WO2022016425A1 - 一种预测癌症预后风险的方法 - Google Patents

一种预测癌症预后风险的方法 Download PDF

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WO2022016425A1
WO2022016425A1 PCT/CN2020/103579 CN2020103579W WO2022016425A1 WO 2022016425 A1 WO2022016425 A1 WO 2022016425A1 CN 2020103579 W CN2020103579 W CN 2020103579W WO 2022016425 A1 WO2022016425 A1 WO 2022016425A1
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gene
cancer
expression level
ch25h
ch25h gene
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PCT/CN2020/103579
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English (en)
French (fr)
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张道允
巩子英
孙永华
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嘉兴允英医学检验有限公司
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Priority to PCT/CN2020/103579 priority Critical patent/WO2022016425A1/zh
Priority to CN202080002636.3A priority patent/CN112119168B/zh
Publication of WO2022016425A1 publication Critical patent/WO2022016425A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/475—Quinolines; Isoquinolines having an indole ring, e.g. yohimbine, reserpine, strychnine, vinblastine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6851—Quantitative amplification
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16H—HEALTHCARE INFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR THE HANDLING OR PROCESSING OF MEDICAL OR HEALTHCARE DATA
    • G16H50/00—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics
    • G16H50/50—ICT specially adapted for medical diagnosis, medical simulation or medical data mining; ICT specially adapted for detecting, monitoring or modelling epidemics or pandemics for simulation or modelling of medical disorders
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present application relates to the field of biotechnology, in particular to a method for predicting the risk of cancer prognosis.
  • Tumor metastasis refers to the process in which tumor cells break away from the original growth site, transport through various pathways, continue to proliferate and grow in organs/tissues far away from the original site in the body, and form tumors of the same nature (metastases).
  • the main ways of tumor metastasis include lymphatic metastasis, hematogenous metastasis and implantation metastasis.
  • tumor metastasis For tumor metastasis, it can be detected clinically by imaging, tumor markers, biopsy biopsy, and cytological or molecular perspectives such as circulating tumor cells (CTC) and circulating tumor DNA (ctDNA) on a regular basis. Biopsy puncture can further confirm whether the tumor has metastasized or recurred, but it is traumatic to the patient and there are certain surgical risks. Exploring the risk of tumor recurrence and metastasis from the perspective of tumor molecular biology is currently a hot research topic. Tumor-specific genomic alterations can be found in cell-free DNA (cfDNA) in patient blood samples. This method can be used as a supplement to biopsy for real-time molecular monitoring. Treat, detect relapse, and track drug resistance.
  • CTC circulating tumor cells
  • ctDNA circulating tumor DNA
  • CTC circulating tumor cells
  • ctDNA circulating tumor DNA
  • a treatment plan can be selected based on cancer prognosis analysis, the frequency of reexamination of patients after treatment, or whether to take other methods to prevent cancer recurrence and metastasis can be determined. Therefore, there is a need to provide a low-cost method that can effectively predict the prognostic risk of cancer.
  • a use of a reagent for detecting the expression level of CH25H gene in the preparation of a kit for predicting cancer prognosis may include detecting the normalized expression level of the CH25H gene in a biological sample obtained from the target subject using the kit.
  • the predicting may also include determining a target parameter based on the normalized expression level of the CH25H gene in the biological sample.
  • the predicting may also include comparing the target parameter to one or more reference thresholds to obtain a comparison result.
  • the prediction may also include determining the likelihood of cancer recurrence or metastasis in the target subject based on the comparison.
  • the one or more reference thresholds include a first reference threshold.
  • the determining the likelihood of cancer recurrence or metastasis based on the comparison result may include determining the likelihood of cancer recurrence or metastasis in the target subject in response to the comparison result being that the target parameter is less than the first reference threshold. Sex is higher.
  • the one or more reference thresholds include a second reference threshold.
  • the predicting the possibility of cancer recurrence or metastasis based on the comparison result may include: in response to the comparison result being that the target parameter is greater than the second reference threshold, predicting the target subject's cancer recurrence or metastasis probability. Less likely.
  • the normalized expression level of the CH25H gene may be determined based on the expression level of the CH25H gene in the target subject and the expression level of an internal reference gene in the target subject.
  • the internal reference gene may include one or more of RPLO, GAPDH, ACTB, B2M, SDHA, HPRT1, ARBP, 18sRNA, 28sRNA.
  • the normalized expression level of the CH25H gene can be determined based on RNA transcripts of the CH25H gene.
  • the detecting the normalized expression level of the CH25H gene in the biological sample may include extracting the RNA of the CH25H gene and the RNA of the internal reference gene from the biological sample.
  • the detection of the normalized expression level of the CH25H gene in the biological sample may also include reverse transcription of the RNA of the CH25H gene and the RNA of the internal reference gene, respectively, to obtain the cDNA of the CH25H gene and the cDNA of the internal reference gene. cDNA.
  • the detecting the normalized expression level of the CH25H gene in the biological sample may further include performing a PCR amplification reaction for the cDNA of the CH25H gene and the cDNA of the internal reference gene.
  • the detection of the normalized expression level of the CH25H gene in the biological sample may include determining the biological sample according to the first cycle threshold of the cDNA of the CH25H gene in the PCR amplification reaction and the second cycle threshold of the cDNA of the internal reference gene. Normalized expression levels of the CH25H gene.
  • the determining a target parameter based on the normalized expression level of the CH25H gene in the biological sample may include: based on the normalized expression level of the CH25H gene in the biological sample of the target subject and normalized expression levels of the CH25H gene in biological samples from a plurality of cancer-free first reference subjects.
  • the one or more reference thresholds may be determined based on normalized expression levels of the CH25H gene in biological samples of a plurality of second reference subjects with cancer.
  • the reagent for detecting the expression level of the CH25H gene may include a specific probe for the CH25H gene.
  • the similarity between the sequence of the specific probe of the CH25H gene and the sequence shown in SEQ ID NO.: 1 can be ⁇ 95%.
  • the reagent for detecting the expression level of the CH25H gene may include specific primers for the CH25H gene.
  • the similarity between the specific primer sequence of the CH25H gene and the sequence shown in SEQ ID NO.: 2 can be ⁇ 95%; and the specific primer sequence of the CH25H gene and SEQ ID NO.: 3 The similarity of the sequences shown can be ⁇ 95%.
  • the cancer may comprise one or more of the following: melanoma, lung cancer, leukemia, stomach cancer, ovarian cancer, pancreatic cancer, breast cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer , cervical cancer or osteosarcoma.
  • the cancer can be non-small cell lung cancer, lung squamous cell carcinoma, lung adenocarcinoma, or small cell lung cancer.
  • the biological sample can be a peripheral blood sample.
  • using the kit to detect the normalized expression level of the CH25H gene in the biological sample obtained from the target subject may further comprise extracting leukocytes from the peripheral blood sample.
  • the using the kit to detect the normalized expression level of the CH25H gene in the biological sample obtained from the target subject may further include using the kit to detect the normalized expression level of the CH25H gene in the leukocytes.
  • a cancer prognosis prediction device may include an acquisition module configured to acquire test data related to the expression level of the CH25H gene in the biological sample of the target subject.
  • the apparatus may include a normalized expression level determination module configured to determine a normalized expression level of the CH25H gene in the biological sample based on the test data.
  • the apparatus may also include a prognostic analysis module.
  • the prognostic analysis module may be configured to determine a target parameter based on the normalized expression level of the CH25H gene in the biological sample.
  • the prognostic analysis module may be configured to compare the target parameter to one or more reference thresholds to obtain a comparison result.
  • the prognostic analysis module may also be configured to determine the likelihood of cancer recurrence or metastasis in the target subject based on the comparison.
  • a computer-readable storage medium stores computer instructions that, when executed by a processor, implement a cancer prognosis prediction method.
  • the cancer prognosis prediction method may include obtaining test data, the test data being correlated with the expression level of the CH25H gene in the biological sample of the target subject.
  • the cancer prognosis prediction method may include determining a normalized expression level of the CH25H gene in the biological sample based on the test data.
