CN113667755A

CN113667755A - Oxaliplatin-related biomarkers

Info

Publication number: CN113667755A
Application number: CN202111147943.3A
Authority: CN
Inventors: 杨承刚; 李昭然; 王丹
Original assignee: Qingdao Yangshen Biomedical Co Ltd
Current assignee: Qingdao Yangshen Biomedical Co Ltd
Priority date: 2021-09-29
Filing date: 2021-09-29
Publication date: 2021-11-19

Abstract

The invention relates to oxaliplatin resistance-related biomarkers. In particular, the invention relates to the use of biomarkers for predicting the sensitivity of colorectal cancer patients to oxaliplatin treatment.

Description

Oxaliplatin-related biomarkers

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to an oxaliplatin drug resistance related biomarker.

Background

Colorectal cancer (colorectal cancer) is one of the most common digestive tract malignant tumors in the world, seriously threatens the life and health of human beings, has the morbidity accounting for the third place of the common malignant tumors, and has the mortality accounting for the third place of cancer death. Colorectal cancer is latent in onset, no obvious clinical symptoms exist in the early stage, and the death rate of colorectal cancer is still as high as 33 percent although the diagnosis and treatment means such as early diagnosis, surgical treatment, new auxiliary treatment and the like of colorectal cancer are obviously improved at present. The main treatment means of early colorectal cancer is surgical treatment, and the 5-year survival rate is about 56-61%. However, many patients have been in the middle and late stages of treatment, and lose the best chance of surgical treatment, and 40-45% of patients relapse after surgery, and lose the chance of re-surgery. Chemotherapy-based combination therapy is becoming the primary treatment for patients with colorectal cancer at middle and advanced stages and recurrence, wherein the chemotherapy regimen based on oxaliplatin is the first-line chemotherapy regimen for the clinical treatment of colorectal cancer at present. And has obvious advantages in anticancer activity. Oxaliplatin, upon alkylation, forms intrastrand and interchain crosslinks with DNA, thereby inhibiting DNA synthesis and replication and promoting apoptosis of tumor cells. However, in the treatment process, the drug resistance of some patients to oxaliplatin severely limits the curative effect of the oxaliplatin. At present, relatively few researches on related molecular markers for predicting oxaliplatin resistance are carried out, and the related molecular markers mainly comprise glutathione transferase, P53, nucleotide excision repair genes ERCC1, ERCC2, XRCCI and the like. However, the molecular markers as related molecular markers of oxaliplatin resistance lack specificity and have certain limitations in clinical application. Therefore, the research on drug-resistant molecular markers specific to oxaliplatin for colorectal cancer has important significance in guiding clinical treatment.

Disclosure of Invention

The present invention relates to the use of biomarkers for predicting the sensitivity of colorectal cancer to treatment with oxaliplatin. In non-limiting embodiments, the invention provides a product for predicting the sensitivity of a colorectal cancer patient to treatment with oxaliplatin, and a device for predicting the sensitivity of a colorectal cancer patient to treatment with oxaliplatin. The invention also provides application of the biomarker in preparing a medicament for improving the sensitivity of a colorectal cancer patient to treatment with oxaliplatin.

The invention provides application of a reagent for detecting the expression level of a biomarker in a sample, wherein the biomarker comprises C9, NPTX2 and/or BEX2, in the preparation of a product for predicting the sensitivity of a colorectal cancer patient to oxaliplatin treatment.

In certain non-limiting embodiments, the reagents include reagents for detecting the level of expression of a biomarker in a sample by polymerase chain reaction, in situ hybridization, gel electrophoresis, sequencing and sequence analysis, microarray analysis, mass spectrometry techniques, gel analysis systems, chromatography, enzyme-linked immunosorbent assays, radioimmunoassays, enzyme immunoassays, western blots, immunoprecipitation, or immunohistochemistry.

In certain embodiments, the reagents comprise primers, probes, microarrays, biomarker-specific antibodies, or biomarker-specific beads.

The invention also provides a product for predicting the sensitivity of colorectal cancer patients to oxaliplatin treatment, which comprises a reagent for detecting the expression level of biomarkers in a sample, wherein the biomarkers comprise C9, NPTX2 and/or BEX 2.

In certain non-limiting embodiments, the product comprises a kit, a chip, a strip. In certain embodiments, the kit comprises a qPCR kit, an immunoblot detection kit, an immunochromatographic detection kit, a flow cytometric assay kit, an immunohistochemical detection kit, an ELISA kit, and an electrochemiluminescent detection kit.

