CN113832228A - Application of biomarker in prediction of sensitivity of rectal cancer to preoperative chemoradiotherapy combined with total rectal resection - Google Patents

Abstract

The invention discloses application of biomarkers in predicting sensitivity of rectal cancer to preoperative radiotherapy and chemotherapy combined with total rectal mesentery resection, wherein the biomarkers are S100P, SIGLEC5 and/or SEMA4B, and the biomarkers have good diagnosis efficiency in predicting sensitivity or responsiveness of a rectal cancer patient to surgical treatment and are high in AUC value through experiments.

Description

Application of biomarker in prediction of sensitivity of rectal cancer to preoperative chemoradiotherapy combined with total rectal resection

Technical Field

The invention belongs to the technical field of biomedicine, and particularly relates to application of biomarkers in predicting sensitivity of rectal cancer to preoperative chemoradiotherapy combined total rectal membranectomy, and more particularly to application of biomarkers S100P, SIGLEC5 and/or SEMA4B in predicting sensitivity of rectal cancer to preoperative chemoradiotherapy combined total rectal membranectomy.

Background

Rectal cancer (Rectal cancer) is one of the most common malignancies of the digestive tract, and refers to the cancer from the dentate line to the junction of the rectosigmoid colon. The morbidity is 3 rd of the malignant tumors in China, the mortality is 5 th of the malignant tumors in China, and the epidemiological characteristics of light morbidity age and high morbidity are achieved. Data show that the middle and lower colorectal cancers are the most common in China and account for more than 80% of all colorectal cancers (Zhou ZG, Wang ZQ. interpretation of guidelines for diagnosis and treatment of mid-low reactive cancer in Europe, America and Japan [ J]Chinese J Experimental Surgery,2009,4(4): 291-. The incidence of rectal cancer is still in the rising stage in our country at present, and as a malignant tumor with high incidence in recent years, the age of onset tends to be younger (Binefa G, Rodrai guez-Morata F, Teule)

et al.Colorectal cancer:from prevention to personalized medicine[J].World journal of gastroenterology:WJG,2014,20(22):6786.)。

The rectal cancer comprises two types of sporadic rectal cancer and hereditary rectal cancer, wherein the hereditary rectal cancer accounts for about 15% -20% of the total number of patients with rectal cancer. Common types of hereditary rectal cancer include hereditary nonpolyposis colorectal cancer (HNPCC) and Familial Adenomatous Polyposis (FAP); non-hereditary rectal cancer is also known as Sporadic rectal cancer (SCC), and this type of cancer accounts for a relatively large proportion of cancers. In modern society, people's eating habits with more eating and less fiber, environmental factors, genetic factors and a plurality of tumor-related factors (including oncogenes, cancer suppressor genes, mismatch repair genes and the like) participate in the pathogenesis of rectal cancer. The pathological types of rectal cancer are various, including adenocarcinoma, adenocarcinoma cancer, undifferentiated carcinoma. The types of rectal cancer include three different types, namely, tumor type, ulcer type and infiltration type, and the prognosis is different. Rectal cancer can spread and metastasize by local infiltration, lymphatic and blood metastases, etc.

Due to the special anatomical location of the rectum, early stage tumors are difficult to detect and have no obvious symptoms, and most patients are diagnosed with middle and advanced stages, wherein about 3/4 is middle and low rectal cancer. The treatment of rectal cancer is performed by radiotherapy, chemotherapy, biological treatment, traditional Chinese medicine treatment and the like except for operation, so that the treatment method is greatly improved, and the treatment mode is changed. In the existing treatment methods for rectal cancer, surgical resection is the only treatment means for radical treatment of rectal cancer, and for middle and late-stage patients, the radical treatment rate is low and the postoperative recurrence rate is high, and the recurrence rate is about 30% according to the report of related documents. On the basis of operation, the radiotherapy and chemotherapy can obviously reduce the local recurrence rate and improve the long-term survival. Therefore, the combined treatment mode of surgery combined with radiotherapy and chemotherapy has become the standard treatment mode of local middle and advanced rectal cancer. Currently, Preoperative chemoradiotherapy combined with total rectal resection (CRT) is the clinically standard treatment regimen for surgically resectable, localized mid-late rectal cancer.

Although preoperative chemoradiotherapy in combination with total rectal resection is a standard method for treating locally advanced rectal cancer patients, it is still difficult to predict which rectal cancer patients will have sensitivity to the combination treatment scheme, and predicting the sensitivity or responsiveness of the rectal cancer patients to the treatment is still a huge challenge, so that the discovery of biomarkers capable of accurately predicting the sensitivity of rectal cancer to the preoperative chemoradiotherapy in combination with total rectal resection treatment and the application of the biomarkers to the screening of the rectal cancer patients sensitive to the preoperative treatment are of great significance to the field.

Disclosure of Invention

In view of the above, the present invention aims to provide an application of the biomarker in predicting the sensitivity of rectal cancer to preoperative radiotherapy and chemotherapy combined with total rectal resection, and provide valuable biological information for the sensitivity of patients to preoperative radiotherapy and chemotherapy combined with total rectal resection.

