CN115125296A - Application of regorafenib drug sensitive marker and related reagent thereof - Google Patents
Application of regorafenib drug sensitive marker and related reagent thereof Download PDFInfo
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Abstract
The invention discloses a regorafenib drug sensitivity marker and related reagent application thereof. The regorafenib drug-sensitive marker is a regorafenib drug-resistant marker or a regorafenib sensitive marker, the regorafenib drug-resistant marker is any one or more of a group of markers including RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1 and WDR86, and the regorafenib sensitive marker is any one or more of TMPRSS6, ENTPD8 and CCDC 159. The regorafenib drug sensitivity marker can be used for predicting the drug effect of regorafenib. The expression of related molecules is detected by sequencing a tissue transcriptome of a liver cancer patient or by PCR, a gene chip, a tissue chip, a NanoString technology and an immunohistochemical method, so that clues can be provided for the application of regorafenib and the accurate treatment of liver cancer.
Description
Technical Field
The invention belongs to the technical field of biomedicine, particularly relates to the field of tumors, and particularly relates to a regorafenib drug sensitivity marker and related reagent application thereof.
Background
The primary liver cancer has the fourth morbidity and the second mortality in the current common malignant tumors of China, and seriously threatens the life and health of people in China. The treatment of liver cancer patients in early stage (A stage of Barcelona 0), surgical resection, liver transplantation and the like can effectively improve the cure rate and survival prognosis of the liver cancer patients. However, since liver cancer is hidden and early symptoms lack specificity, many patients are diagnosed with liver cancer and are in the middle or late stage. For a long time, the liver cancer of middle and late stages generally shows poor prognosis and low survival rate due to few drug treatment schemes and low clinical effective rate.
Regorafenib is an oral multi-target kinase inhibitor, can inhibit VEGFR-1, 2, 3, TIE-2, BRAF, KIT, RET, PDGFR and FGFR, and has a structure similar to that of sorafenib. Regorafenib is FDA approved for treatment of hepatocellular carcinoma patients who have received sorafenib treatment. Studies have shown that sorafenib-regorafenib sequential treatment can provide limited prolongation of patient overall survival. Regorafenib, as a second-line drug for liver cancer treatment, has a limited clinical effective rate of only 11% although the therapeutic effect of liver cancer can be improved to a certain extent, and most people are not suitable for use, so that many ineffective treatment phenomena occur. Therefore, a drug sensitive molecule typing scheme for supplementing regorafenib is needed to improve the curative effect of liver cancer treatment.
Disclosure of Invention
In order to overcome the problems in the prior art, the first object of the present invention is to provide a regorafenib drug-sensitive marker, which can be used for prompting regorafenib drug-resistant and sensitive people and providing a basis for accurate treatment of liver cancer. The second purpose of the invention is to provide the application of the regorafenib drug-sensitive marker in the research of regorafenib drug application for non-treatment purposes. A third object of the present invention is to provide some applications of the regorafenib marker-related agent, which includes: quantitative reagents, inhibitors, promoters and the like for detecting the expression of the liver cancer cell line, and the application of the related reagents can fill the blank of the field of accurate diagnosis and treatment of liver cancer.
The purpose of the invention is realized as follows:
first aspect of the invention: high expression of a group of gene drug resistance markers, namely RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1 and WDR86, is found to be related to regorafenib drug resistance. The genes TMPRSS6, ENTPD8 and CCDC159 are found to be sensitive to the action of regorafenib. Therefore, the gene expression can be used as a potential drug sensitive biomarker for guiding the treatment of the primary liver cancer. Namely, the invention provides a regorafenib drug-sensitive marker which is a regorafenib drug-resistant marker or a regorafenib sensitive marker; the regorafenib drug resistance marker is any one or more of a group of markers including RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1 and WDR 86; the regorafenib sensitive marker is any one or more of TMPRSS6, ENTPD8 and CCDC 159. The regorafenib drug sensitive marker is RNA or reverse transcribed cDNA thereof, or protein.
The specific screening process is as follows:
the invention collects the operation samples of a plurality of cases of primary liver cancer patients, samples the samples at multiple points, and successfully establishes a liver cancer organoid biological library covering hundreds of sites by a three-dimensional culture method. The cohort confirmed that the organoids retained the pathological features of the patient by Hematoxylin-Eosin (HE) staining. Through immunofluorescence and immunohistochemical staining of the organoids and corresponding patient specimens, the molecular markers of the liver cancer organoids and the clinical liver cancer patients are also consistent.