  • the cancer prognosis prediction method may include determining a target parameter based on the normalized expression level of the CH25H gene in the biological sample.
  • the cancer prognosis prediction method may further include comparing the target parameter to one or more reference thresholds to obtain a comparison result.
  • the cancer prognosis prediction method may further include determining the likelihood of cancer recurrence or metastasis in the target subject based on the comparison result.
  • a cancer prognosis prediction method can include obtaining test data that correlates with the expression level of the CH25H gene in the biological sample of the target subject.
  • the method may include determining a normalized expression level of the CH25H gene in the biological sample based on the test data.
  • the method can include determining a target parameter based on the normalized expression level of the CH25H gene in the biological sample.
  • the method may also include comparing the target parameter to one or more reference thresholds to obtain a comparison result.
  • the method may further include determining the likelihood of cancer recurrence or metastasis in the target subject based on the comparison.
  • a method for treating cancer or preventing recurrence and metastasis of cancer can include detecting the normalized expression level of the CH25H gene in a biological sample obtained from the subject of interest.
  • the method can include determining a target parameter based on the normalized expression level of the CH25H gene in the biological sample.
  • the method may also include comparing the target parameter to one or more reference thresholds.
  • the method may further comprise administering to the subject a composition comprising an agent that increases the expression level of the CH25H gene or that increases the activity of the CH25H protein in response to the target parameter being less than the reference threshold.
  • the agent further comprises at least one of an interferon, a Toll-like receptor 4 agonist.
  • FIG. 1 is a schematic diagram of an application scenario of a cancer prognosis risk prediction system 100 according to some embodiments of the present application;
  • FIG. 2 is a schematic diagram of the architecture of a computing device 200 according to some embodiments of the present application.
  • FIG. 3 is a block diagram of a cancer prognosis risk prediction system according to some embodiments of the present application.
  • Figure 4 is a method of predicting cancer prognosis risk according to some embodiments of the present application.
  • FIG. 5 is a flow chart for detecting the normalized expression level of CH25H gene in a biological sample according to some embodiments of the present application.
  • system means for distinguishing different components, elements, parts, parts or assemblies at different levels.
  • device means for converting signals into signals.
  • unit means for converting signals into signals.
  • module means for converting signals into signals.
  • a "subject” (may also be referred to as an "individual", “subject”) is a subject to be detected or tested.
  • the subject can be a vertebrate.
  • the vertebrate is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates) and rodents (eg, mice and rats).
  • the mammal can be a human.
  • a "target subject” refers to a subject who has cancer and has not yet received treatment (eg, chemotherapy), or has had (or currently has) cancer and has received treatment.
  • cholesterol hydroxylase -25- (cholesterol-25-hydroxylase, CH25H ) is a multi-transmembrane 31.6kDa size of the endoplasmic reticulum (endoplasmic reticulum, ER) enzymes, its main function is O 2, As an additional substrate, NADPH is a cofactor to catalyze the production of 25-hydroxycholesterol (25-hydroxycholesterol, 25HC) from cholesterol.
  • 25HC belongs to endogenous oxysterols among the many oxidation products of cholesterol, and is a soluble factor that controls sterol biosynthesis by regulating sterol regulatory element-binding protein (SREBP) and nuclear receptors. 25HC can play a role in many aspects, for example, play an important role in anti-virus, play an important role in immunity and so on.
  • primer refers to a single-stranded polynucleotide capable of hybridizing to a nucleic acid and allowing the polymerization of complementary nucleic acids, usually by providing a free 3'-OH group.
  • PCR polymerase chain reaction
  • Quantitative real-time PCR may also be referred to as qPCR, RT-PCR.
  • qPCR is a method of detecting the total amount of product after each polymerase chain reaction (PCR) cycle by fluorescent chemicals in a DNA amplification reaction. Quantitative analysis.
  • the present application proposes that the risk of metastasis or recurrence of cancer in a subject can be predicted based on the normalized gene expression level of CH25H in a biological sample of the subject. According to the risk of cancer metastasis or recurrence, measures such as interventional treatment or adjustment of the frequency of post-treatment review can be taken.
  • the invention is expected to improve the survival rate of cancer patients and has important clinical application value.
  • the target parameter of the CH25H gene when the target parameter of the CH25H gene is less than the first reference threshold, it is determined that the target subject has a high possibility of cancer recurrence or metastasis; when the target parameter of the CH25H gene is greater than the second reference threshold, the target is determined The subject's cancer was less likely to recur or metastasize.
  • TEVs tumor-derived extracellular vesicles
  • These vesicles contain a cocktail of molecules that "acclimate" healthy cells in preparation for the seeding and reproduction of cancer cells, promoting tumor growth, recurrence and metastasis. But not all healthy cells that come into contact with these vesicles are acclimated, and there are ways to prevent this acclimation process.
  • the 25-hydroxycholesterol produced by CH25H can inhibit the absorption of vesicles by normal cells, thus inhibiting tumor recurrence and metastasis.
  • FIG. 1 is a schematic diagram of an application scenario of a cancer prognosis risk prediction system 100 according to some embodiments of the present application.
  • the cancer prognostic risk prediction system 100 may include a processing device 110 , a network 120 and a memory 130 .
  • the memory 130 may store the basic information, disease history, treatment plan and other data of the target subject (eg, the target subject 140 ), and may also store the gene expression information of the target subject 140 , such as the target subject 140 . Normalized expression levels of the subject's CH25H gene and internal reference genes, as well as target parameters, etc.
  • the memory 130 may also store data such as one or more reference thresholds.
  • biological samples of the target subject may be kept in specialized storage facilities for further processing, such as RNA extraction, and the like.
  • biological sample 145 may be a tissue sample or a bodily fluid sample.
  • the biological sample 145 may be blood, such as peripheral blood.
  • the processing device 110 may be used to process and analyze relevant information to generate prognostic prediction results.
  • the processing device 110 may obtain relevant information and/or data (eg, the first reference threshold, the second reference threshold, the normalized expression level of the CH25H gene in the biological sample, etc.) from the memory 130, or may directly Obtain relevant information and/or data obtained by processing the biological sample 145 of the target subject 140 by staff or other equipment.
  • Processing device 110 may process data and/or information obtained from memory 130 .
  • the processing device 110 may be capable of determining instructions for a target parameter of the CH25H gene in the biological sample 145 of the target subject 110, for comparing the target parameter with one or more reference thresholds, and for obtaining a cancer prognosis prediction based on the comparison results result.
  • processing device 110 may be a single processor or a group of processors.
  • the processor farm may be centralized or distributed (eg, processing device 110 may be a distributed system).
  • processing device 110 may be local or remote.
  • processing device 110 may obtain information and/or data from memory 130 via network 120 .
  • processing device 110 may be implemented on a cloud platform.
  • the cloud platform may include a private cloud, a public cloud, a hybrid cloud, a community cloud, a distributed cloud, an inter-cloud, multiple clouds, etc., or any combination of the foregoing examples.
  • Processing device 110 may include a central processing unit (CPU), application specific integrated circuit (ASIC), application specific instruction processor (ASIP), graphics processing unit (GPU), physical processor (PPU), digital signal processor (DSP), field Programmable gate array (FPGA), programmable logic circuit (PLD), controller, microcontroller unit, reduced instruction set computer (RISC), microprocessor, etc. or any combination of the above.
  • CPU central processing unit
  • ASIC application specific integrated circuit
  • ASIP application specific instruction processor
  • GPU graphics processing unit
  • PPU physical processor
  • DSP digital signal processor
  • FPGA field Programmable gate array
  • PLD programmable logic circuit
  • controller microcontroller unit, reduced instruction set computer (RISC), microprocessor, etc. or any combination of the above.
  • Network 120 may provide a channel for information exchange.
  • information may be exchanged between processing device 110 and memory 130 over network 120 .
  • processing device 110 may receive data in memory 130 over network 120 .
  • information about target subject 140 and/or biological sample 145 may be transmitted to processing device 110 and/or memory 130 via network 120 .
  • information of the target subject 140 (such as the normalized expression level of the CH25H gene, etc.) may be transmitted to the processing device 110 via the network 120 .