In certain embodiments, the kit comprises instructions for predicting whether a colorectal cancer patient is susceptible to treatment with oxaliplatin.

In certain embodiments, the product further comprises a reagent for processing the sample.

The invention also provides application of the biomarker in preparing a medicament for improving the sensitivity of a colorectal cancer patient to oxaliplatin treatment, wherein the biomarker comprises C9, NPTX2 and/or BEX 2.

In certain non-limiting embodiments, the drug comprises a C9 inhibitor, NPTX2 promoter, and/or BEX2 inhibitor.

The invention also provides a device for predicting the sensitivity of a colorectal cancer patient to treatment with oxaliplatin, comprising:

a) an analyzer unit comprising one or more detection reagents that specifically bind to a biomarker, the unit being adapted to determine the amount of the one or more biomarkers in the sample; and

b) an analyzer unit for comparing the one or more measured quantities with one or more reference quantities, thereby diagnosing lung cancer, said unit comprising a database of one or more reference quantities and a computer-implemented algorithm for performing the comparison;

the biomarkers include C9, NPTX2, and/or BEX 2.

Drawings

FIG. 1 is a boxplot of the differential expression of C9;

fig. 2 is a boxplot of the differential expression of NPTX 2;

FIG. 3 is a box line plot of the differential expression of BEX 2;

FIG. 4 is a ROC plot of C9 predicting susceptibility of colorectal cancer patients to treatment with oxaliplatin;

FIG. 5 is a ROC plot of NPTX2 predicting colorectal cancer patient sensitivity to oxaliplatin treatment;

FIG. 6 is a ROC plot of BEX2 predicting colorectal cancer patient sensitivity to oxaliplatin treatment;

FIG. 7 is a ROC plot of the combination of C9 and NPTX2 predicting the sensitivity of colorectal cancer patients to treatment with oxaliplatin;

FIG. 8 is a ROC plot of NPTX2 and BEX2 in combination predicting the sensitivity of colorectal cancer patients to oxaliplatin treatment;

FIG. 9 is a ROC plot of the prediction of colorectal cancer patient's sensitivity to oxaliplatin treatment by the combination of C9 and BEX 2;

FIG. 10 is a ROC plot of C9+ NPTX2+ BEX2 in combination predicting the sensitivity of colorectal cancer patients to oxaliplatin treatment.

Detailed Description

For purposes of clearly explaining the present invention, and not by way of limitation, the detailed description of the invention is divided into the following subsections:

a. biomarkers

b. Biomarker detection

c. Reagent kit

d. Medicine

Biomarkers

The term "biomarker" refers to a biological molecule present in an individual at different concentrations that can be used to predict the disease state of the individual. Biomarkers can include, but are not limited to, nucleic acids, proteins, and variants and fragments thereof. A biomarker may be DNA comprising all or part of a nucleic acid sequence encoding the biomarker, or the complement of such a sequence. Biomarker nucleic acids useful in the present invention are considered to include DNA and RNA comprising all or part of any nucleic acid sequence of interest.

In the present invention, the biomarkers include C9, NPTX2 and/or BEX 2.

The term "and/or" as used herein in phrases such as "a and/or B" is intended to include both a and B; a or B; a (alone); and B (alone). Likewise, the term "and/or" as used in phrases such as "A, B and/or C" is intended to encompass each of the following embodiments: A. b and C; A. b or C; a or C; a or B; b or C; a and C; a and B; b and C; a (alone); b (alone); and C (alone).

In the present invention, biomarkers such as C9(gene ID: 735), NPTX2(gene ID: 4885), BEX2(gene ID: 84707), including gene and its encoded protein and homologs, mutations, and isoforms, are included. The term encompasses full-length, unprocessed biomarkers, as well as any form of biomarker that results from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of the biomarkers.

In certain embodiments, the level of the biomarker is compared to a reference control level. A "reference control level" or "reference control expression level" used interchangeably herein can be established using, for example, a reference control sample. Non-limiting examples of reference control samples include colorectal cancer patient samples that are sensitive to oxaliplatin treatment.

The term "patient" refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, dogs, cats, rodents, and the like. Further, the patient is a human patient. The term "patient" encompasses individuals with cancer (e.g., colorectal cancer), including those individuals who have undergone or who are candidates for resection (surgery) to remove cancerous tissue.