And selecting nearly hundreds of sites, sequencing exons and transcriptomes of the tissues and the corresponding organoids, and analyzing genome characteristics of the tissues and the corresponding organoids. The molecular characteristics of the organoid biological bank highly-reserved tissue samples are verified from multiple dimensions such as somatic mutation, copy number variation, transcriptome similarity and the like. In addition, the large-scale liver cancer tissues and organoid queues established by the team are consistent with the previous liver cancer research, and have high mutation of the liver cancer. In addition, the liver cancer data of the research reflects the inter-tumor (inter-tumor) and intra-tumor heterogeneity of different degrees, and provides a basis for researching the drug sensitivity heterogeneity of the liver cancer.
In order to research the drug sensitive molecular typing of the liver cancer targeting drug regorafenib, transcriptome sequencing is carried out on a plurality of organoid sites, and through Lasso model and biological information analysis, sensitive markers of regorafenib are analyzed to be TMPRSS6, ENTPD8 and CCDC159, and drug resistant markers are analyzed to be RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1 and WDR 86. The training set AUC was 0.82. Then, by the same method, a plurality of liver cancer specimens are collected to perform organoid culture, drug sensitivity test and transcriptome sequencing, and molecular typing research is performed to obtain a verification set of regorafenib of 0.80.
Second aspect of the invention: provides the application of the regorafenib drug sensitive marker in the research of regorafenib drugs for non-treatment purposes. Including basic research application in some drug effects and application in drug effect determination of regorafenib improvement research.
In a third aspect of the invention: the application of the quantitative reagent of the expression quantity of the regorafenib drug sensitivity marker in preparing a regorafenib drug sensitivity detection reagent is provided, and the drug sensitivity detection is drug resistance detection or drug sensitivity detection.
The detected sample is a liver cancer tissue sample, and the quantitative reagent is any one of a transcriptome sequencing reagent, a PCR (polymerase chain reaction), a gene chip, a tissue chip, a NanoString technology and an immunohistochemical reagent.
The invention provides a regorafenib drug sensitivity evaluation kit, which comprises a quantitative reagent for detecting the expression quantity of a regorafenib drug marker, wherein the drug sensitivity evaluation is drug resistance evaluation or drug sensitivity evaluation.
In addition, the invention also provides a regorafenib drug sensitivity analysis system for primary liver cancer, which comprises the following components:
the target expression quantity detection device comprises: detecting the expression amount of the regorafenib drug-sensitive marker in a sample; the sample is liver cancer tissue of a patient;
drug sensitivity analysis device: determining the administration effect of the liver cancer based on the expression amount of the regorafenib drug sensitivity marker;
a result output device: and the drug sensitivity analysis device is used for outputting the result obtained by the analysis of the drug sensitivity analysis device, wherein the drug sensitivity analysis is drug resistance analysis or sensitivity analysis.
The fourth aspect of the present invention: provides a pharmaceutical composition for treating liver cancer, regorafenib and an inhibitor or an accelerator of the regorafenib drug sensitive marker.
One scheme is as follows: the pharmaceutical composition comprises regorafenib and an inhibitor of the regorafenib drug-sensitive marker, wherein the regorafenib drug-sensitive marker is a regorafenib drug-resistant marker. The pharmaceutical composition enhances the drug effect of regorafenib on a specific patient by inhibiting the expression of regorafenib drug resistance genes.
The other scheme is as follows: the pharmaceutical composition comprises regorafenib and an accelerator for the regorafenib drug-sensitive marker, wherein the regorafenib drug-sensitive marker is a regorafenib sensitive marker. The pharmaceutical composition enhances the drug effect of regorafenib on a specific patient by promoting the expression of regorafenib sensitive genes.
The invention has the advantages and beneficial effects that:
a group of genes (RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1, WDR86) are determined to be related to regorafenib resistance; the TMPRSS6, ENTPD8 and CCDC159 genes are determined to be related to regorafenib sensitivity. These markers can be used to predict the efficacy of regorafenib. The expression of related molecules is detected by tissue sequencing of a liver cancer patient or RT-qPCR and immunohistochemical methods, so that clues can be provided for the application of regorafenib and the accurate treatment of liver cancer.
Drawings
The invention is further illustrated by the following examples in conjunction with the drawings.
FIG. 1 is a graph of the pathological characteristics and tissue markers of an organoid-preserved patient from a hepatocellular carcinoma (HCC) patient. Wherein, figure 1a HE and immunohistochemical staining of different sites of hcc patients; figure 1b microscopic status and HE staining of hcc patient organoids; FIG. 1C immunofluorescence staining of organoids; figure 1d immunohistochemical staining of organoids.
FIG. 2 shows exon, transcriptome profiles of organoid-preserved patients. Among them, figure 2A, figure 2b. exon sequencing revealed that organoids retained the mutational characteristics of the patient. Transcriptome sequencing showed that organoids retained the transcriptome profile of patients, fig. 2C, fig. 2d.