The above object of the present invention is achieved by the following technical solutions:

in a first aspect, the invention provides the use of a reagent for detecting the level of biomarker expression in a sample in the manufacture of a product for predicting the sensitivity or responsiveness of a patient with rectal cancer to a combination of preoperative chemoradiotherapy and total rectal resection.

Further, the biomarker is S100P, SIGLEC5, and/or SEMA 4B.

The biomarkers provided by the invention comprise genes S100P, SIGLEC5 and SEMA4B, and the information of the genes S100P, SIGLEC5 and SEMA4B is respectively as follows:

the Gene S100P (S100 calcium binding protein P) has a Gene ID of 6286 in NCBI and is located in the 6-band 1 sub-band of the short arm 1 region of chromosome 4;

the Gene SIGLEC5(sialic acid binding Ig like lectin 5) has a Gene ID of 8778 in NCBI and is located on 3, 4, 1, and 1 sub-bands of the long arm 1 region of chromosome 19;

the Gene SEMA4B (semaphorin 4B) has a Gene ID of 10509 in NCBI and is located in the 6-band 1 sub-band of long arm 2 region of chromosome 15.

Further, the biomarkers include S100P, SIGLEC5, SEMA4B, S100P + SIGLE C5 combination, S100P + SEMA4B combination, SIGLEC5+ SEMA4B combination, S100P + SIGLEC5+ S EMA4B combination;

preferably, the biomarkers comprise a S100P + SIGLEC5 combination, a S100P + SEMA4B combination, a SIGLEC5+ SEMA4B combination, a S100P + SIGLEC5+ SEMA4B combination.

Further, the reagent includes a reagent for detecting the mRNA expression level of the biomarker in the sample, and a reagent for detecting the expression level of a polypeptide and/or protein encoded by the biomarker in the sample.

Further, the agent is selected from:

a probe that specifically recognizes the biomarker; or

Primers that specifically amplify the biomarkers; or

Ligands, antibodies, antibody fragments, affinity proteins that specifically bind to the biomarkers.

Further, the product comprises reagents for detecting the expression level of the biomarkers in the sample by real-time quantitative PCR, RT-PCR, immunodetection, in situ hybridization and chip;

preferably, the sample is derived from tissue.

Further, the real-time quantitative PCR as referred to in the present invention refers to a real-time quantitative polymerase chain reaction, PCR generally employs a plurality of cycles of denaturation, annealing of primer pairs to opposite strands, and primer extension to exponentially increase the copy number of a target nucleic acid sequence; RT-PCR, which is referred to herein as reverse transcription-polymerase chain reaction, uses reverse transcriptase to prepare complementary DNA (cDNA) from mRNA, and the cDNA is then amplified by PCR to produce multiple copies of the DNA.

Further, the immunoassay described in the present invention refers to a protein immunoassay, including a sandwich immunoassay, such as a sandwich ELISA, in which two antibodies recognizing different epitopes on a biomarker are used for the detection of the biomarker; radioimmunoassay (RIA), direct, indirect or contrast enzyme-linked immunosorbent assay (ELISA), Enzyme Immunoassay (EIA), Fluorescence Immunoassay (FIA), western blot, immunoprecipitation, and any particle-based immunoassay (e.g., using gold, silver or latex particles, magnetic particles, or quantum dots). The immunoassay may be performed, for example, in a microtiter plate or strip format.

Further, the in situ hybridization in the present invention refers to a process of hybridizing specifically labeled nucleic acids with known sequences as probes with nucleic acids in cells or tissue sections, thereby performing accurate quantitative localization of specific nucleic acid sequences, and the in situ hybridization can be performed on cell samples or tissue samples.

Further, the chip detection in the present invention refers to gene chip detection, also called DNA chip detection or biochip detection, which refers to fixing a large number of probe molecules on a support, hybridizing with a labeled sample, and analyzing the sequence and number of target molecules by detecting the intensity and distribution of hybridization signals.

In a second aspect of the invention, a kit is provided for predicting the sensitivity or responsiveness of a patient with rectal cancer to a preoperative chemoradiotherapy in combination with total rectal resection.

Further, the kit comprises reagents for detecting the expression level of biomarkers S100P, SIGLEC5, and/or SEMA4B in a sample;

preferably, the reagent for detecting the expression level of the biomarker in the sample comprises a reagent for detecting the expression level of mRNA of the biomarker in the sample, and a reagent for detecting the expression level of polypeptide and/or protein encoded by the biomarker in the sample.

Further, the reagent for detecting the mRNA expression level of the biomarker in the sample comprises a probe for specifically recognizing the biomarker, a primer for specifically amplifying the biomarker;

preferably, the reagent for detecting the expression level of the polypeptide and/or protein encoded by the biomarker in the sample comprises a ligand, an antibody fragment, an affinity protein that specifically binds to the biomarker.

Further, the kit comprises a qPCR kit, an ELISA kit, an immunoblotting detection kit, an immunohistochemical detection kit, an immunochromatography detection kit, a flow cytometry analysis kit and an electrochemiluminescence detection kit.

Further, the primers contained in the kit for specifically amplifying the biomarkers may be prepared by chemical synthesis, appropriately designed by referring to known information using methods well known to those skilled in the art, and prepared by chemical synthesis.