As used herein, the term "sample" refers to a biological sample obtained or derived from a source of interest as described herein. In some embodiments, the source of interest comprises an organism, such as an animal or human. In some embodiments, the biological sample comprises a biological tissue or fluid. In some embodiments, the biological sample may be or comprise bone marrow; blood; blood cells; ascites fluid; tissue or fine needle biopsy samples; a body fluid containing cells; free floating nucleic acids; sputum; saliva; (ii) urine; cerebrospinal peritoneal fluid; pleural fluid; feces; lymph; a skin swab; orally administering the swab; a nasal swab; washings or lavages such as catheter lavages or bronchoalveolar lavages; (ii) an aspirate; scraping scraps; bone marrow specimen; a tissue biopsy specimen; a surgical specimen; feces, other body fluids, secretions and/or excretions; and/or cells therein, and the like. In some embodiments, the biological sample is or comprises cells obtained from an individual. In some embodiments, the sample is a "primary sample" obtained directly from a source of interest by any suitable means. For example, in some embodiments, the primary biological sample is obtained by a method selected from the group consisting of: biopsies (e.g., fine needle aspirates or tissue biopsies), surgical tissue, collection of bodily fluids (e.g., blood, lymph, stool, etc.), and the like.

Biomarker detection

Methods of qualitatively and quantitatively detecting and/or determining the expression level of C9 nucleic acids, NPTX2 nucleic acids, BEX2 nucleic acids include, but are not limited to, Polymerase Chain Reaction (PCR) including conventional PCR, qPCR, and digital PCR, in situ hybridization (such as, but not limited to, fluorescence in situ hybridization ("FISH")), gel electrophoresis, sequencing and sequence analysis, microarray analysis, and other techniques known in the art.

In certain embodiments, the detection method can be real-time PCR (RT-PCR), quantitative PCR, fluorescent PCR, RT-MSP (RT methylation specific polymerase chain reaction), PicoGreenTM (Molecular Probes, Eugene, Oreg.) detection of DNA, radioimmunoassay, OR direct radiolabeling of DNA. For example, but not limited to, the nucleic acid biomarkers can be reverse transcribed into cDNA, followed by polymerase chain reaction (RT-PCR); alternatively, a single enzyme may be used in both steps, as described in U.S. Pat. No. 5,322,770, or as described in r.l. marshall et al, PCR Methods and Applications 4: 80-84(1994), the biomarkers can be reverse transcribed into cDNA and then subjected to the symmetric gap ligase chain reaction (RT-AGLCR).

In certain embodiments, real-time quantitative polymerase chain reaction (qRT-PCR) is used to assess mRNA levels of the biomarkers. The levels of the biomarker and the control mRNA can be quantified in the cancer tissue of a colorectal cancer patient to be evaluated or in cancer tissue of a colorectal cancer patient sensitive to treatment with oxaliplatin. In certain embodiments, the level of one or more biomarkers can be quantified in a biological sample.

In certain embodiments, in situ hybridization visualization may be used, wherein a radiolabeled antisense RNA probe is hybridized to a thin section of a biological sample, e.g., a biopsy sample, washed, cleaved with rnase, and exposed to a sensitive emulsion for autoradiography. The sample may be stained with hematoxylin to reveal the histological composition of the sample, and dark field imaging with appropriate filters to reveal the developed emulsion. Non-radioactive labels such as digoxigenin may also be used.

In certain non-limiting embodiments, the assessment of nucleic acid biomarker expression can be performed by Fluorescence In Situ Hybridization (FISH). FISH is a technique that can directly identify specific regions of DNA or RNA in a cell, thus enabling visual determination of biomarker expression in a tissue sample. FISH is a straightforward in situ technique that can be relatively fast and sensitive, and also can be automated. Immunohistochemistry may be combined with FISH methods when the expression level of the biomarker is difficult to determine by FISH alone.

In certain embodiments, the expression of a nucleic acid biomarker can be detected on a DNA array, chip, or microarray. Oligonucleotides corresponding to the biomarkers are immobilized on a chip, and the chip is then hybridized with labeled nucleic acids of a biological sample obtained from the subject, such as a tumor tissue sample. The sample comprising the biomarker transcript obtains a positive hybridization signal. Methods for preparing DNA arrays and their use are well known in the art. (see, e.g., U.S. Pat. Nos. 6,618,6796, 6,379,897, 6,664,377, 6,451,536, 548,257; U.S. Pat. application Nos. 20030157485 and Schena et al 1995Science 20: 467-. Continuous analysis of gene expression (SAGE) can also be performed (see, e.g., U.S. patent application No. 20030215858).