  • network 120 may be any type of wired or wireless network.
  • network 120 may include a cable network, wireline network, fiber optic network, telecommunications network, intranet, Internet, local area network (LAN), wide area network (WAN), wireless area network (WLAN), metropolitan area network (MAN) ), Public Switched Telephone Network (PSTN), Bluetooth network, ZigBee network, Near Field Communication (NFC) network, etc. or any combination of the above.
  • LAN local area network
  • WAN wide area network
  • WLAN wireless area network
  • MAN metropolitan area network
  • PSTN Public Switched Telephone Network
  • PSTN Public Switched Telephone Network
  • Bluetooth network ZigBee network
  • NFC Near Field Communication
  • Memory 130 may be used to store data and/or instruction sets. In some embodiments, memory 130 may store data obtained from processing device 110 . In some embodiments, memory 130 may store information and/or instructions for execution or use by processing device 110 to perform the example methods described herein. In some embodiments, memory 130 may store gene expression data. In some embodiments, memory 130 may include mass storage, removable memory, volatile read-write memory, read-only memory (ROM), the like, or any combination thereof. In some embodiments, memory 130 may be implemented on a cloud platform. For example only, cloud platforms may include private clouds, public clouds, hybrid clouds, community clouds, distributed clouds, intermediate clouds, multi-clouds, etc., or any combination thereof. In some embodiments, memory 130 may be part of processing device 110 .
  • target subject 140 may be a subject who has had cancer and has received treatment. Treatment may include, but is not limited to, tumor resection surgery, chemotherapy, local radiation therapy, biological immunotherapy, targeted therapy, drug therapy, and the like. In some embodiments, target subject 110 may have one or more cancers.
  • the cancer can include breast cancer, triple negative breast cancer, metaplastic breast cancer (MpBC), leukemia, gastric cancer, ovarian cancer, prostate cancer, bladder cancer, rectal cancer, liver cancer, cervical cancer, lung squamous cell carcinoma, Lung adenocarcinoma, head and neck squamous cell carcinoma (HNSCC), human papillomavirus (HPV) positive HNSCC, HPV negative/TP53 mutant HNSCC, metastatic HNSCC, oropharyngeal HNSCC, non-oropharyngeal HNSCC, melanoma, lumen A breast cancer, luminal B breast cancer, HER2+ breast cancer, microsatellite instability high (MSI-H) colorectal cancer, microsatellite stable colorectal cancer (MSS), non-small cell lung cancer (NSCLC), small cell lung cancer , chordoma, or adrenocortical carcinoma.
  • MpBC metaplastic breast cancer
  • MpBC metaplastic breast cancer
  • the cancer can be breast cancer, colon cancer, lung cancer, pancreatic cancer, prostate, Merkel cells, ovary, liver, endometrium, bladder, kidney or cancer of unknown primary (CUP).
  • Sarcomas can be liposarcoma, osteosarcoma, extraskeletal myxoid chondrosarcoma, or uterine sarcoma.
  • the sarcoma comprises alpha alveolar soft tissue sarcoma (ASPS), angiosarcoma, breast angiosarcoma, chondrosarcoma, chordoma, clear cell sarcoma, hyperplastic small round cell tumor (DSRCT), epithelioid cell hemangioendothelioma ( EHE), epithelioid sarcoma, endometrial stromal sarcoma (ESS), Ewing sarcoma, fibromatosis, fibrosarcoma, giant cell tumor, leiomyosarcoma (LMS), uterine LMS, liposarcoma, malignant fibrous histiocytoma (MFH) /UPS), malignant peripheral nerve sheath tumor (MPNST), osteosarcoma, perivascular epithelioid cell tumor (PEComa), rhabdomyosarcoma, solitary fibroma (SFT), synovial s
  • APS
  • biological sample 145 can be used to reflect information about the CH25H gene in target subject 140 .
  • the biological sample may comprise a tissue sample or a bodily fluid sample.
  • the bodily fluid sample may include one or a combination of peripheral blood, tissue fluid, lymph or cerebrospinal fluid samples.
  • the bodily fluid sample may include a mammalian blood sample, tissue fluid sample, or lymphatic fluid sample.
  • the mammal can be a human.
  • the biological sample may be peripheral blood, such as venous blood.
  • information about the target subject 140 and/or biological sample 145 may be transmitted to one or more of the cancer prognostic risk prediction system 100 by manual (eg, manual input by staff) or machine (eg, equipment, etc.) components (eg, processing device 110, memory 130).
  • FIG. 2 is a schematic diagram of the architecture of a computing device 200 according to some embodiments of the present application.
  • computing device 200 may include processor 210 , memory 220 , input/output interface 230 , and communication port 240 .
  • Processing device 110 and/or memory 130 may be implemented on the computing device 200 .
  • a cancer prognosis prediction apparatus may be implemented in the computing device 200 .
  • processing device 110 may be implemented on computing device 200 and configured to perform the functions of processing device 110 herein.
  • the processing device 110 may detect normalized expression levels of the CH25H gene in a biological sample obtained from a target subject.
  • the processing device 110 may determine the target parameter based on the normalized expression level of the CH25H gene in the biological sample. As another example, the processing device 110 may compare the target parameter to one or more reference thresholds to obtain a comparison, and based on the comparison, determine the likelihood of cancer recurrence or metastasis in the target subject.
  • the processor 210 may execute computational instructions (program code) and perform the functions of the processing device 110 described herein.
  • Computing instructions may include programs, objects, components, data structures, procedures, modules, and functions (functions refer to the specific functions described in this application).
  • the processor 210 may process instructions in the cancer prognosis risk prediction system 100 to predict the effect of cancer prognosis.
  • processor 210 may include a microcontroller, microprocessor, reduced instruction set computer (RISC), application specific integrated circuit (ASIC), application specific instruction set processor (ASIP), central processing unit (CPU) , Graphics Processing Units (GPUs), Physical Processing Units (PPUs), Microcontroller Units, Digital Signal Processors (DSPs), Field Programmable Gate Arrays (FPGAs), Advanced RISC Machines (ARMs), Programmable Logic Devices, and Any circuits, processors, etc., or any combination thereof, that perform one or more functions.
  • RISC reduced instruction set computer
  • ASIC application specific integrated circuit
  • ASIP application specific instruction set processor
  • CPU Central processing unit
  • GPUs Graphics Processing Units
  • PPUs Physical Processing Units
  • Microcontroller Units Microcontroller Units
  • DSPs Digital Signal Processors
  • FPGAs Field Programmable Gate Arrays
  • ARMs Advanced RISC Machines
  • Programmable Logic Devices and Any circuits, processors, etc., or any combination thereof
  • Memory 220 may store data/information obtained from any component of cancer prognostic risk prediction system 100 .
  • memory 220 may include mass storage, removable storage, volatile read and write memory, read only memory (ROM), etc., or any combination thereof.
  • Exemplary mass storage may include magnetic disks, optical disks, solid state drives, and the like.
  • Removable storage may include flash drives, floppy disks, optical disks, memory cards, U disks, compact disks, removable hard disks, and the like.
  • Volatile read and write memory may include random access memory (RAM).
  • RAM may include dynamic RAM (DRAM), double rate synchronous dynamic RAM (DDRSDRAM), static RAM (SRAM), thyristor RAM (T-RAM), zero capacitance (Z-RAM), and the like.
  • DRAM dynamic RAM
  • DDRSDRAM double rate synchronous dynamic RAM
  • SRAM static RAM
  • T-RAM thyristor RAM
  • Z-RAM zero capacitance
  • ROM may include mask ROM (MROM), programmable ROM (PROM), erasable programmable ROM (PEROM), electrically erasable programmable ROM (EEPROM), compact disk ROM (CD-ROM), and digital versatile disk ROM Wait.
  • MROM mask ROM
  • PROM programmable ROM
  • PEROM erasable programmable ROM
  • EEPROM electrically erasable programmable ROM
  • CD-ROM compact disk ROM
  • digital versatile disk ROM Wait digital versatile disk ROM Wait.
  • the input/output interface 230 may be used to input or output signals, data or information.