Further, the antibody specifically binding to the biomarker contained in the kit may use an antibody or a fragment thereof of any structure, size, immunoglobulin class, origin, etc., as long as it binds to the target protein.

Further, the antibody or fragment thereof described in the present invention may be monoclonal or polyclonal. An antibody fragment refers to a portion of an antibody or a peptide comprising a portion of an antibody that retains the binding activity of the antibody to an antigen. Antibody fragments may include F (ab ') 2, Fab', Fab, single chain fv (scfv), disulfide-bonded fv (dsfv) or polymers thereof, dimerized V regions (diabodies), or peptides containing CDRs. Antibodies can be obtained by methods well known to those skilled in the art. For example, mammalian cell expression vectors that retain all or part of the target protein or incorporate polynucleotides encoding them are prepared as antigens. After immunizing an animal with an antigen, immune cells are obtained from the immunized animal and myeloma cells are fused to obtain hybridomas. The antibody is then collected from the hybridoma culture. Finally, a monoclonal antibody against the marker protein can be obtained by subjecting the obtained antibody to antigen-specific purification using the marker protein or a portion thereof used as an antigen.

In a third aspect of the invention, a pharmaceutical composition for increasing the sensitivity or responsiveness of a patient with rectal cancer to a combination of preoperative chemoradiotherapy and total rectal resection is provided.

Further, the pharmaceutical composition comprises an agent that reduces the expression level of S100P, an agent that promotes the expression level of SIGLEC5, and/or an agent that reduces the expression level of SEMA 4B.

Further, the pharmaceutical composition also comprises a pharmaceutically acceptable carrier and/or an auxiliary material.

Further, in the case of pharmaceutical compositions, a safe and effective amount of the inhibitor of the present invention is administered to a human, wherein the safe and effective amount is typically at least about 100 micrograms per kilogram of body weight for oral administration. The particular dosage will also take into account factors such as the route of administration, the health of the patient, etc., which are within the skill of the skilled practitioner.

Furthermore, the medicine of the invention can be prepared into various dosage forms according to requirements. Including but not limited to: tablets, solutions, granules, patches, ointments, capsules, aerosols or suppositories for transdermal, mucosal, nasal, buccal, sublingual or oral use.

Further, the route of administration of the agents of the present invention is not limited, including but not limited to: intravenous, intraperitoneal, intraocular, intraarterial, intrapulmonary, oral, intravesicular, intramuscular, intratracheal, subcutaneous, transdermal, pleural, 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.

Further, 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.

In a fourth aspect, the invention provides the use of a biomarker for the manufacture of a pharmaceutical composition for increasing the sensitivity or responsiveness of a patient with rectal cancer to a preoperative chemoradiotherapy in combination with total rectal resection therapy.

Further, the biomarker is S100P, SIGLEC5, and/or SEMA 4B.

A fifth aspect of the invention provides the use of the biomarkers S100P, SIGLEC5, and/or SEMA4B in the construction of a computational model or a system embedded with said computational model for predicting the sensitivity or responsiveness of a patient with rectal cancer to a pre-operative chemoradiotherapy in combination with a total rectal resection treatment;

preferably, the computational model is operated by bioinformatics methods with the expression levels of biomarkers S100P, SIGLEC5, and/or SEMA4B as input variables to output the sensitivity or responsiveness of the rectal cancer patient to the treatment of preoperative chemoradiotherapy in combination with total rectal resection.

In a sixth aspect of the invention, a system is provided for predicting the sensitivity or responsiveness of a patient with rectal cancer to a combination of preoperative chemoradiotherapy and total rectal resection.

Further, the system comprises:

(1) an evaluation device: comprises a control unit and a storage unit, and is used for predicting and evaluating the sensitivity or the responsiveness of a rectal cancer patient to preoperative radiotherapy and chemotherapy combined with total rectal mesenterectomy treatment;

(2) information communication terminal apparatuses communicatively connected to each other: for providing data on the expression level of biomarkers S100P, SIGLEC5, and/or SEMA4B in a sample from a patient with rectal cancer;

preferably, the control unit of the evaluation device comprises four units:

1) a data receiving unit: for receiving data on the expression levels of the biomarkers S100P, SIGLEC5, and/or SEMA4B in the sample from the patient with rectal cancer transmitted from the information communication terminal device;

2) a discrimination value calculation unit: calculating a discrimination value based on discrimination of the expression levels of the biomarkers S100P, SIGLEC5, and/or SEMA4B received by the data receiving unit and the expression levels of the biomarkers S100P, SIGLEC5, and/or SEMA4B stored in the storage unit as explanatory variables;

3) discrimination value criterion evaluation unit: evaluating the sensitivity or responsiveness of the rectal cancer patient to preoperative chemoradiotherapy in combination with total rectal mesenterectomy treatment based on the discrimination value calculated by the discrimination value calculation unit;

4) an evaluation result transmitting unit: which transmits the result of predictive evaluation of the rectal cancer patient obtained by the discrimination value criterion evaluation unit to the information communication terminal device.