In certain embodiments, to monitor the expression level of the nucleic acid biomarker mRNA, mRNA can be extracted from a biological sample to be tested, reverse transcribed, and a fluorescently labeled cDNA probe can be generated. The labeled cDNA probes are then applied to a microarray capable of hybridizing to the biomarkers, the probes are allowed to hybridize to the microarray and the slide is scanned to measure fluorescence intensity. The intensity correlates with the hybridization intensity and the expression level of the biomarker.

Types of probes for detecting nucleic acid biomarkers include cDNA, ribonucleic acid probes, synthetic oligonucleotides, and genomic probes. The type of probe used is generally determined on a case-by-case basis, for example, as ribonucleic acid probes for in situ hybridization and cDNA for northern blotting. In certain non-limiting embodiments, the probes are directed to nucleotide regions that are unique to a particular biomarker RNA. The probes may be as short as desired to differentially recognize specific biomarker mRNA transcripts, and may be as short as, for example, 15 bases. Probes of at least 17 bases, 18 bases and 20 bases may also be used. In certain embodiments, the primers and probes specifically hybridize under stringent conditions to a nucleic acid fragment having a nucleotide sequence corresponding to the target gene. The term "stringent conditions" as used herein means that hybridization will occur only if there is at least 95% or at least 97% identity between the sequences.

The form of the probe label may be any suitable form, such as using, for example³²P and³⁵a radioisotope, or fluorophore of S. Labeling with a radioisotope can be achieved by using an appropriately labeled base, whether the probe is chemically synthesized or biosynthesized.

Methods for detecting and/or determining the level of protein biomarkers, such as C9 protein, NPTX2 protein, BEX2 protein, are well known to those skilled in the art and include, but are not limited to, mass spectrometry techniques, one or two dimensional gel analysis systems, chromatography, enzyme linked immunosorbent assays (ELISA), Radioimmunoassays (RIA), Enzyme Immunoassays (EIA), western blotting, immunoprecipitation, and immunohistochemistry. These methods use antibodies or antibody equivalents to detect proteins, or use biophysical techniques. Antibody arrays or protein chips may also be used.

In certain non-limiting embodiments, the detection method for measuring protein biomarker expression comprises the steps of: contacting a biological sample, such as a tissue sample, with an antibody or variant (e.g., fragment) thereof that selectively binds to a biomarker, and detecting whether the antibody or variant thereof binds to the sample. The method may further comprise contacting the sample with a second antibody, such as a labeled antibody. The method may also include, for example, one or more washing steps to remove one or more reagents.

In certain non-limiting embodiments, western blotting can be used to detect and quantify biomarker protein expression levels. Cells or tissues may be homogenized in lysis buffer to form a lysate, then subjected to SDS-PAGE and blotted to a membrane, such as a nitrocellulose filter. The antibody (unlabeled) is then contacted with the membrane and passed through a secondary immunizing agent such as labeled protein A or anti-immunoglobulin (suitable labels include¹²⁵I. Horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used. In certain embodiments, immunodetection with anti-biomarker antibodies can be performed using an enhanced chemiluminescence system (e.g., from perkinelmer life Sciences, Boston, Mass.). The membrane can then be peeled off and re-blotted with a control antibody specific for the control protein.

Immunohistochemistry may be used to detect the expression and/or presence of a biomarker, for example, in a biopsy sample. Suitable antibodies can be contacted with, for example, a thin layer of cells, followed by washing to remove unbound antibody, and then contacted with a labeled secondary antibody. The labeling may be by fluorescent markers, enzymes such as peroxidase, avidin or radioactive labels. The test can be scored visually using a microscope and the results can be quantified. Machine-based or automated imaging systems may also be used to measure immunostaining results for biomarkers.

Various automated sample processing, scanning and analysis systems suitable for immunohistochemistry are available in the art. Such Systems may include automated staining (see, e.g., Benchmark Systems, ventana medical Systems, Inc.) and microscopic scanning, computerized image analysis, serial section comparison (to control variables in the orientation and size of the sample), digital report generation, and archiving and tracking of samples, such as slides on which tissue sections are placed. Cell imaging systems are commercially available that combine conventional optical microscopy with digital image processing systems to perform quantitative analysis of cells and tissues, including immunostained samples. See, for example, the CAS-200 system (Becton, Dickinson & Co.).