  • input/output interface 230 may be used to enable user (eg, target subject 110 , user of cancer prognosis risk prediction system 100 , etc.) to interact with processing device 110 .
  • the user may input characteristic information of the tumor patient through the input/output interface 230 .
  • input/output interface 230 may include input devices and output devices. Exemplary input devices may include a keyboard, mouse, touch screen, microphone, etc., or any combination thereof. Exemplary output devices may include display devices, speakers, printers, projectors, etc., or any combination thereof. Exemplary display devices may include liquid crystal displays (LCDs), light emitting diode (LED) based displays, flat panel displays, curved displays, television equipment, cathode ray tubes (CRTs), etc., or any combination thereof.
  • LCDs liquid crystal displays
  • LED light emitting diode
  • CRTs cathode ray tubes
  • Communication port 240 may connect to network 120 for data communication. Connections can be wired, wireless, or a combination of both. Wired connections may include electrical cables, fiber optic cables, or telephone lines, among others, or any combination thereof. Wireless connections may include Bluetooth, WiFi, WiMax, WLAN, ZigBee, mobile networks (eg, 3G, 4G or 5G, etc.), etc., or any combination thereof.
  • the communication port 240 may be a standardized port such as RS232, RS485, or the like. In some embodiments, communication port 240 may be a specially designed port.
  • the cancer prognosis risk prediction system may include an acquisition module 310 , a normalized expression level determination module 320 , and a prognosis analysis module 330 .
  • the acquisition module 310 may be used to acquire test data, the test data being related to the expression level of the CH25H gene in the biological sample of the target subject.
  • Blood sample test data may comprise data of the target subject, RNA gene CH25H data (e.g., concentration, nucleotide sequence, etc.), cDNA data (e.g., concentration, nucleotide sequence, CT values, 2 ⁇ CT, ⁇ CT CH25H gene ), RNA and cDNA data reference gene of data values, 2 d ⁇ CT the like (e.g., concentration, nucleotide sequence, CT values, 2 ⁇ CT, ⁇ CT value, 2 d ⁇ CT etc.) and the like.
  • the normalized expression level module 320 determines the normalized expression level of the CH25H gene in the biological sample based on the test data.
  • the normalized expression level of the CH25H gene is determined based on the expression level of the CH25H gene in the target subject and the expression level of the internal reference gene in the target subject.
  • the internal reference gene includes one or more of RPLO, GAPDH, ACTB, B2M, SDHA, HPRT1, ARBP, 18sRNA, 28sRNA and the like.
  • the normalized expression level of the CH25H gene is determined based on the RNA transcript of the CH25H gene.
  • the RNA of the CH25H gene and the RNA of the internal reference gene can be extracted from the biological sample.
  • the biological sample can be peripheral blood.
  • leukocytes can be extracted from peripheral blood, and then the RNA of the CH25H gene and the RNA of the internal reference gene can be extracted.
  • the RNA of the CH25H gene and the RNA of the internal reference gene can be respectively reverse transcribed to obtain the cDNA of the CH25H gene and the cDNA of the internal reference gene.
  • a PCR amplification reaction can be performed for the cDNA of the CH25H gene and the cDNA of the internal reference gene.
  • the difference between the first cycle threshold and the second cycle threshold can be determined.
  • the normalized expression level of the CH25H gene in the biological sample can be represented by the difference between the first cycle threshold and the second cycle threshold.
  • the prognostic analysis module 330 can determine the target parameter based on the normalized expression level of the CH25H gene in the biological sample.
  • the target parameter is determined based on the normalized expression level of the CH25H gene in the biological sample of the target subject and the normalized expression level of the CH25H gene in the biological sample of a plurality of first reference subjects without cancer.
  • the prognostic analysis module 330 may also be used to compare the target parameter to one or more reference thresholds to obtain comparison results.
  • the reference thresholds may include a first reference threshold and a second reference threshold.
  • the one or more reference thresholds are determined based on normalized expression levels of the CH25H gene in biological samples of a plurality of second reference subjects with cancer.
  • the prognostic analysis module 33 can also be used to predict the likelihood of cancer recurrence or metastasis in the target subject based on the comparison. In some embodiments, in response to the comparison being that the target parameter is less than the first reference threshold, it is determined that the target subject is more likely to have cancer recurrence or metastasis. In response to the comparison being that the target parameter is greater than the second reference threshold, the target subject is predicted to be less likely to have cancer recurrence or metastasis.
  • system and its modules shown in FIG. 3 may be implemented in various ways.
  • the system and its modules may be implemented in hardware, software, or a combination of software and hardware.
  • the hardware part can be realized by using dedicated logic;
  • the software part can be stored in a memory and executed by a suitable instruction execution system, such as a microprocessor or specially designed hardware.
  • a suitable instruction execution system such as a microprocessor or specially designed hardware.
  • the methods and systems described above may be implemented using computer-executable instructions and/or embodied in processor control code, for example on a carrier medium such as a disk, CD or DVD-ROM, such as a read-only memory (firmware) ) or a data carrier such as an optical or electronic signal carrier.
  • the system and its modules of the present application can not only be implemented by hardware circuits such as very large scale integrated circuits or gate arrays, semiconductors such as logic chips, transistors, etc., or programmable hardware devices such as field programmable gate arrays, programmable logic devices, etc. , can also be implemented by, for example, software executed by various types of processors, and can also be implemented by a combination of the above-mentioned hardware circuits and software (eg, firmware).
  • the acquisition module 310, the normalized expression level determination module 320, and the prognosis analysis module 330 may be different modules in a system, or may be a module that implements the functions of two or more modules described above.
  • the acquisition module 310 and the normalized expression level determination module 320 may also be one module, and this module may simultaneously have the functions of acquiring test data and determining the normalized expression level of the CH25H gene in the biological sample.
  • each module may share one storage module, and each module may also have its own storage module. Such deformations are all within the protection scope of the present application.
  • Figure 4 is a method for predicting cancer prognosis risk according to some embodiments of the present application.
  • a computing device the computing device 200 shown in FIG. 2 , the processing device in FIG. 1 .
  • at least a portion of the steps in flow 400 may be implemented as an instruction (eg, an application program) stored in memory 130, memory 220.
  • Processing device 110 in FIG. 1 , processor 210 and/or modules in FIG. 2 may execute the instructions, and upon execution of the instructions, processing device 110 , processor 210 and/or modules may be configured to perform flow 400 .
  • the operation of the procedure shown below is for illustration purposes only. In some embodiments, process 400 may be accomplished using one or more additional operations not described and/or one or more operations not described. Additionally, the order of operations of the processes shown in FIG. 4 and described below is not intended to be limiting.
  • the kit can be used to detect the normalized expression level of the CH25H gene in the biological sample obtained from the target subject.
  • the biological sample may be peripheral blood, such as venous blood.
  • the peripheral blood of the target subject can be collected by a blood collection method.
  • blood collection methods may include negative pressure blood collection methods, skin blood collection methods, and venous blood collection methods.
  • the biological sample may be obtained at the hospital of the target subject.
  • the biological sample can be stored in a cryopreservation device and removed for testing when needed. For detailed information about target subjects and biological samples, reference may be made to FIG. 1 and its description, which will not be repeated here.
  • the likelihood of cancer recurrence or metastasis can be predicted for a target subject after the target subject has received cancer treatment.
  • the subject may be provided with interventional treatment, or the subject may be advised to increase the frequency of re-examination.
  • the possibility of cancer recurrence or metastasis can be predicted for the target subject, so as to provide a certain reference value for clinical selection of a treatment plan.
  • the kit can be used to detect the expression level of the CH25H gene in the biological sample.
  • the expression level of the CH25H gene can be determined based on the RNA transcript of the CH25H gene.
  • normalization can be performed, that is, the expression level of the internal reference gene is used as a reference to characterize the normalized expression level of the CH25H gene.
  • the expression level of CH25H gene can be determined by measuring the content of CH25H mRNA.
  • the term "internal reference gene” is a gene that is stably expressed in an organism or cell, and whose expression level is almost unchanged, and is used as a control for the expression level of the CH25H gene.