Compared with the prior art, the invention has the advantages and beneficial effects that:

the invention discovers for the first time that the genes S100P, SIGLEC5 and/or SEMA4B can be used for predicting the sensitivity or the responsiveness of a rectal cancer patient to the treatment of preoperative radiotherapy and chemotherapy combined with total rectal mesenterectomy, and proved by experiments, the genes S100P, SIGLEC5 and/or SEMA4B have better diagnostic efficacy for predicting the sensitivity or the responsiveness of a rectal cancer patient to surgical treatment, AUC values are all higher, can be used for clinically and auxiliarily evaluating the sensitivity of a rectal cancer patient to preoperative radiotherapy and chemotherapy combined total rectal mesentery resection treatment, further screening out rectal cancer groups which benefit from survival in the preoperative radiochemotherapy and the full rectal mesenterectomy combined treatment, realizing personalized treatment, providing a new strategy and thought for clinically treating rectal cancer, meanwhile, a candidate target is provided for developing a medicine for improving the treatment sensitivity of a rectal cancer patient to preoperative radiotherapy and chemotherapy combined with total rectal mesenterectomy.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

fig. 1 shows a graph of differential expression of genes S100P, SIGLEC5, SEMA4B in training sets between responders who were sensitive to preoperative chemoradiotherapy combined with total mesentery resection therapy and non-responders who were not sensitive to preoperative chemoradiotherapy combined with total mesentery resection therapy, wherein a is: S100P, panel B: SIGLEC5, panel C: SEMA 4B;

fig. 2 shows a graph of differential expression of genes S100P, SIGLEC5, SEMA4B between responders validated to be sensitive to preoperative chemoradiotherapy combined with total mesenterectomy therapy and non-responders not sensitive to preoperative chemoradiotherapy combined with total mesenterectomy therapy, wherein a is: S100P, panel B: SIGLEC5, panel C: SEMA 4B;

FIG. 3 shows ROC plots for predicting sensitivity of colorectal cancer patients to treatment with preoperative chemoradiotherapy combined with total colorectal resection using genes S100P, SIGLEC5, SEMA4B in training sets, wherein, Panel A: S100P, panel B: SIGLEC5, panel C: SEMA 4B;

FIG. 4 shows a ROC plot of the prediction of susceptibility of rectal cancer patients to treatment with preoperative chemoradiotherapy in combination with total coliform resection using the genes S100P + SIGLEC5+ SEMA4B in combination in a training set;

fig. 5 shows ROC plots for predicting sensitivity of rectal cancer patients to treatment with preoperative chemoradiotherapy in combination with total rectal membranectomy in validation set using genes S100P, SIGLEC5, SEMA4B, wherein, a plot: S100P, panel B: SIGLEC5, panel C: SEMA 4B;

FIG. 6 shows a ROC plot using genes S100P + SIGLEC5+ SEMA4B in validation sets to predict sensitivity of colorectal cancer patients to treatment with preoperative chemoradiotherapy in combination with total colorectal resection.

Detailed Description

The present invention is further illustrated by the following examples, which should be construed as being merely illustrative and not limitative of the remainder of the disclosure. As will be understood by those of ordinary skill in the art: various changes, modifications, substitutions and alterations can be made to the embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents. The following examples are examples of experimental methods not indicating specific conditions, and the detection is usually carried out according to conventional conditions or according to the conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs, and any methods and materials similar or equivalent to those described herein may be applied.

In order to further clarify the content of the present invention, some of the scientific terms referred to in the present invention are explained as follows.

In the context of the present invention, the term "biomarker", like "marker", "molecular marker", "marker molecule", "genetic marker", refers to an indicator of a patient's phenotype, in a particular embodiment of the present invention, a marker that is differentially expressed between a rectal cancer patient (responder) that is sensitive to treatment with preoperative chemoradiotherapy in combination with total mesenterectomy and a rectal cancer patient (non-responder) that is not sensitive to treatment with preoperative chemoradiotherapy in combination with total mesenterectomy, including but not limited to: genes, proteins, small molecule metabolites, carbohydrates, glycolipid-based molecules, and the like, preferably, the marker is a gene.

In the context of the present invention, the term "expression level", as well as "expression level of a biomarker", "expression level of a molecular marker", "expression level of a gene marker", "expression level of a marker molecule", refers to the level of mRNA expression of a biomarker according to the present invention in a sample, and/or the level of polypeptide and/or protein expression encoded by a biomarker according to the present invention in a sample.

In the context of the present invention, the term "sample", as well as "sample", "subject sample", "sample of a patient having rectal cancer", refers to a composition obtained or derived from a subject of interest (patient having rectal cancer) comprising cellular entities and/or other molecular entities to be characterized and/or identified, for example, on the basis of physical, biochemical, chemical and/or physiological characteristics. The sample may be obtained from blood of a subject (patient with rectal cancer) and other fluid samples of biological origin and tissue samples, such as biopsy tissue samples or tissue cultures or cells derived therefrom. The source of the tissue sample may be solid tissue, such as from a fresh, frozen and/or preserved organ or tissue sample, biopsy tissue or aspirate; blood or any blood component; a body fluid; cells from any time of pregnancy or development of the individual; or plasma. The term "sample" includes a biological sample that has been treated in any way after it has been obtained, e.g., by treatment with a reagent, stabilization, or enrichment for certain components (e.g., proteins or polynucleotides), or embedded in a semi-solid or solid matrix for sectioning purposes. Samples described in the present invention include, but are not limited to: tissue, whole blood, blood-derived cells, serum, plasma, lymph, synovial fluid, cell extracts and combinations thereof, preferably, the sample is a tissue.