FIG. 3 is organoid susceptibility testing. Fig. 3A is a drug sensitivity curve for regorafenib, fig. 3B, tested for 7 liver cancer targeting drugs for 376 organoids.
Figure 4 is a molecular profile of regorafenib drug sensitivity. Figure 4a. drug sensitive molecular typing of regorafenib. Figure 4b. AUC for regorafenib training set and validation set. Fig. 4c is a graph of regorafenib drug-resistant target interaction network.
Fig. 5 shows the confirmation of regorafenib molecular typing in 100 liver cancer tissues.
Detailed Description
The technical scheme of the invention is further explained by combining experimental data.
Example 1
Screening regorafenib drug sensitivity markers:
in the embodiment, a liver cancer organoid biological library covering 376 loci is successfully established by a 3D culture method. Taking a surgical specimen of a patient with Hepatocellular carcinoma (HCC) as an example, the inventors confirmed that the organoids retained the pathological features of the patient by HE staining (fig. 1A, 1B). By immunofluorescence and immunohistochemical staining of organoids and corresponding patient specimens, the hepatocellular carcinoma patients and the organoid high-school HCC diagnosis markers AFP and hepatocyte are positive (figures 1C and 1D), and the cholangiocarcinoma marker EPCAM is negative and is consistent with the molecular marker of the liver cancer patients in clinic. The organoid system established in this example well retains the pathological characteristics of the patient.
In this example, 99 sites were selected, and exon and transcriptome sequencing of tissues and corresponding organoids was performed, and genomic features of tissues and corresponding organoids were analyzed. The molecular characteristics of the organoid biological bank highly reserved tissue samples are verified from multiple dimensions such as somatic mutation, copy number variation, transcriptome similarity and the like. The large-scale liver cancer tissue and organoid cohort established by the team is consistent with the previous liver cancer research, and has high mutation of liver cancer (fig. 2A-B). At the transcriptome level, organoids and tissues of corresponding patients also had a high correlation (FIGS. 2C-D).
In this example, first-line liver cancer drugs sorafenib and ranvatinib, commonly used two/three-line drugs regorafenib, apatinib and bevacizumab, and drug sensitivity tests of Pemigatinib, bile duct cancer IDH1 mutated drug Ivosidenib for bile duct cancer FGFR2 fusion were performed on 376 organoids. This example plots the drug sensitivity curve of the first line drug regorafenib (fig. 3A) and a heat map of all targeted drugs (fig. 3B).
In order to study the drug sensitive molecular typing of the liver cancer targeting drug regorafenib, the present example performed transcriptome sequencing on 106 organoid sites, and by Lasso model and biological information analysis, the sensitive markers of regorafenib were TMPRSS6, ENTPD8, and CCDC159, and the drug-resistant markers were RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1, and WDR86 (fig. 4A). The training set AUC was 0.82 (fig. 4B). Then, by the same method, 106 liver cancer specimens were collected for organoid culture, susceptibility testing, and transcriptome sequencing, and molecular typing studies were performed, resulting in a validation set of regorafenib of 0.80 (fig. 4B). And reveals a regorafenib drug-resistant target interaction network diagram (fig. 4C).
This example demonstrates the efficacy of this drug sensitive molecule typing in the tissues of 100 patients with liver cancer. Regorafenib has a susceptibility molecular profile AUC of 0.79 in liver cancer tissues (fig. 5).
Example 2
The use of regorafenib markers in the study of regorafenib drugs for non-therapeutic purposes. In this example, the activity of regorafenib was studied: selecting specific liver cancer tissues, reacting different batches of regorafenib with the selected liver cancer tissues, and determining the quantitative expression of one or more of regorafenib drug sensitive molecules (such as RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1 and WDR86) screened in example 1, thereby judging the biological activity of each batch of regorafenib.
Example 3
The embodiment provides an RT-qPCR regorafenib drug sensitivity assessment kit, which comprises a quantitative reagent for detecting the expression level of the regorafenib drug sensitivity marker (different kits can be any one of RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1, WDR86, TMPRSS6, ENTPD8 and CCDC 159) screened in the embodiment 1. The kit comprises an upstream primer and a downstream primer of the RNA of the regorafenib drug sensitive marker. The kit disclosed by the invention is combined with a common RNA extraction reagent and a reverse transcription reagent, so that the expression quantity of the regorafenib drug sensitivity marker in a sample can be specifically detected, and the kit is favorable for judging the sensitivity of a liver cancer patient to regorafenib in advance. The detection can be carried out immediately after the liver cancer tissue is obtained by the operation or pathological puncture of the liver cancer patient, the curative effect of the medicine can be evaluated before the regorafenib is used, and a personalized and accurate treatment scheme is provided for the patient.