  • leukocytes can be extracted from a biological sample (eg, peripheral blood), eg, by natural sedimentation, accelerated erythrocyte sedimentation, and the like. RNA is then extracted from the leukocytes, eg, by an RNA extraction kit. In some embodiments, RNA can be extracted directly from biological samples (eg, peripheral blood). Details on extracting RNA can be found elsewhere in this disclosure, for example in step 501 in Figure 5 and its description.
  • the subject's biological sample can contain target RNA, which can be derived from the CH25H gene.
  • target RNA can be transcribed from the CH25H gene.
  • Biological samples also contain internal reference RNA.
  • the internal reference RNA can be RNA transcribed from the internal reference gene.
  • Internal reference genes can include but are not limited to ribosomal protein (RPLO), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), ⁇ -actin (ACTB), 18sRNA, 28sRNA, ⁇ 2 microglobulin (B2M), succinate dehydrogenase subunit A (SDHA), one or more of hypoxanthine phosphoribosyltransferase 1 (hypoxanthine phosphoribosyltransferase 1, HPRT1), region binding protein (attachment region binding protein, ARBP), etc. kind.
  • RPLO ribosomal protein
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • ACTB ⁇ -actin
  • 18sRNA 18sRNA
  • 28sRNA ⁇ 2 microglobulin
  • B2M ⁇ 2 microglobulin
  • SDHA succinate dehydrogenase subunit A
  • the reference RNA can be transcribed from the
  • kits can include RNA extraction kits, reverse transcription kits, expression level detection kits (eg, qPCR kits).
  • RNA content eg, RNA of CH25H and internal reference genes
  • RNA extraction kits can include RNA and reverse transcription kits.
  • RNA can be reverse transcribed into cDNA (eg, cDNA for CH25H and reference genes) by reverse transcription kits.
  • the normalized expression level of CH25H gene can be detected by qPCR kit based on real-time fluorescence quantitative PCR technology.
  • the qPCR kit includes reagents for detecting the expression level of the CH25H gene, which includes a specific probe for the CH25H gene and a specific primer for the CH25H gene. Details regarding the detection of normalized expression levels of the CH25H gene in biological samples can be found elsewhere in this disclosure, eg, in Figure 2 and its description.
  • the normalized expression level of the CH25H gene can be determined.
  • the quantitative methods of QPCR can be divided into absolute quantification and relative quantification. Absolute quantification is the exact content of the copy value of the target gene (ie CH25H gene) in the sample by the external standard curve established by the standard of the target gene. Relative quantification is used to analyze changes in the expression of a gene in a specific sample relative to a reference sample (eg, an untreated control). For example only, a relative quantitative approach can be used in this application to determine the normalized expression level of the CH25H gene.
  • the calculation methods used for relative quantification are the standard curve method and the comparative cycle threshold (CT) method.
  • the standard curve method is to obtain relative quantitative results by making a standard curve of calibrators (samples used as a basis for comparing results).
  • the comparative CT method is to obtain the normalized expression level of the CH25H gene based on comparing the CT values of the target gene and the internal reference gene.
  • the normalized expression level of the CH25H gene can be determined by the difference between the CT value of the internal reference gene and the CT value of the CH25H gene.
  • the normalized expression level of the CH25H gene can be expressed by ⁇ CT.
  • ⁇ CT can be determined by the following formula (1):
  • ⁇ CT CT internal reference - CT CH25H (1)
  • CT internal reference represents the CT value of the internal reference gene in the same biological sample
  • CT CH25H represents the CT value of the CH25H gene in the biological sample.
  • the normalized expression level of the CH25H gene can also be represented by - ⁇ CT.
  • the normalized expression level of the CH25H gene can be determined by the ratio of the CT value of the internal reference gene to the CT value of the CH25H gene. For example, it can be expressed as 2 CT internal reference /2 CT CH25H . 2 CT internal reference /2 CT CH25H equivalent Therefore, the normalized expression level of the CH25H gene can also be expressed as 2 ⁇ CT . In some embodiments, the normalized expression level of the CH25H gene can also be represented by 2- ⁇ CT.
  • the expression level of the CH25H gene in addition to detecting the mRNA expression level of the CH25H gene using qPCR technology, can also be represented by detecting the protein expression level of the CH25H gene.
  • detecting the protein expression level of the CH25H gene use an ELISA kit to detect the protein content of the CH25H gene expression.
  • a sandwich ELISA principle can be used, with capture antibodies pre-coated on 96-well micro ELISA plates and biotin-conjugated antibodies as detection antibodies. The enzyme standard, test sample and biotin-conjugated detection antibody are then added to the wells, incubated, and washed. Horseradish peroxidase-labeled streptavidin is added and unbound conjugate is washed away with wash buffer.
  • TMB 3,3',5,5'-Tetramethylbenzidine
  • step 401 may be repeated multiple times to detect multiple values of the normalized expression level of the CH25H gene in the biological sample obtained from the target subject, and after determining the average value according to the multiple values, the subsequent step 403 This average value is used in -407 to predict the likelihood of cancer recurrence or metastasis in the target subject.
  • the test data obtained in step 401 and related to the expression level of the CH25H gene in the biological sample of the target subject may be sent to the processor 110 and/or the memory 130 .
  • the test data may include a first cycle threshold corresponding to the CH25H gene and a second cycle threshold corresponding to an internal reference gene.
  • the processor 110 may determine the normalized expression level based on the first cycle threshold and the second cycle threshold.
  • target parameters may be determined based on the normalized expression level of the CH25H gene in the biological sample.
  • Target parameters can be used to compare with reference thresholds to predict the patient's risk of tumor metastasis or recurrence.
  • steps 403-407 may be performed manually by a user.
  • steps 403 - 407 may be performed by processing device 110 .
  • step 403 may be accomplished by prognostic analysis module 330 .
  • the processing device 110 may be based on a normalized expression level of the CH25H gene in the biological sample of the target subject (hereinafter referred to as "sample normalized expression level") and a plurality of first references without cancer
  • the target parameter is determined by the normalized expression level of the CH25H gene in the subject's biological sample.
  • the first reference subject can be a healthy subject (eg, a human) without cancer and can be used as a control group.
  • the method for detecting the normalized expression level of the CH25H gene in the biological sample (for example, peripheral blood) of the first reference subject and the method for detecting the normalized expression level of the CH25H gene in the biological sample of the target subject in step 401 similar, and will not be repeated here.
  • an average of the normalized expression levels of a plurality of first reference subjects can be determined as a control normalized expression level.
  • the first reference subjects may be grouped according to characteristic information (eg, age, gender, physical health, etc.). For example, a set of first reference subjects may be in the age range of 30-50. As another example, a set of first reference subjects may be all male or female, or half male and half female. As another example, a set of first reference subjects may be subjects with chronic diseases (eg, diabetes). In some embodiments, for each group of first reference subjects, an average value of control-normalized expression levels can be determined separately.
  • one or more values of the control normalized expression levels described above may be stored in a storage device (eg, storage device 130).
  • the processing device 110 may retrieve one or more numerical values against the normalized expression level from the storage device. For example, the processing device 110 may select a corresponding control-normalized expression level value based on characteristic information of the target subject (eg, at least one of age, gender, physical health, etc.). The processing device 110 may further determine the target parameter based on the sample normalized expression level and the control normalized expression level.
  • target parameters can be determined based on normalized expression levels expressed in ⁇ CT.
  • the target parameter can be determined by comparing a sample normalized expression level to a control normalized expression level.
  • the target parameter may be represented by d ⁇ CT.
  • d ⁇ CT can be determined by the following formula (2):
  • ⁇ CT sample represents the normalized expression level in the biological sample in the target subject
  • ⁇ CT control represents the normalized expression level in the biological sample in the control group.
  • the target parameter may be determined based on an exponential function, for example, a normalized expression level determined based on a difference in CT values may be used as an index.
  • the target parameter may be 2 d ⁇ CT .
  • the target parameter may be ed ⁇ CT .
  • the target parameter may be 2- d ⁇ CT .
  • the target parameter may be e -d ⁇ CT .