In the context of the present invention, the term "primer", as well as "amplification primer", refers to a nucleic acid fragment comprising 5-100 nucleotides, preferably said primer or amplification primer comprises 15-30 nucleotides capable of initiating an enzymatic reaction (e.g., an enzymatic amplification reaction).

In the context of the present invention, the term "probe" refers to a nucleic acid sequence comprising at least 5 nucleotides, e.g.comprising 5 to 100 nucleotides, which probe is capable of hybridizing under the specified conditions with the expression product of the target gene or with the amplification product of this expression product to form a complex. The hybridization probes may also include labels for detection. Such labels include, but are not limited to, labels for fluorescent quantitative PCR or fluorescent in situ hybridization.

In the context of the present invention, the term "kit" refers to an article of manufacture (e.g., a package or container) comprising probes for specifically detecting the biomarker genes or proteins of the present invention;

in certain embodiments, the article of manufacture is marketed, distributed, or sold as a unit for performing the methods of the present invention. Such kits may comprise carrier means compartmentalized to receive, in close confinement, one or more container means (e.g., vials, tubes, etc.), each container means comprising one of the separate components to be used in the method. For example, one of the container means may comprise a probe that carries or can carry a detectable label. Such probes may be polynucleotides specific for polynucleotides of one or more genes comprising gene expression characteristics. Where the kit utilizes nucleic acid hybridization to detect a target nucleic acid, the kit can also have a container containing one or more nucleic acids for amplifying the target nucleic acid sequence and/or a container containing a reporter means, such as a biotin-binding protein, such as avidin or streptavidin, bound to a reporter molecule, such as an enzymatic, fluorescent, or radioisotope label;

a kit will generally comprise the above-described container and one or more additional containers containing commercially and user-desired materials, including buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. A label may be present on the container to indicate the particular application of the composition, and may also indicate the direction of in vivo or in vitro use, such as those described above. Other optional components of the kit include one or more buffers (e.g., blocking buffer, wash buffer, substrate buffer, etc.), other reagents (e.g., substrate chemically altered by enzymatic labeling), epitope retrieval solutions, control samples (positive and/or negative controls), control sections, and the like.

In the context of the present invention, the term "area under the working characteristic curve (AUC) of a subject" is an indicator of the performance or accuracy of a diagnostic procedure. The accuracy of a diagnostic method is best described by its Receiver Operating Characteristics (ROC). ROC plots are line graphs of all sensitivity/specificity pairs derived from continuously varying decision thresholds across the entire data range observed. In an embodiment of the present invention, the diagnostic efficacy of the biomarkers S100P, SIGLEC5, SEMA4B for predicting the sensitivity or responsiveness of the patient with rectal cancer to the treatment of the combination of preoperative chemoradiotherapy and total rectal resection can be evaluated according to the area under the working characteristic curve (AUC) of the subject, wherein if the AUC > 0.6, the biomarkers have certain diagnostic efficacy and can be used as the markers for predicting the sensitivity or responsiveness of the patient with rectal cancer to the treatment of the combination of preoperative chemoradiotherapy and total rectal resection; if AUC is greater than 0.7, the biomarker has relatively good diagnostic efficacy and can be used as a marker for predicting the sensitivity or responsiveness of a rectal cancer patient to preoperative chemoradiotherapy combined with total rectal resection; if the AUC is more than 0.8, the biomarker has better diagnostic efficacy and can be used as a marker for predicting the sensitivity or responsiveness of a rectal cancer patient to preoperative radiotherapy and chemotherapy combined with total rectal resection.

In the context of the present invention, the term "reactive" refers to a patient/subject having, suspected of having, or predisposed to having rectal cancer that shows a response to a treatment regimen combining preoperative chemoradiotherapy with total mesenterectomy. A skilled artisan, in accordance with the methods described herein, will readily determine whether a patient/subject treated with preoperative chemoradiotherapy in combination with total rectal resection exhibits a response.

In the context of the present invention, the term "sensitive" means that a patient/subject having, suspected of having, or prone to having rectal cancer shows in some way a positive response to treatment with preoperative chemoradiotherapy in combination with total mesenterectomy. One skilled in the art will readily determine whether a patient/subject treated with preoperative chemoradiotherapy in combination with total rectal resection will exhibit a response in accordance with the methods described herein.

In the context of the present invention, the detection technique for detecting the level of biomarker expression refers to detection using a variety of detection techniques known to those of ordinary skill in the art, including, but not limited to: nucleic acid sequencing technology, nucleic acid hybridization technology, nucleic acid amplification technology and immunodetection technology.