Labeled antibodies to the biomarkers may also be used for imaging purposes, e.g., to detect the presence of the biomarkers in cells of a subject. Suitable labels include radioisotopes, iodine (A), (B), (C), (D) and D)¹²⁵I、¹²¹I) Carbon (C)¹⁴C) Sulfur (S), (S)³⁵S), tritium (³H) Indium (I) and (II)¹¹²In) and technetium (99mTc), fluorescent labels such as fluorescein and rhodamine, and biotin. The immunoenzymatic interaction can be visualized using different enzymes such as peroxidase, alkaline phosphatase or different chromogens such as DAB, AEC or Fast Red. The labeled antibody or antibody fragment will preferentially aggregate at the location of the cell containing the biomarker. The labeled antibody or variant thereof, e.g., an antibody fragment, can then be detected using known techniques.

Antibodies include any antibody, whether natural or synthetic, full length or fragments thereof, monoclonal or polyclonal, that binds sufficiently strongly and specifically to the biomarker to be detected. The antibody may have up to about 10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11M and 10^-12K of M_d. The phrase "specifically binds" refers to, for example, the binding of an antibody to an epitope or antigen or antigenic determinant in a manner that allows for the use ofA second preparation of the same or similar epitope, antigen or antigenic determinant displaces or competes for the binding.

Antibodies and derivatives thereof that may be used include polyclonal or monoclonal antibodies, synthetic and engineered antibodies, chimeric, human, humanized, primatized (CDR-grafted), veneered (veneered) or single chain antibodies, phase produced antibodies (e.g., from a phage display library), and functional binding fragments of antibodies. For example, antibody fragments capable of binding a biomarker, or portion thereof, including but not limited to Fv, Fab ', and F (ab') 2 fragments may be used. Such fragments may be produced by enzymatic cleavage or by recombinant techniques

In certain non-limiting embodiments, agents other than antibodies that specifically bind to polypeptides, such as peptides, are used. The specifically bound peptides can be identified by any method known in the art, such as a peptide phage display library. Typically, one reagent capable of detecting the biomarker polypeptide may be used, thereby detecting the presence of the biomarker and/or quantifying.

In addition, biomarkers can be detected using mass spectrometry such as MALDI/TOF (time of flight), SELDI/TOF, liquid chromatography-mass spectrometry (LC-MS), gas chromatography-mass spectrometry (GC-MS), high performance liquid chromatography-mass spectrometry (HPLC-MS), capillary electrophoresis-mass spectrometry, nuclear magnetic resonance spectroscopy, or tandem mass spectrometry (e.g., MS/MS, ESI-MS/MS, etc.).

Mass spectrometry Methods are well known in the art and have been used to quantify and/or identify biomolecules such as proteins (see, e.g., Li et al (2000) Tibtech 18: 151-. Furthermore, mass spectrometry techniques have been developed which allow at least partial de novo sequencing of isolated proteins. Chait et al, Science 262: 89-92 (1993); keough et al, proc.natl.acad.sci.usa.96: 7131-6 (1999); for review see Bergman, EXS 88: 133-44(2000).

Detecting the presence of a biomarker or other substance typically includes detecting signal intensity. This may in turn reflect the quantity and nature of the polypeptide bound to the substrate. For example, in certain embodiments, the signal intensities of the peaks from the spectra of the first and second samples may be compared (e.g., visually or by computer analysis) to determine the relative amounts of specific biomarkers. Software programs such as the biomerker Wizard program (cipergen Biosystems, inc., Fremont, Calif.) may be used to assist in the analysis of mass spectra.

Other methods for determining the expression of nucleic acid and/or protein biomarkers in a sample are described, for example, in U.S. patent nos. 6,271,002; U.S. patent nos. 6,218,122; U.S. patent nos. 6,218,114; and U.S. patent No. 6,004,755; and Wang et al, j.clin.oncol., 22 (9): 1564-1671 (2004); and Schena et al, Science, 270: 467-; all of which are incorporated herein by reference in their entirety.

Reagent kit

In certain non-limiting embodiments, the invention provides a kit for predicting the sensitivity of a colorectal cancer patient to treatment with oxaliplatin, comprising reagents for detecting the biomarkers set forth in the preceding section. The kit further comprises instructions or support materials describing the use of the kit to determine whether oxaliplatin is likely to produce an anti-cancer effect in colorectal cancer, and/or a website or publication describing the same.