  • the target parameter may be determined based on normalized expression levels expressed in 2 ⁇ CT.
  • the parameter of interest can be determined by the ratio of the sample normalized expression level to the control normalized expression level. For example, it can be determined by the following formula (3):
  • Target parameter 2 ⁇ CT samples /2 ⁇ CT controls (3)
  • 2 ⁇ CT sample represents the normalized expression level of the CH25H gene in the biological sample of the target subject
  • 2 ⁇ CT control represents the normalized expression level of the CH25H gene in the biological sample of the first reference subject.
  • a processing device may compare the target parameter to one or more reference thresholds to obtain a comparison result.
  • the reference threshold may be determined based on normalized expression levels of the CH25H gene in biological samples of a plurality of second reference subjects with cancer.
  • a second reference subject refers to a subject who has had cancer and received treatment. And, for the second reference subject, it is known whether the cancer recurs or metastasizes within a certain period of time after receiving the treatment. The time period can be three years, five years, etc.
  • the method for detecting the normalized expression level of the CH25H gene in the biological sample of the second reference subject is similar to the method for detecting the normalized expression level of the CH25H gene in the biological sample of the target subject in step 401, and will not be repeated here. .
  • control parameters can be determined for each second reference subject in the metastatic and non-metastatic groups.
  • the method for determining the reference parameter is similar to the method for determining the target parameter, and details are not described herein again.
  • reference thresholds can be determined based on the control parameters in the two groups.
  • the reference threshold includes a first reference threshold and a second reference threshold. For example, the average value of the control parameters of the metastatic group can be used as the first reference threshold value, and the average value of the control parameters of the tumor non-metastatic group can be used as the second reference threshold value.
  • the target parameters in the two groups of second reference subjects may be determined according to step 403, thereby determining the first and second reference thresholds.
  • said first reference threshold may 0.48,0.5,0.52,0.54,0.6 the like
  • the second reference threshold may 0.9,0.95,1,1.05 Wait.
  • the first reference threshold may be 0.5
  • the second reference threshold may be 1.
  • the first reference threshold may be -1.2, -1, -0.95, -0.82, -0.7, -0.6, and so on.
  • the second reference threshold may be 0, 0.2, 0.39, 0.46, 0.6, 0.8, and the like.
  • the first reference threshold may be -1 and the second reference threshold may be 0.
  • the processing device 110 may determine the likelihood of recurrence or metastasis of the target subject's cancer based on the comparison.
  • the target subject in response to the comparison being that the target parameter is less than the first reference threshold, it is determined that the target subject is more likely to have cancer recurrence or metastasis. In some embodiments, in response to the comparison being that the target parameter is greater than a third threshold, it is determined that the target subject is more likely to have cancer recurrence or metastasis.
  • the high likelihood of cancer recurrence or metastasis may refer to a greater than 70%, 75%, or 80% probability of cancer recurrence or metastasis, or the like.
  • the target subject in response to the comparison being that the target parameter is greater than the second reference threshold, the target subject is predicted to be less likely to have cancer recurrence or metastasis. In some embodiments, in response to the comparison being that the target parameter is less than the fourth reference threshold, the target subject is predicted to be less likely to have cancer recurrence or metastasis.
  • the low probability of cancer recurrence or metastasis corresponds to a cancer recurrence or metastasis probability of less than 40%, 35%, or 30%, etc.
  • a numerical value of the likelihood of cancer recurrence or metastasis can be determined by a machine learning model.
  • characteristic information of the target patient eg, test data of the target subject, gender, age, type of cancer the target subject has or is currently suffering from, the target subject has other diseases, etc.
  • the prediction result can be expressed as: whether the tumor has recurrence or metastasis; the possibility of recurrence or metastasis (eg, higher or lower, specific percentage value, etc.).
  • the machine learning model may be a supervised learning model.
  • the machine learning model may include one or a combination of a support vector machine model, a decision tree model, a neural network model, a nearest neighbor classifier, and the like.
  • a machine learning model can be trained using test data of multiple second subjects, characteristic information of multiple second subjects, and whether there is a recurrence or metastasis.
  • FIG. 5 is a flow chart for detecting the normalized expression level of CH25H gene in a biological sample according to some embodiments of the present application.
  • a computing device the computing device 200 shown in FIG. 2 , the processing device in FIG. 1 .
  • at least a portion of the steps in process 500 may be implemented as an instruction (eg, an application program) stored in memory 130, memory 220.
  • Processing device 110 in FIG. 1 , processor 210 and/or modules in FIG. 2 may execute the instructions, and upon execution of the instructions, processing device 110 , processor 210 and/or modules may be configured to perform flow 500 .
  • process 500 may be accomplished using one or more additional operations not described and/or one or more operations not described. Additionally, the order of operations of the processes shown in FIG. 5 and described below is not intended to be limiting.
  • step 501 the RNA of the CH25H gene and the RNA of the internal reference gene can be extracted from the biological sample.
  • Methods for extracting RNA can include guanidine isothiocyanate cesium chloride ultracentrifugation method, guanidine hydrochloride-organic solvent method, lithium chloride-urea method, hot phenol method, rapid extraction method, cytoplasmic RNA extraction method, phenol-lithium chloride method Simultaneous extraction of cellular RNA and DNA, one-step rapid hot phenol extraction method.
  • RNA can be extracted from a biological sample (e.g., peripheral blood) by the method of extracting RNA.
  • the steps of extracting target RNA and internal reference RNA may include at least high temperature denaturation, extraction, precipitation, washing and solubilization.
  • target RNA and reference RNA can be extracted directly from biological samples (eg, peripheral blood). There is no need to first extract leukocytes from biological samples, and then extract target RNA and internal reference RNA from leukocytes.
  • leukocytes may be first extracted from the biological sample, eg, by natural sedimentation, accelerated erythrocyte sedimentation, and the like. Then, target RNA and internal reference RNA were extracted from leukocytes.
  • the kit for extracting target RNA and internal reference RNA can be the kits shown in Table 1. It should be noted that the reagents in Table 1 are only examples, and those skilled in the art can change and modify the types and amounts of reagents.
  • Buffer A 40ml Tris, NaCl, SDS Buffer B 15ml Chloroform Buffer C 15ml Glycogen, Sodium Acetate Buffer E 30ml anhydrous ethanol Buffer F 30ml anhydrous ethanol Buffer G 15ml H 2 O T1 centrifuge tube 50 polypropylene T2 centrifuge tube 100 polypropylene
  • step 503 the RNA of the CH25H gene and the RNA of the internal reference gene can be reverse transcribed respectively to obtain the cDNA of the CH25H gene and the cDNA of the internal reference gene.
  • Reverse transcription can take the RNAs transcribed from the target gene and the reference gene as templates, and synthesize complementary single-stranded DNA (cDNA) by reverse transcriptase.
  • cDNA complementary single-stranded DNA
  • the specific reverse transcription primer used for reverse transcription of the target RNA may include: nucleotides with a similarity of ⁇ 70% to the sequence shown in SEQ ID NO.: 4, and SEQ ID NO.: 4 Nucleotides with a similarity of ⁇ 75% to the sequence shown, nucleotides with a similarity of ⁇ 80% to the sequence shown in SEQ ID NO.: 4, and a similarity of ⁇ 85 to the sequence shown in SEQ ID NO.: 4 % of nucleotides, nucleotides with a similarity of ⁇ 90% to the sequence shown in SEQ ID NO.: 4, and nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 4.
  • the specific reverse transcription primer used for reverse transcription of the internal reference RNA may include: nucleotides with a similarity of ⁇ 70% to the sequence shown in SEQ ID NO.: 5, and SEQ ID NO.: Nucleotides with a similarity degree of ⁇ 75% to the sequence shown in 5, nucleotides with a similarity degree of ⁇ 80% with the sequence shown in SEQ ID NO.: 5, and a similarity degree of ⁇ 80% with the sequence shown in SEQ ID NO.: 5 85% of the nucleotides, nucleotides with a similarity of ⁇ 90% to the sequence shown in SEQ ID NO.: 5, and nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 5.