Illustrative, non-limiting examples of nucleic acid sequencing techniques include, but are not limited to: chain terminator (Sanger) sequencing and dye terminator sequencing. One of ordinary skill in the art will recognize that RNA is typically reverse transcribed into DNA prior to sequencing because it is less stable in cells and more susceptible to nuclease attack in experiments. Another illustrative, non-limiting example of a nucleic acid sequencing technique includes: next generation sequencing (deep sequencing/high throughput sequencing), a high throughput sequencing technology is a sequencing-by-synthesis technology based on unimolecular cluster, and is based on a proprietary reversible termination chemical reaction principle. Random fragments of genome DNA are attached to an optically transparent glass surface during sequencing, hundreds of millions of clusters are formed on the glass surface after the DNA fragments are extended and subjected to bridge amplification, each cluster is a monomolecular cluster with thousands of identical templates, and then four kinds of special deoxyribonucleotides with fluorescent groups are utilized to sequence the template DNA to be detected by a reversible edge-to-edge synthesis sequencing technology.

Illustrative non-limiting examples of nucleic acid hybridization techniques include, but are not limited to: in Situ Hybridization (ISH), microarrays, and Southern or Northern blots. In Situ Hybridization (ISH) is a hybridization of specific DNA or RNA sequences in a tissue section or section using a labeled complementary DNA or RNA strand as a probe (in situ) or in the entire tissue if the tissue is small enough (whole tissue embedded ISH). DNA ISH can be used to determine the structure of chromosomes. RNA ISH is used to measure and locate mRNA and other transcripts (e.g., ncRNA) within tissue sections or whole tissue embedding. Sample cells and tissues are typically treated to fix the target transcript in situ and to increase probe access. The probe is hybridized to the target sequence at high temperature, and then excess probe is washed away. The localization and quantification of base-labeled probes in tissues labeled with radiation, fluorescence or antigens is performed using autoradiography, fluorescence microscopy or immunohistochemistry, respectively. ISH can also use two or more probes labeled with radioactive or other non-radioactive labels to detect two or more transcripts simultaneously.

Illustrative non-limiting examples of nucleic acid amplification techniques include, but are not limited to: polymerase Chain Reaction (PCR), reverse transcription polymerase chain reaction (RT-PCR), Transcription Mediated Amplification (TMA), Ligase Chain Reaction (LCR), Strand Displacement Amplification (SDA), and Nucleic Acid Sequence Based Amplification (NASBA). One of ordinary skill in the art will recognize that certain amplification techniques (e.g., PCR) require reverse transcription of RNA into DNA prior to amplification (e.g., RT-PCR), while other amplification techniques directly amplify RNA (e.g., TMA and NASBA).

Illustrative non-limiting examples of immunodetection techniques include, but are not limited to: assays performed at the protein level by Western blotting and ELISA-based detection, using immunoagglutination, immunoprecipitation (e.g. immunodiffusion, immunoelectrophoresis, immunoadhesion), Western blotting techniques (e.g. (in situ) immunocytochemistry, affinity chromatography, enzyme immunoassays), and the like. The amount of purified polypeptide in solution can also be determined by physical means, such as photometry. Methods for quantifying a particular polypeptide in a mixture typically rely on specific binding, e.g., of an antibody. Specific detection and quantification methods that exploit antibody specificity include immunoassay methods. Example 1 Gene Screen associated with sensitivity of rectal cancer to treatment with preoperative chemoradiotherapy in combination with total rectal resection

1. Data source

The data used in the study are all from the Gene Expression Omnibus database (GEO database), and related searches are carried out in the GEO database by taking 'Rectal cancer', 'preferential chemical therapy (CRT) and preliminary medical retrieval' as key words, after the study on the level of excluding cell lines or animals and the study of a single sample, two sets of GEO data sets are included in the study of the invention, namely GSE35452 and GS933E 75 respectively;

the GSE35452 dataset was from the GPL570 platform and comprised rectal cancer tissue samples from 46 patients with rectal cancer, 22 of whom were non-responders (NR) who were not responsive to preoperative chemoradiotherapy combined with total rectal resection therapy, and 24 of whom were responders (reponders, R) who were sensitive to preoperative chemoradiotherapy combined with total rectal resection;

the GSE93375 dataset contained rectal cancer tissue samples from 42 patients with rectal cancer, 25 of whom were non-responders (NR) who were not sensitive to preoperative chemoradiotherapy combined with total rectal resection, and 17 of whom were responders (responders, R) who were sensitive to preoperative chemoradiotherapy combined with total rectal resection;

wherein, Mandel classification is adopted to classify responders and non-responders, and the specific classification standard of the Mandel classification is as follows: TRG1 (pathologically complete response, pCR), TRG2 (scattered in tumor cells), TRG3 (partial response, mainly fibrosis), TRG4 (partial response, mainly tumor cells) and TRG5 (no regressive changes), TRG1 and TRG2 were classified as responders (R), i.e. patients with rectal cancer that were susceptible to treatment with preoperative chemoradiotherapy in combination with total rectal resection, whereas TRG3, TRG4 and TRG5 were classified as non-responders (NR), i.e. patients with rectal cancer that were not susceptible to treatment with preoperative chemoradiotherapy in combination with total rectal resection.

2. Data packet

Taking the data set GSE35452 downloaded from the GEO database as a training set, wherein the sample size is NR: r is 22: 24;

taking the data set GSE93375 downloaded from the GEO database as a verification set, the sample size is NR: r ═ 25: 17.

