CN117778553A - Kit for predicting liver cancer hand-foot syndrome caused by sorafenib and application of kit - Google Patents
Kit for predicting liver cancer hand-foot syndrome caused by sorafenib and application of kit Download PDFInfo
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- CN117778553A CN117778553A CN202311141265.9A CN202311141265A CN117778553A CN 117778553 A CN117778553 A CN 117778553A CN 202311141265 A CN202311141265 A CN 202311141265A CN 117778553 A CN117778553 A CN 117778553A
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- MLDQJTXFUGDVEO-UHFFFAOYSA-N BAY-43-9006 Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 MLDQJTXFUGDVEO-UHFFFAOYSA-N 0.000 title claims abstract description 30
- 239000005511 L01XE05 - Sorafenib Substances 0.000 title claims abstract description 30
- 229960003787 sorafenib Drugs 0.000 title claims abstract description 30
- 208000002375 Hand-Foot Syndrome Diseases 0.000 title claims abstract description 19
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- 201000007270 liver cancer Diseases 0.000 title claims abstract description 7
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 16
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- 206010067484 Adverse reaction Diseases 0.000 description 5
- 208000020061 Hand, Foot and Mouth Disease Diseases 0.000 description 5
- 208000025713 Hand-foot-and-mouth disease Diseases 0.000 description 5
- 230000006838 adverse reaction Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 4
- 206010020772 Hypertension Diseases 0.000 description 3
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 3
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- 206010038389 Renal cancer Diseases 0.000 description 2
- 208000006265 Renal cell carcinoma Diseases 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
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- 208000008960 Diabetic foot Diseases 0.000 description 1
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- CPYIZQLXMGRKSW-UHFFFAOYSA-N zinc;iron(3+);oxygen(2-) Chemical compound [O-2].[O-2].[O-2].[O-2].[Fe+3].[Fe+3].[Zn+2] CPYIZQLXMGRKSW-UHFFFAOYSA-N 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The application provides a kit for predicting liver cancer hand-foot syndrome caused by sorafenib and application thereof, wherein the kit comprises a reagent for detecting genotypes at one or more SNP loci selected from rs448012, rs2745557 and rs20417.
Description
Technical Field
The application belongs to the field of molecular biological detection and cancer treatment. Specifically, the application provides a kit for predicting liver cancer hand-foot syndrome caused by sorafenib and application thereof.
Background
Sorafenib (Sorafenib) is a multi-kinase inhibitor of the RAF/MEK/ERK pathway, and the major use that has been approved today is to treat liver and kidney cancers that lack surgical opportunities, clinically belonging to the most common targeted drug categories for such patients. Known side effects of sorafenib include hypertension, gastrointestinal discomfort, hand-foot syndrome, skin damage, and hematopoietic problems, among others.
The hand-foot syndrome caused by sorafenib is more serious, and the medication of patients can be influenced in many cases; on the other hand, there are studies showing that hand-foot syndrome is a manifestation of the responsiveness of patients to sorafenib to some extent. Therefore, from the point of view of guiding medication or from the point of view of predicting the efficacy of sorafenib, it is necessary to study and predict the molecular mechanism and related SNP sites of hand-foot syndrome caused by sorafenib.
Although the anti-tumor mechanism of sorafenib has been studied intensively, the mechanism of side effects and the method for predicting sorafenib are not fully studied, and no mature prediction method and reagent are available.
Disclosure of Invention
Aiming at the problems, the applicant selects a larger amount of sample adverse reactions and control samples to construct a DNA pool, and selects rs448012 on VEGFR3 from the DNA pool; rs2745557 and rs20417 on COX-2 are used as SNP loci for predicting hand-foot syndrome side effects caused by sorafenib, and a kit and a detection method are provided on the basis.
In one aspect, the application provides a kit for predicting that sorafenib causes liver cancer hand-foot syndrome, wherein the kit comprises reagents for detecting genotypes at one or more SNP loci selected from rs448012, rs2745557 and rs20417.
In another aspect, the application provides the use of an agent for detecting genotypes at one or more SNP sites selected from rs448012, rs2745557, rs20417 in the preparation of a kit for predicting hand-foot syndrome side effects caused by sorafenib.
Further, the reagent for detecting the genotype at one or more SNP loci selected from rs448012, rs2745557 and rs20417 is selected from reagents for direct sequencing after amplification, taqman detection, SNPshot detection, PCR-RFLP, PCR-SSCP, gene chip and mass spectrometry.
Further, the reagent for detecting the genotype at one or more SNP loci selected from rs448012, rs2745557 and rs20417 comprises a primer with a sequence of SEQ ID NO. 1-6.