Types of kits include, but are not limited to, packaged biomarker-specific probes and primer sets (e.g., TaqMan probe/primer sets), arrays/microarrays, biomarker-specific antibodies, biomarker-specific beads, which further comprise one or more probes, primers, or other reagents for detecting one or more biomarkers of the invention.

In certain non-limiting embodiments, the present invention provides kits for predicting the sensitivity of a colorectal cancer patient to treatment with oxaliplatin, comprising means for detecting the presence of a nucleic acid biomarker.

In a specific non-limiting embodiment, the kit can comprise a pair of oligonucleotide primers suitable for Polymerase Chain Reaction (PCR) or nucleic acid sequencing for detecting the nucleic acid biomarker to be identified. A pair of primers may comprise nucleotide sequences complementary to the biomarkers described above and of sufficient length to selectively hybridize to the biomarkers. Alternatively, complementary nucleotides can selectively hybridize to specific regions sufficiently close to the 5 'and/or 3' positions of the biomarker for PCR and/or sequencing. Multiple biomarker-specific primers may be included in the kit to detect more than one biomarker simultaneously. The kit may further comprise one or more polymerases, reverse transcriptases, and nucleotide bases, wherein the nucleotide bases may also be detectably labeled.

In certain non-limiting embodiments, the length of the primer may be at least about 10 nucleotides or at least about 15 nucleotides or at least about 20 nucleotides, and/or up to about 200 nucleotides or up to about 150 nucleotides or up to about 100 nucleotides or up to about 75 nucleotides or up to about 50 nucleotides.

In another non-limiting embodiment, the oligonucleotide primers can be immobilized on a solid surface, substrate, or support, such as on a nucleic acid microarray, wherein the location at which each oligonucleotide primer binds to the solid surface or support is known and identifiable. The oligonucleotides may be immobilized to a substrate, such as glass, plastic, paper, nylon, or other type of membrane, filter, chip, bead, or any other suitable solid support. The polynucleotide may be synthesized directly on the substrate, or synthesized separately from the substrate and then immobilized on the substrate. Arrays are prepared using known methods.

In a specific non-limiting embodiment, the kit may comprise at least one nucleic acid probe suitable for in situ hybridization or in situ fluorescence hybridization for detecting the biomarker to be identified. Such kits typically comprise one or more oligonucleotide probes specific for various biomarkers. The means for testing multiple biomarkers may optionally be contained in a single kit.

In certain embodiments, a kit can include containers (including microtiter plates suitable for use in the automated practice of the present methods), each container having one or more of the various reagents (typically in concentrated form) used in the method, including, for example, pre-fabricated microarrays, buffers, appropriate nucleoside triphosphates (e.g., dATP, dCTP, dGTP, and dTTP, or rATP, rCTP, rGTP, and UTP), reverse transcriptases, DNA polymerases, RNA polymerases, and one or more probes and primers of the invention (e.g., poly (T) or random primers of appropriate length linked to a promoter that reacts with RNA polymerase).

In a non-limiting embodiment, the present invention provides a kit for predicting the sensitivity of a colorectal cancer patient to treatment with oxaliplatin comprising means for detecting the level of a protein biomarker.

In non-limiting embodiments, the kit may comprise at least one antibody or antigen-binding fragment thereof for use in the immunoassay of the biomarker to be identified. Polyclonal and monoclonal antibodies that specifically target biomarkers can be prepared using conventional immunological techniques as are generally known to those skilled in the art. The immunodetection reagents of the kit may include a detectable label associated with or linked to a given antibody or antigen itself. Such detectable labels include, for example, chemiluminescent or fluorescent molecules (rhodamine, luciferin, green fluorescent protein, luciferase, Cy3, Cy5, or ROX), radioactive labels (R: (R) (R))³H、³⁵S、³²P、¹⁴C or¹³¹I) Or enzymes (alkaline phosphatase, horseradish peroxidase).

In another non-limiting embodiment, one or more biomarker-specific antibodies can be provided bound to a solid support, such as a column matrix, an array, or a well of a microtiter plate. Alternatively, the support may be provided as a separate element of the kit.

In certain non-limiting embodiments, when the array is used by the measurement means in the kit, the set of biomarkers described above may constitute at least 10% or at least 20% or at least 30% or at least 40% or at least 50% or at least 60% or at least 70% or at least 80% of the species of biomarkers present on the microarray.