3. data pre-processing

The raw data of the training set and the verification set downloaded from the GEO database are standardized. The gene expression profile of the gene expression matrix files in the downloaded data sets GSE35452 and GSE93375 is annotated by adopting annotation files, gene probes are converted into gene symbols, wherein the multiple probes correspond to the same gene, and the average value is taken as the expression quantity of the gene.

4. Differential expression analysis

Performing differential expression analysis on the preprocessed data in the data sets GSE35452 and GSE93375 by using a "limma" package in an R language, wherein the screening criteria of the differential expression genes are as follows: value < 0.05.

5. Results of the experiment

The results show that 915 differentially expressed genes obtained by screening in the training set, 646 differentially expressed genes obtained by screening in the verification set and 13 differentially expressed genes which are shared by the two data sets and have consistent expression are obtained, wherein the differentially expressed genes S100P, SIGLEC5 and SEMA4B obtained by screening are shown in fig. 1A, fig. 1B and fig. 1C in the training set, the differentially expressed genes S100P and SEMA4B in the verification set are shown in fig. 2A, fig. 2B and fig. 2C, the differentially expressed genes S100P and SEMA4B in the patients sensitive to the preoperative chemoradiotherapy and total rectal mesenterectomy combined treatment are down-regulated, the differentially expressed genes SIGLEC5 in the patients sensitive to the preoperative chemoradiotherapy and total rectal mesenterectomy combined treatment are up-regulated, and the differences have statistical significance (P < 0.05).

Example 2 verification of the diagnostic efficacy of genes S100P, SIGLEC5, and SEMA4B for predicting the sensitivity of patients with rectal cancer to preoperative chemoradiotherapy in combination with total rectal resection

1. Experimental methods

Performing Receiver Operating Characteristic (ROC) analysis on the genes S100P, SIGLEC5, SEMA4B selected in example 1 and differentially expressed between responders and non-responders, using the R package "pROC" (version 1.15.0), calculating the area under the operating characteristic curve (AUC) of the subject to evaluate the accuracy of the genes S100P, SIGLEC5, SEMA4B, S100P + SIGLEC5, S100P + SEMA4B, SIGLEC5+ SEMA4B, S100P + SIGLEC5+ SEMA4B in training and validation sets, respectively, for predicting the sensitivity of rectal cancer patients to preoperative chemoradiotherapy combined with total rectal resection, wherein the AUC values range from 0 to 1;

wherein, when judging the diagnosis efficacy of the individual genes (S100P, SIGLEC5 and SEMA4B) in the training set and the verification set for predicting the treatment sensitivity of the rectal cancer patients to the preoperative chemoradiotherapy and the combination of the colorectal resection, the expression quantity of the individual genes is directly adopted for analysis, the level corresponding to the point with the maximum Youden index is selected as the cutoff value, and the genes with the AUC of 0.5< AUC <0.8 are used for the combined analysis;

when the diagnosis efficiency of the gene combination (S100P + SIGLEC5, S100P + SEMA4B, SIGLEC5+ SEMA4B, S100P + SIGLEC5+ SEMA4B) on the prediction of the sensitivity of the rectal cancer patient to the preoperative chemoradiotherapy combined total rectal membranous resection is judged in a training set and a verification set, Logitics regression analysis is carried out on the expression level of each gene, the probability that each rectal cancer individual is sensitive to the preoperative chemoradiotherapy combined total rectal membranous resection is calculated through a fitted regression curve, different probability division threshold values are determined, and the accuracy, the specificity, the sensitivity and the like of the gene combination for predicting the sensitivity of the rectal cancer patient to the preoperative chemoradiotherapy combined total rectal membranous resection are calculated according to the determined probability division threshold values.

2. Results of the experiment

The results show that the diagnostic efficacy of the gene combination of S100P + SIGLEC5, S100P + SEMA4B, SIGLEC5+ SEMA4B, S100P + SIGLEC5+ SEMA4B for predicting the sensitivity of a rectal cancer patient to the treatment of the preoperative chemoradiotherapy combined with total rectal resection is significantly better than that of the single gene of S100P, SIGLEC5, SEMA4B, and that the AUC values of the genes for predicting the sensitivity of a rectal cancer patient to the treatment of the preoperative chemoradiotherapy combined with total rectal resection in the training set or in the validation set are higher (see table 1, table 2, fig. 3A, fig. 3B, fig. 3C, fig. 4, fig. 5A, fig. 5B, fig. 5C, fig. 6), indicating that the genes of S100P, SIGLEC5, and/or SEMA4B can be used as biomarkers for predicting the sensitivity of a rectal cancer patient to the preoperative chemoradiotherapy combined with total rectal resection.

TABLE 1 AUC values of genes in training set for prediction of susceptibility of rectal cancer patients to preoperative chemoradiotherapy combined with total rectal mesenterectomy treatment

Gene	AUC value
		S100P	0.67992
SIGLEC5	0.65152
		SEMA4B	0.75379
S100P+SIGLEC5	0.68939
		S100P+SEMA4B	0.76894
SIGLEC5+SEMA4B	0.79924
		S100P+SIGLEC5+SEMA4B	0.81629

TABLE 2 Gene AUC values in validation set for predicting susceptibility of rectal cancer patients to preoperative chemoradiotherapy combined with total rectal mesenterectomy treatment

Gene	AUC value
		S100P	0.70588
SIGLEC5	0.70588
		SEMA4B	0.68706
S100P+SIGLEC5	0.83059
		S100P+SEMA4B	0.74118
SIGLEC5+SEMA4B	0.71529
		S100P+SIGLEC5+SEMA4B	0.85177

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall into the protection scope of the claims of the present invention.

