the 6 genetic markers in the peripheral blood samples. The following Table 1 lists the fluorescent quantitative PCR reaction system. The following Table 2 lists the primer and probe sequences designed for the feature genes of lung cancer.
5) Diagnosis of results of the samples to be detected: According to the relative mRNA contents of the 6 genetic markers of CKLF, GLRX, HSP90AA1, NDUFB2, RPL24 and UQCRQ in the peripheral blood samples detected by real-time fluorescent quantitative PCR, and calculate the logistic regression log-likelihood ratio of the samples through the corresponding micronodular lung cancer identifying models; the detection result with the X value ≥0 is identified as positive, namely lung cancer; and the detection result with the X value <0 is identified as negative, namely not lung cancer;
2. Results
The peripheral blood samples were collected from a total of 144 cases of malignant micronodular lung cancer (Lung Cancer), 39 cases of benign micro-nudules (Benign) and 107 healthy subjects (Health), the relative expressions of the 6 genetic markers of micronodular lung cancer and the reference gene of GAPDH in peripheral blood were detected using fluorescent quantitative PCR, the logistic regression log-likelihood ratio X value of each sample was calculated, and if the X value is ≥0, it is considered as positive detection result, otherwise it is considered as negative detection result. By comparing the detection results with the pathological detection results, it is obtained that the feature genetic markers of lung cancer of the invention can better identify malignant micro-nodules (micronodular lung cancer) and benign micro-nodules, it has the sensitivity and specificity both higher than 75% for the detection of micronodular lung cancer, and see
The above embodiments only express the execution modes of the invention in more specific and detailed description, but they can't be understood as the limitation to the scope of the invention patent accordingly. It shall be indicated that for the common technicians of this field, under the premise without separating from the concept of the invention, several deformations and improvements can also be obtained, which are all within the protective range of the invention. Therefore, the protective range of the invention patent shall be subject to the claims.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201610353398.6 | May 2016 | CN | national |
| 201810661113.4 | Jun 2018 | CN | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2017/083019 | May 2017 | US |
| Child | 16196163 | US |