In certain non-limiting embodiments, the kits of the present disclosure may comprise one or more probes, primers, antibodies, or other detection reagents for detecting the level of expression of a biomarker in a sample. For example, the kit may comprise an antibody or fragment thereof for detecting the protein expression level of a biomarker in a biological sample.

In certain non-limiting embodiments, the kits of the present disclosure may comprise one or more probes, primers, antibodies, or other detection reagents for detecting the level of C9 expression in a sample. For example, the kit may comprise an antibody or fragment thereof for detecting the protein expression level of C9 in a biological sample.

In certain non-limiting embodiments, the kits of the present disclosure may comprise one or more probes, primers, antibodies, or other detection reagents for detecting the expression level of NPTX2 in a sample. For example, the kit may comprise an antibody or fragment thereof for detecting the protein expression level of NPTX2 in a biological sample.

In certain non-limiting embodiments, the kits of the present disclosure may comprise one or more probes, primers, antibodies, or other detection reagents for detecting the expression level of BEX2 in a sample. For example, the kit may comprise an antibody or fragment thereof for detecting the protein expression level of BEX2 in a biological sample.

The kit may further comprise means for allowing a comparison between the biomarker levels in the cancer sample and the biomarker levels in the reference control sample. For example, but not limited to, the kits of the present disclosure comprise one or more probes, primers, antibodies, or other detection reagents for detecting a reference protein or mRNA, which can be used to normalize the expression levels of one or more biomarkers in a sample to allow comparison. Non-limiting examples of reference proteins such as housekeeping proteins include alpha-or beta-tubulin, actin, filaggrin, vinculin, and GADPH.

In certain non-limiting embodiments, the kit can further comprise instructions for using the kit to detect the biomarker of interest.

Medicine

The invention provides application of biomarkers in preparing a medicament for improving the sensitivity of colorectal cancer patients to oxaliplatin treatment, wherein the biomarkers comprise C9, NPTX2 and/or BEX 2.

Non-limiting examples of inhibitors of the invention include compounds, molecules, chemicals, polypeptides, proteins that inhibit and/or decrease the expression and/or activity of a biomarker. Non-limiting examples include ribozymes, antisense oligonucleotides, shRNA molecules, and siRNA molecules that specifically inhibit the expression or activity of a biomarker. One non-limiting example of a biomarker inhibitor includes an antisense, shRNA, or siRNA nucleic acid sequence that is homologous to at least a portion of the biomarker nucleic acid sequence, wherein the portion has a homology of at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 98% with respect to the biomarker nucleic acid sequence, wherein the percent homology can be determined by, for example, BLAST or FASTA software. In certain non-limiting embodiments, the complementary portion may constitute at least 10 nucleotides or at least 15 nucleotides or at least 20 nucleotides or at least 25 nucleotides or at least 30 nucleotides, and the length of the antisense nucleic acid, shRNA or siRNA molecule may be at most 15 or at most 20 or at most 25 or at most 30 or at most 35 or at most 40 or at most 45 or at most 50 or at most 75 or at most 100 nucleotides. The antisense, shRNA or siRNA molecule may comprise DNA or atypical or non-naturally occurring residues such as, but not limited to, phosphorothioate residues.

The promoter in the present invention refers to any substance that can improve the stability of the biomarker or its expression product, up-regulate the expression of the biomarker, increase the effective action time of the biomarker, or promote the transcription of the biomarker.

In certain non-limiting embodiments, the medicament further comprises pharmaceutically acceptable carriers, diluents, fillers, binders, and other excipients.

Therapeutically inert inorganic or organic carriers known to those skilled in the art include, but are not limited to, lactose, corn starch or derivatives thereof, talc, vegetable oils, waxes, fats, polyols such as polyethylene glycol, water, sucrose, ethanol, glycerol and the like, various preservatives, lubricants, dispersants, flavoring agents. Moisturizers, antioxidants, sweeteners, colorants, stabilizers, salts, buffers and the like may also be added as needed to aid in the stability of the formulation or to aid in the enhancement of the activity or its bioavailability or to produce an acceptable mouthfeel or odor in the case of oral administration, and the formulations that may be used in such medicaments may be in the form of their original compounds as such, or optionally in the form of their pharmaceutically acceptable salts.