Example 4
This example provides an RT-qPCR regorafenib drug sensitivity assessment kit, which includes a quantification reagent for detecting an expression level of the regorafenib sensitive drug-resistant marker (any one of RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1, and WDR86) selected in example 1. The kit comprises an upstream primer and a downstream primer of the RNA of the regorafenib sensitive drug-resistant marker. The kit disclosed by the invention is combined with a common RNA extraction reagent and a reverse transcription reagent, can be used for specifically detecting the expression quantity of the regorafenib sensitive drug-resistant marker in a sample, and is beneficial to judging the drug resistance of a liver cancer patient to regorafenib in advance. The method can be used for immediately measuring the liver cancer tissue obtained by the operation or pathological puncture of a liver cancer patient, and can evaluate the curative effect of the medicine before regorafenib is used, so that a personalized and accurate treatment scheme is provided for the patient.
Example 5
The embodiment provides a regorafenib drug resistance analysis system for primary liver cancer, which includes:
the RT-qPCR target expression quantity detection device comprises: the kit is used for detecting the expression level of regorafenib drug-resistant markers (any one of RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1 and WDR86) in a sample; the sample is liver cancer tissue of a patient; the reagent adopted by the target expression quantity detection device is an RT-qPCR quantitative reagent, and comprises upstream and downstream primers of the gene;
drug resistance analysis device: determining the administration effect of the liver cancer based on the expression level of regorafenib drug resistance markers RPL39 and the like;
a result output device: used for outputting the result analyzed by the drug resistance analysis device.
The curative effect of the medicine can be evaluated before the regorafenib is used by a patient, and a personalized and accurate treatment scheme is provided for the patient.
Example 6
A pharmaceutical composition for treating liver cancer comprises regorafenib and an inhibitor of regorafenib drug-resistant markers (any one or more of RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1 and WDR 86). The pharmaceutical composition can be an injection preparation, is used in treatment of liver cancer patients, and can reduce the drug resistance of regorafenib of the patients.
Finally, it should be noted that the above is only intended to illustrate the technical solution of the present invention and not to limit it, and although the present invention has been described in detail with reference to the preferred arrangement, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Claims (10)
1. A regorafenib drug sensitivity marker characterized by: the regorafenib drug-sensitive marker is a regorafenib drug-resistant marker or a regorafenib sensitive marker; the regorafenib drug-resistant marker is any one or more of RPL39, KIF9, GPC2, NKPD1, PNMAL1, ATP1A3, OSR2, ADCYAP1R1 and WDR86, and the regorafenib sensitive marker is any one or more of TMPRSS6, ENTPD8 and CCDC 159.
2. A regorafenib drug sensitivity marker according to claim 1 further comprising: is RNA or its reverse transcribed cDNA, or protein.
3. Use of the regorafenib drug-sensitive marker of claim 1 in research on the administration of regorafenib for non-therapeutic purposes.
4. Application of a quantitative reagent for detecting the expression amount of a regorafenib drug sensitivity marker in claim 1 in preparation of a regorafenib drug sensitivity detection reagent, wherein the drug sensitivity detection is drug resistance detection or drug sensitivity detection.
5. The use of claim 4, wherein the sample to be detected is liver cancer tissue sample, and the quantitative reagent is any one of transcriptome sequencing reagent, PCR, gene chip, tissue chip, NanoString technology and immunohistochemical reagent.
6. A regorafenib drug sensitivity assessment kit is characterized in that: comprises a quantitative reagent for detecting the expression amount of the regorafenib drug sensitivity marker in claim 1, wherein the drug sensitivity is evaluated by drug resistance evaluation or drug sensitivity evaluation.
7. A regorafenib drug sensitivity analysis system for primary liver cancer is characterized by comprising:
the target expression quantity detection device comprises: for detecting the expression level of the regorafenib drug-sensitive marker of claim 1 in a sample; the sample is liver cancer tissue of a patient;
drug sensitivity analysis device: determining the administration effect of the liver cancer based on the expression amount of the regorafenib drug sensitivity marker;
a result output device: and the drug sensitivity analysis device is used for outputting the result obtained by the analysis of the drug sensitivity analysis device, wherein the drug sensitivity analysis is drug resistance analysis or sensitivity analysis.
8. A pharmaceutical composition for treating liver cancer, which is characterized in that: an inhibitor or an enhancer comprising regorafenib and a regorafenib drug sensitivity label according to claim 1.
9. The pharmaceutical composition for treating liver cancer according to claim 8, wherein: an inhibitor comprising regorafenib and the regorafenib drug marker of claim 1, wherein the regorafenib drug marker is a regorafenib drug-resistant marker.
10. The pharmaceutical composition for treating liver cancer according to claim 8, wherein: an enhancer comprising regorafenib and the regorafenib drug marker of claim 1, wherein the regorafenib drug marker is a regorafenib sensitive marker.
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