  • the CH25H gene and internal reference gene can be reverse transcribed by the reverse transcription kit in Table 2. It should be noted that the reverse transcription kits in Table 2 are only examples, and those skilled in the art can change and modify the types and amounts of reagents.
  • a PCR amplification reaction may be performed for the cDNA of the CH25H gene and the cDNA of the internal reference gene.
  • the PCR amplification reaction of the target cDNA is real-time fluorescence quantitative PCR.
  • the basic principle of PCR amplification is: using single-stranded DNA (cDNA) as a template, 4 kinds of dNTPs as substrates, and in the presence of primers at the 3' end of the template, the complementary chain is extended with an enzyme, and the cycle is repeated many times. It can amplify a small amount of template DNA to a great extent.
  • a microcentrifuge tube add two primers complementary to the known sequences at both ends of the DNA fragment to be amplified, an appropriate amount of buffer, a small amount of DNA membrane plate, four dNTP solutions, heat-resistant Taq DNA polymerase, Mg 2+ etc.
  • the above solution is first heated to denature the template DNA at high temperature, and the double-strand is unwound into a single-strand state; then the temperature of the solution is lowered, so that the synthetic primer is paired with its target sequence at a low temperature to form a partial double-strand, which is called annealing; Then the temperature is raised to a suitable temperature, under the catalysis of TaqDNA polymerase, using dNTP as raw material, the primer is extended in the 5' ⁇ 3' direction to form a new DNA fragment, which can be used as the template for the next round of reaction, so Repeatedly changing the temperature, a cycle composed of high temperature denaturation, low temperature renaturation and suitable temperature extension, and repeated cycles, enables the rapid amplification of the target gene.
  • the reagent for detecting the expression level of the CH25H gene includes a primer specific for the CH25H gene.
  • the similarity between the specific primer sequence of the CH25H gene and the sequence shown in SEQ ID NO.: 2 is greater than or equal to 70%, 75%, 80%, 85%, 90% or 95%; or the The similarity between the specific primer sequence of CH25H gene and the sequence shown in SEQ ID NO.:3 is greater than or equal to 70%, 75%, 80%, 85%, 90% or 95%.
  • the specific primer sequence of the CH25H gene can include a forward primer sequence and a reverse primer sequence, the forward primer sequence corresponds to SEQ ID NO.: 2, and the reverse primer sequence corresponds to SEQ ID NO.: 3.
  • the PCR amplification reaction of the internal reference cDNA is real-time quantitative PCR (Q-PCR).
  • the qPCR-specific primers used for amplification of the internal reference cDNA may include: nucleotides with a similarity of ⁇ 70% to the sequence shown in SEQ ID NO.: 6, which are similar to those shown in SEQ ID NO.: 6 Nucleotides with sequence similarity ⁇ 75%, nucleotides with SEQ ID NO.: 6 similarity ⁇ 80%, nucleotides with SEQ ID NO.: 6 similarity ⁇ 85% nucleotides, nucleotides with a similarity of ⁇ 90% to the sequence shown in SEQ ID NO.: 6, nucleotides with a similarity of ⁇ 95% to the sequence shown in SEQ ID NO.: 6; or with SEQ ID NO.
  • the specific primer sequence of the internal reference gene can include a forward primer sequence and a reverse primer sequence, the forward primer sequence corresponds to SEQ ID NO.: 6, and the reverse primer sequence corresponds to SEQ ID NO.: 7.
  • the reagent for detecting the expression level of the CH25H gene comprises a specific probe for the CH25H gene.
  • the probes used for the amplification of the target cDNA may include: nucleotides with a similarity of ⁇ 70% with the sequence shown in SEQ ID NO.: 1, and nucleotides with a similarity of ⁇ 75% with the sequence shown in SEQ ID NO.: 1 Nucleotides, nucleotides with a similarity of ⁇ 80% to the sequence shown in SEQ ID NO.: 1, nucleotides with a similarity of ⁇ 85% to the sequence shown in SEQ ID NO.: 1, and SEQ ID NO.
  • the probes used for amplification of the internal reference cDNA may include: nucleotides with a similarity of ⁇ 70% to the sequence shown in SEQ ID NO.: 8, and nucleotides with a similarity to the sequence shown in SEQ ID NO.: 8 Nucleotides with a similarity of ⁇ 75%, nucleotides with a similarity of ⁇ 80% with the sequence shown in SEQ ID NO.: 8, and nucleosides with a similarity of ⁇ 85% with the sequence shown in SEQ ID NO.: 8 Acid, nucleotides with ⁇ 90% similarity with the sequence shown in SEQ ID NO.:8, nucleotides with ⁇ 95% similarity with the sequence shown in SEQ ID NO.:8.
  • qPCR of CH25H gene and internal reference gene can be performed by the qPCR kit in Table 3. It should be noted that the qPCR kits in Table 3 are only examples, and those skilled in the art can change and modify the types and amounts of reagents.
  • the normalized expression level of the CH25H gene in the biological sample can be determined according to the first cycle threshold of the cDNA of the CH25H gene in the PCR amplification reaction and the second cycle threshold of the cDNA of the internal reference gene.
  • the amount of target cDNA and the amount of internal reference cDNA can be characterized as a cycle threshold (CT) of the target cDNA (also referred to as a first cycle threshold) and a CT value of an internal reference cDNA (also referred to as a second cycle threshold) cycle threshold).
  • CT value refers to the cycle threshold experienced when the fluorescent signal in each reaction tube reaches the set threshold.
  • the first cycle threshold of the cDNA of the CH25H gene in the PCR amplification reaction (which can be expressed as CT CH25H ) and the second cycle threshold of the cDNA of the internal reference gene (which can be expressed as CT internal reference ) determine the CH25H gene in the biological sample.
  • Normalizing the expression level includes determining the difference between the first cycle threshold and the second cycle threshold (which can be expressed as ⁇ CT), and using the difference between the first cycle threshold and the second cycle threshold to represent the normalized expression of the CH25H gene in the biological sample level (eg, ⁇ CT or 2 ⁇ CT ).
  • a use of a reagent for detecting the expression level of CH25H gene in the preparation of a kit for predicting cancer prognosis can include using the kit to detect a normalized expression level of the CH25H gene in a biological sample obtained from a target subject; determining a target parameter based on the normalized expression level of the CH25H gene in the biological sample; combining the target parameter with one or more A number of reference thresholds are compared to obtain a comparison result; based on the comparison result, the likelihood of cancer recurrence or metastasis in the target subject is determined.
  • a cancer prognosis prediction device may include an acquisition module configured to acquire test data related to the expression level of the CH25H gene in the biological sample of the target subject; a normalized expression level determination module configured to obtain test data based on the test data , determine the normalized expression level of the CH25H gene in the biological sample; the prognosis analysis module is configured to determine the target parameter based on the normalized expression level of the CH25H gene in the biological sample; the target parameter is combined with one or more reference thresholds A comparison is made to obtain a comparison; and based on the comparison, a likelihood of cancer recurrence or metastasis in the target subject is determined.
  • a computer-readable storage medium stores computer instructions, and when the computer instructions are executed by the processor, implements a method for predicting cancer prognosis, characterized in that, the method for predicting cancer prognosis includes: acquiring test data, the test data being related to the biological characteristics of the target subject The expression level of the CH25H gene in the sample is related; based on the test data, determine the normalized expression level of the CH25H gene in the biological sample; determine the target parameter based on the normalized expression level of the CH25H gene in the biological sample; and the target parameter and a The term or multiple reference thresholds are compared to obtain a comparison result; and based on the comparison result, the likelihood of cancer recurrence or metastasis in the target subject is determined.
  • a cancer prognosis prediction method may include: acquiring test data, the test data being correlated with the expression level of the CH25H gene in the biological sample of the target subject, and detecting the standard expression level of the CH25H gene in the biological sample; and determining the CH25H gene in the biological sample based on the test data.
  • a normalized expression level of a gene based on the normalized expression level of the CH25H gene in the biological sample, determine a target parameter; compare the target parameter with one or more reference thresholds to obtain a comparison result; and based on the comparison result, determine Likelihood of cancer recurrence or metastasis in the target subject.