Further, the reagent for detecting genotype at one or more SNP loci selected from rs448012, rs2745557, rs20417 further comprises SatI, taq alpha I and HhaI endonuclease.
Further, the reagent for detecting the genotype at one or more SNP loci selected from the group consisting of rs448012, rs2745557 and rs20417 further comprises PCR buffer, dNTP, DNA polymerase and MgCl 2 。
In another aspect, the present application provides a non-diagnostic method of predicting hand-foot syndrome side effects caused by sorafenib, the method comprising the step of detecting genotypes at one or more SNP sites selected from rs448012, rs2745557, rs20417.
Further, in the method, the primer with the sequence of SEQ ID NO.1-6 is used for detecting the genotype by a PCR-RFLP method.
Further, in the method, if one of the CC genotype at rs448012, the GG genotype at rs2745557 and the rs20417 GG/GC genotype is detected, the sample-derived person is predicted to have a risk of hand-foot syndrome side effects caused by sorafenib.
The adverse effects of the present application may be those of the invention in patients treated with sorafenib for liver cancer, such as hepatocellular carcinoma, renal carcinoma, and other cancers. The kit methods of the present application can be used in combination with other methods and kits for predicting adverse effects of sorafenib.
The methods and kits of the present application may employ various SNP detection methods known and developed in the art, such as direct sequencing after amplification, taqman method detection, SNPshot detection, PCR-RFLP, PCR-SSCP, gene chip detection, mass spectrometry typing methods, and the like.
Specific types of reagents include, but are not limited to, primers, probes, gene chips, electrophoresis chips, fluorescent labels, polymerases, and the like; the design of chips, probes, primers, etc. can be realized by those skilled in the art based on books, design sites/tools, and technical services of professional companies in the art such as molecular cloning.
Detailed Description
Example 1 case Condition and treatment protocol
Study cases were from naval 971 hospital and other two-way hospitals, for a total of 161 patients with intermediate and advanced hepatocellular carcinoma who had not undergone surgery or intervention. Of these, 84 men and 77 women had an average age of 54.7.+ -. 9.5 years. The selected patients have no obvious hand and foot skin problems (including common urticaria, dermatitis, pompholyx, warts, psoriasis, vitiligo, diabetic foot, wound difficult to heal and the like) in the past cases and in the self-description.
Sorafenib (Bayer, duoJimei, 200 mg/tablet) was prescribed after diagnostic evaluation by a qualified physician in all cases, using 2 tablets each time, standard using method twice daily; during the administration period, if normal life is not affected, other skin medicines except calamine lotion (physical therapy) are not used.
Hand-foot-and-mouth syndrome determination uses the National Cancer Institute (NCI) grading criteria as follows:
TABLE 1 grading Standard of hand-foot-and-mouth syndrome by the National Cancer Institute (NCI)
Among 161 patients, 1 patient dies due to other reasons in a 2-month monitoring period, 5 patients stop taking medicine due to side effects such as frequent occurrence of hypertension or dizziness and vomiting, and 1 patient stops taking medicine due to severe hand-foot syndrome; of the 154 patients who completed the monitoring period, 31 cases of hand-foot-and-mouth syndrome were totally monitored, and the rest included 1 case of patients who stopped medication due to severe hand-foot syndrome and 1 case of grade 2 hand-foot-and-mouth syndrome recorded in patients who stopped medication due to hypertension, 33 cases were totally recorded, 20 cases for men and 13 cases for women.
Example 2 SNP screening
DNA was extracted from whole blood samples of the study cases described in example 1 (OD 260/OD280 greater than 1.7, less than 1.9); constructing a DNA mixing pool of a hand-foot-and-mouth syndrome group and a hand-foot-and-mouth syndrome group; samples were processed, hybridized, stained, scanned, and quality controlled according to the instructions of Affymetrix SNP Array 6.0.0 chips. Processing CEL files obtained from the chip by using SNPMaP software package, calculating RAS of each site, and performing t-test to analyze gene frequency difference between the hand-foot-mouth syndrome group and the hand-foot-mouth syndrome group, wherein Bonferroni corrects; statistical software used SPSS20.0, double sided p <0.05.
GWAS analysis found the following significant differential SNP sites: rs448012 on VEGFR 3; rs2745557, rs20417 on COX-2.