Claims (10)

1. Use of a reagent for detecting the level of expression of a biomarker in a sample, wherein the biomarker is S100P, SIGLEC5, and/or SEMA4B, in the manufacture of a product for predicting the sensitivity or responsiveness of a patient with rectal cancer to a combination of preoperative chemoradiotherapy and total rectal mesenterectomy therapy.

2. The use of claim 1, wherein the reagents comprise reagents for measuring the level of mRNA expression of the biomarker in the sample, and reagents for measuring the level of expression of polypeptides and/or proteins encoded by the biomarker in the sample.

3. Use according to claim 1 or 2, wherein said agent is selected from:

a probe that specifically recognizes the biomarker; or

Primers that specifically amplify the biomarkers; or

Ligands, antibodies, antibody fragments, affinity proteins that specifically bind to the biomarkers.

4. The use of claim 1, wherein the product comprises reagents for detecting the level of expression of the biomarker in a sample by real-time quantitative PCR, RT-PCR, immunodetection, in situ hybridization, chip;

preferably, the sample is derived from tissue.

5. A kit for predicting the sensitivity or responsiveness of a patient with rectal cancer to treatment with preoperative chemoradiotherapy in combination with total rectal resection, comprising reagents for detecting the expression level of a biomarker in a sample, said biomarker being S100P, SIGLEC5, and/or SEMA 4B;

preferably, the reagent for detecting the expression level of the biomarker in the sample comprises a reagent for detecting the expression level of mRNA of the biomarker in the sample, and a reagent for detecting the expression level of polypeptide and/or protein encoded by the biomarker in the sample.

6. The kit of claim 5, wherein the reagents for detecting the mRNA expression level of a biomarker in the sample comprise a probe that specifically recognizes the biomarker, a primer that specifically amplifies the biomarker;

preferably, the reagent for detecting the expression level of the polypeptide and/or protein encoded by the biomarker in the sample comprises a ligand, an antibody fragment, an affinity protein that specifically binds to the biomarker.

7. A pharmaceutical composition for increasing the sensitivity or responsiveness of a patient with rectal cancer to treatment with preoperative chemoradiotherapy in combination with total rectal resection, comprising an agent that decreases the expression level of S100P, an agent that increases the expression level of SIGLEC5, and/or an agent that decreases the expression level of SEMA 4B.

8. Use of a biomarker for the manufacture of a pharmaceutical composition for increasing the sensitivity or responsiveness of a patient with rectal cancer to treatment with preoperative chemoradiotherapy in combination with total rectal resection, wherein the biomarker is S100P, SIGLEC5, and/or SEMA 4B.

9. Use of biomarkers S100P, SIGLEC5, and/or SEMA4B in the construction of a computational model or a system embedded with said computational model for predicting the sensitivity or responsiveness of a patient with rectal cancer to a preoperative chemoradiotherapy in combination with total mesenterectomy treatment;

preferably, the computational model is operated by bioinformatics methods with the expression levels of biomarkers S100P, SIGLEC5, and/or SEMA4B as input variables to output the sensitivity or responsiveness of the rectal cancer patient to the treatment of preoperative chemoradiotherapy in combination with total rectal resection.

10. A system for predicting the sensitivity or responsiveness of a patient with rectal cancer to a pre-operative chemoradiotherapy in combination with a total rectal resection treatment, the system comprising:

(1) an evaluation device: comprises a control unit and a storage unit, and is used for predicting and evaluating the sensitivity or the responsiveness of a rectal cancer patient to preoperative radiotherapy and chemotherapy combined with total rectal mesenterectomy treatment;

(2) information communication terminal apparatuses communicatively connected to each other: for providing data on the expression level of biomarkers S100P, SIGLEC5, and/or SEMA4B in a sample from a patient with rectal cancer;

preferably, the control unit of the evaluation device comprises four units:

1) a data receiving unit: for receiving data on the expression levels of the biomarkers S100P, SIGLEC5, and/or SEMA4B in the sample from the patient with rectal cancer transmitted from the information communication terminal device;

2) a discrimination value calculation unit: calculating a discrimination value based on discrimination of the expression levels of the biomarkers S100P, SIGLEC5, and/or SEMA4B received by the data receiving unit and the expression levels of the biomarkers S100P, SIGLEC5, and/or SEMA4B stored in the storage unit as explanatory variables;

3) discrimination value criterion evaluation unit: evaluating the sensitivity or responsiveness of the rectal cancer patient to preoperative chemoradiotherapy in combination with total rectal mesenterectomy treatment based on the discrimination value calculated by the discrimination value calculation unit;

4) an evaluation result transmitting unit: which transmits the result of predictive evaluation of the rectal cancer patient obtained by the discrimination value criterion evaluation unit to the information communication terminal device.

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