The medicine of the present invention may be prepared into various preparation forms. Including, but not limited to, tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

The route of administration of the medicament of the present invention is not limited, and includes, but is not limited to, intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, transpleural, topical, inhalation, transmucosal, cutaneous, gastrointestinal, intraarticular, intraventricular, rectal, vaginal, intracranial, intraurethral, intrahepatic, intratumoral. In some cases, the administration may be systemic. In some cases topical administration.

The dose of the drug of the present invention is not limited as long as the desired effect is obtained, and can be appropriately determined depending on the symptoms, sex, age, and the like.

The following examples are presented to describe certain preferred embodiments of the invention and certain aspects of the invention and should not be construed as limiting the scope of the invention. The following examples are presented to further detail the embodiments of the present invention in conjunction with the attached tables and figures.

Example 1 differential expression of genes

The invention downloads GSE28702 from a GEO database, and the sample size of a GSE28702 data set is FOLFOX sensitive group: the FOLFOX resistant medicine group is 42:41.

Differential expression analysis was performed on the GSE28702 dataset using the R package "limma" (version 3.36.5) to yield 50 differentially expressed genes, with the screening criteria: pvalue <0.05, | logFC | > 0.5. The expression levels of the differentially expressed genes C9, NPTX2 and BEX2 involved in the invention are shown in Table 1 and figures 1-3, compared with the drug resistant group FOLFOX, NPTX2 is up-regulated in the sensitive group FOLFOX, and C9 and BEX2 are down-regulated in the sensitive group FOLFOX.

TABLE 1 differentially expressed genes

Gene	logFC	t	P.Value	Up/Down
					C9	-0.601	-2.190	0.031	Down
NPTX2	0.656	2.970	0.004	Up
					BEX2	-0.574	-3.540	0.001	Down

Example 2 diagnostic efficacy

The Receiver Operating Curve (ROC) was plotted using the R package "pROC" (version 1.15.0) and the AUC values, sensitivity and specificity were analyzed, the results are shown in table 2 and fig. 4-10.

TABLE 2 biomarker/biomarker combination diagnostic potency data

The results prove that the biomarkers related to the invention have good diagnostic efficacy in predicting the sensitivity of colorectal cancer patients to oxaliplatin treatment, and the diagnostic efficacy of the biomarker combination is superior to that of a single biomarker.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. Use of a reagent for detecting the level of expression of a biomarker in a sample, wherein the biomarker comprises C9, NPTX2 and/or BEX2, for the manufacture of a product for predicting the sensitivity of a colorectal cancer patient to treatment with oxaliplatin.

2. The use of claim 1, wherein the reagent for detecting the expression level of the biomarker in the sample comprises a reagent for detecting the expression level of the biomarker in the sample by polymerase chain reaction, in situ hybridization, gel electrophoresis, sequencing and sequence analysis, microarray analysis, mass spectrometry, gel analysis systems, chromatography, enzyme-linked immunosorbent assay, radioimmunoassay, enzyme immunoassay, western blot, immunoprecipitation, or immunohistochemistry.

3. The use of claim 1 or 2, wherein the reagent for detecting the expression level of the biomarker in the sample comprises a primer, a probe, a microarray, a biomarker-specific antibody, or a biomarker-specific bead.

4. A product for predicting the sensitivity of a patient with colorectal cancer to treatment with oxaliplatin, said product comprising reagents for detecting the expression level of biomarkers in a sample, said biomarkers comprising C9, NPTX2 and/or BEX 2.

5. The product of claim 4, wherein the product comprises a kit, chip, strip.

6. The product of claim 5, wherein the kit comprises a qPCR kit, an immunoblot detection kit, an immunochromatographic detection kit, a flow cytometric assay kit, an immunohistochemical detection kit, an ELISA kit, and an electrochemiluminescent detection kit.

7. A product according to claim 5 wherein said kit comprises instructions for predicting whether a colorectal cancer patient is susceptible to treatment with oxaliplatin.

8. The product of claim 4, further comprising reagents for processing the sample.

9. Use of a biomarker comprising C9, NPTX2 and/or BEX2, preferably a drug comprising a C9 inhibitor, NPTX2 promoter and/or BEX2 inhibitor, in the manufacture of a medicament for increasing the sensitivity of a patient with colorectal cancer to treatment with oxaliplatin.

10. A device for predicting the sensitivity of a colorectal cancer patient to treatment with oxaliplatin, comprising:

the biomarkers include C9, NPTX2, and/or BEX 2.