Example 3 distribution of related SNP locus genotypes in cases
The venous blood of each case is taken, genomic DNA is extracted, and SNP locus genotype is detected by using a PCR-RFLP method. The primer sequences used were partially modified by themselves, with reference to the prior art, as shown in Table 2 below:
TABLE 2 primer sequences and endonucleases for detecting SNP loci
The distribution of the genotypes of the relevant SNP loci in the cases is shown in tables 3 to 5 below:
TABLE 3 distribution of rs448012 genotypes in cases
TABLE 4 distribution of rs2745557 genotypes in cases
TABLE 5 distribution of rs20417 genotypes in cases
The above data demonstrate. The sites have the capability of predicting adverse reactions of the sorafenib hand-foot-and-mouth disease singly or jointly, and according to statistical results of different combinations of genotypes, the applicant gives reference standards for predicting adverse reactions of the sorafenib hand-foot-and-mouth disease: one of the CC genotype at rs448012, the GG genotype at rs2745557, and the rs20417 GG/GC genotype is detected (risk); reference standard for predicting adverse reactions of sorafenib severe hand-foot-and-mouth disease: the GG genotype at rs2745557 or the rs20417 GG genotype (high risk) was detected.
Example 4 clinical validation of predictive methods
In the latter stage, 72 patients (70 hepatocellular carcinoma and 2 renal carcinoma) were selected for clinical use, and no obvious hand-foot skin problem was found in the selected patients in the past and from the description. Sorafenib (Bayer, duoJimei, 200 mg/tablet) was used, with the method of use being 2 tablets each time, standard twice daily. The hand-foot-and-mouth disease judgment standard is shown in the embodiment 1, the prediction method is as described in the embodiment 3, and the clinical observation period is 2 months.
According to the judgment standard given in example 3, 27 patients with adverse reaction risks of sorafenib hand-foot-and-mouth disease are detected in total, wherein 18 patients with high risk are at risk, and the general risk is 9; 45 risk-free patients, after 2 months of clinical observation, developed results as follows (all patients completed treatment this time, no dislocation):
table 6 predicts clinical observations for each group
The result shows that the incidence rate of the hand-foot-mouth syndrome side effect in the risk-free group is 6.7%, which is far lower than the clinically recorded 15-24% of the hand-foot-mouth syndrome side effect proportion; the incidence of (high + general) hand-foot-and-mouth syndrome side effects in the risk group is 66.7%; 77.8% more in the high risk group; the above data further demonstrate the reliability of the predictive methods of the present application.
Claims (9)
1. The kit for predicting liver cancer hand-foot syndrome caused by sorafenib is characterized by comprising a reagent for detecting genotypes at one or more SNP loci selected from rs448012, rs2745557 and rs20417.
2. Application of a reagent for detecting genotypes at one or more SNP loci selected from rs448012, rs2745557 and rs20417 in preparation of a kit for predicting hand-foot syndrome side effects caused by sorafenib.
3. The kit of claim 1 or the use of claim 2, wherein the reagent for detecting the genotype at one or more SNP sites selected from the group consisting of rs448012, rs2745557, rs20417 is selected from the group consisting of reagents for direct sequencing after amplification, taqman detection, SNPshot detection, PCR-RFLP, PCR-SSCP, gene chip, mass spectrometry.
4. A kit or use according to claim 3, wherein the reagents for detecting genotypes at one or more SNP sites selected from rs448012, rs2745557, rs20417 comprise primers of sequence SEQ ID No. 1-6.
5. The kit or use of claim 4, wherein the reagent for detecting genotype at one or more SNP sites selected from the group consisting of rs448012, rs2745557, rs20417 further comprises SatI, taq ai and HhaI endonucleases.
6. The kit or use according to claim 5, wherein the reagent for detecting genotype at one or more SNP sites selected from the group consisting of rs448012, rs2745557, rs20417 further comprises PCR buffer, dNTP, DNA polymerase, mgCl 2 。
7. A non-diagnostic method for predicting hand-foot syndrome side effects caused by sorafenib, said method comprising the step of detecting genotypes at one or more SNP sites selected from rs448012, rs2745557, rs20417.
8. The method of claim 7, wherein the method uses primers having sequences of SEQ ID NO.1-6 to detect genotype by PCR-RFLP method.
9. The method of claim 8, wherein the method predicts that the sample-derived human is at risk of hand-foot syndrome side effects caused by sorafenib if one of the CC genotype at rs448012, the GG genotype at rs2745557, and the rs20417 GG/GC genotype is detected.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311141265.9A CN117778553A (en) | 2023-09-06 | 2023-09-06 | Kit for predicting liver cancer hand-foot syndrome caused by sorafenib and application of kit |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311141265.9A CN117778553A (en) | 2023-09-06 | 2023-09-06 | Kit for predicting liver cancer hand-foot syndrome caused by sorafenib and application of kit |
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| CN117778553A true CN117778553A (en) | 2024-03-29 |
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| CN202311141265.9A Pending CN117778553A (en) | 2023-09-06 | 2023-09-06 | Kit for predicting liver cancer hand-foot syndrome caused by sorafenib and application of kit |
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- 2023-09-06 CN CN202311141265.9A patent/CN117778553A/en active Pending
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