  • a method for treating cancer or preventing cancer recurrence and metastasis may include: detecting the normalized expression level of the CH25H gene in the biological sample obtained from the target subject; determining a target parameter based on the normalized expression level of the CH25H gene in the biological sample; combining the target parameter with one or more A plurality of standard reference thresholds are compared; in response to the target parameter being less than the reference standard threshold, the subject is administered a composition comprising an agent that increases the expression level of the CH25H gene or that increases the activity of the CH25H protein.
  • the target subject when a high probability of cancer recurrence or metastasis is predicted, the target subject can be provided with interventional treatment, and the frequency of re-examination can also be increased.
  • drug therapy may be provided to the target subject, eg, administration of a composition to the target subject.
  • the composition contains an agent capable of increasing the expression level of the CH25H gene or increasing the activity of the CH25H protein.
  • the agent can include reserpine.
  • the agent may also include an interferon or a Toll-like receptor 4 (TLR4) agonist, and the like.
  • TLR4 agonists can include, but are not limited to, lipopolysaccharide (LPS), lipid A, and derivatives thereof.
  • the composition may be administered alone or in combination with other anticancer drugs (eg, monoclonal antibodies, antitumor antibiotics, etc.).
  • test materials used in the following examples were purchased from conventional biochemical reagent companies unless otherwise specified.
  • the quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.
  • Example 1 Extracting the target RNA and internal reference RNA.
  • RNA-containing centrifuge tube On ice, extract the supernatant and place it in a T2 centrifuge tube (divide into two tubes, each tube is about 500ul), add 50ul of Buffer C and 1mL of absolute ethanol, and invert it upside down until it is evenly mixed. Placed at -80°C and precipitated for 2 hours.
  • Example 2 The target RNA and internal reference RNA were reverse transcribed into target cDNA and internal reference cDNA, respectively.
  • the reverse transcription system is as follows:
  • RNA Take 11.5 ⁇ L of sample RNA, 1 ⁇ L of super RT reverse transcriptase, 1 ⁇ L of reverse transcription primer, 2 ⁇ L of dNTP mixture, 4 ⁇ L of buffer, and 0.5 ⁇ L of RNase inhibitor, add them to a sterile centrifuge tube, and mix well.
  • the reverse transcription primer of the target RNA is: ccacattgtctgctcccaca (SEQ ID NO.: 4)
  • the reverse transcription primer of the internal reference RNA is: atgtcgaagaagcccaaaga (SEQ ID NO.: 5).
  • the reverse transcription product can be directly used in the subsequent PCR amplification reaction.
  • Example 3 The target cDNA and the internal reference cDNA were subjected to PCR amplification reaction.
  • the PCR amplification system is as follows:
  • the specific primers of the target gene CH25H are: aaggtgcaccaccagaactc (SEQ ID NO.: 2, forward primer), atgtcgaagaagcccaaaga (SEQ ID NO.: 3, reverse primer).
  • the specific primers for the internal reference gene RPLO are: gcgacctggaagtccaacta (SEQ ID NO.: 6, forward primer), ccacattgtctgctcccaca (SEQ ID NO.: 7, reverse primer).
  • the sequence of the specific probe of the target gene CH25H is: caacgcagtatatgagc (SEQ ID NO.: 1)
  • the sequence of the specific probe of the internal reference gene RPLO is: cttaagatcatccaactattg (SEQ ID NO.: 8).
  • the PCR amplification reaction conditions are as follows:
  • Example 4 Determining the expression levels of target RNA and internal reference RNA of the first reference subject.
  • the amplification curve refers to the curve made with the cycle number as the abscissa and the real-time fluorescence intensity during the reaction as the ordinate.
  • the baseline refers to a line with little change in fluorescence intensity and close to a straight line during the first few cycles of the PCR amplification reaction.
  • the fluorescence threshold was 10 times the standard deviation of the fluorescence intensity in the first 3-15 cycles of PCR, and the fluorescence threshold was set at the exponential phase of PCR amplification.
  • the CT value represents the number of cycles that each PCR reaction tube undergoes when the fluorescent signal reaches the set threshold. According to the adjusted threshold and baseline, the CT values of the CH25H gene and the reference gene RPLO in the blood samples of healthy people were determined and averaged, and the average value of the CT values of the CH25H gene and the reference gene of healthy people was used as a control.
  • CT values of CH25H gene and internal reference gene of some of the first reference subjects are shown in Table 5 below:
  • CT values of the CH25H gene and the internal reference gene RPLO in the blood samples of the first reference subject were averaged as a control. From this table, the normalized expression levels of the first reference subject are known. By way of example only, normalized expression levels may be expressed using ⁇ CT or 2 ⁇ CT . ⁇ CT is -12.29,2 ⁇ CT is 0.000199683.
  • Example 5 Determine the expression levels of target RNA and internal reference RNA in the sample.
  • the target parameter d ⁇ CT CH25H, 2 d ⁇ CT and 2 ⁇ CT value sample / 2 ⁇ CT control when the value is greater than 0 d ⁇ CT, showing the expression level of the second reference CH25H subject genes relative to the healthy Gene expression is high; otherwise, it is low.
  • the 2 d ⁇ CT was greater than 1
  • the expression level of the CH25H gene in the second reference subject was high relative to the expression level of the gene in healthy people; otherwise, it was low.
  • the 2 ⁇ CT sample / 2 ⁇ CT control is greater than 1, the expression of the CH25H gene in the second reference subject is high relative to the expression of the gene in healthy people; otherwise, it is low.
  • 2 ⁇ CT sample / 2 ⁇ CT control can also be expressed as That is 2 d ⁇ CT .
  • 2 ⁇ CT samples / 2 ⁇ CT and 2 d ⁇ CT control mode of expression but the same result.
  • Embodiment 6 The relationship between the patient's metastasis and the comparison result of the target parameter and the reference threshold.
  • the mRNA expression of CH25H gene on the leukocytes of patients with metastasis was significantly decreased, and there was a significant difference (p ⁇ 0.01%).
  • Biomarkers For example, when the target parameter of CH25H is represented by d ⁇ CT, the first reference threshold is -1. When d ⁇ CT is less than -1, the expression of CH25H gene is low and the possibility of cancer metastasis is high; the second reference threshold is 0, When d ⁇ CT is greater than 0, the expression of CH25H gene is higher, and the possibility of cancer metastasis is lower.
  • the first reference threshold is 0.5; when 2 d ⁇ CT is less than 0.5, the expression of CH25H gene is low, and the possibility of cancer metastasis is high; the second reference threshold is 1 , when the 2 d ⁇ CT was greater than 1, the expression of the CH25H gene was higher, and the possibility of cancer metastasis was lower.
  • the first reference threshold is 0.5.
  • the second reference threshold value 1 when the sample 2 ⁇ CT / 2 ⁇ CT control is greater than 1, higher gene expression level CH25H, lower likelihood of cancer metastasis.
  • a method for predicting cancer prognosis disclosed in this application may bring beneficial effects including but not limited to: (1) CH25H gene can be used as a marker for predicting cancer prognosis, and can monitor tumor recurrence and metastasis in real time and non-invasively after surgery; ( 2) By comparing the target parameters of the CH25H gene with the reference threshold, the possibility of tumor recurrence and metastasis can be accurately predicted, which can help to select the treatment plan for patients; (3) If the possibility of recurrence and metastasis is high, The frequency of follow-up can be increased after surgery, radiotherapy, or chemotherapy, and adjuvant therapy can be performed before and after surgery, radiotherapy, or chemotherapy.

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Abstract

一种检测CH25H基因表达水平的试剂在制备用于癌症预后预测的试剂盒中的一种用途。所述预测包括：使用所述试剂盒检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平；基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数；将所述目标参数与一项或多项参考阈值进行比较以获取比较结果；以及基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。还公开了一种癌症预后预测装置、计算机可读存储介质、一种癌症预后预测方法、一种治疗癌症或预防癌症复发转移的方法。

Description

一种预测癌症预后风险的方法 技术领域
本申请涉及生物技术领域，特别涉及一种预测癌症预后风险的方法。
背景技术
癌症具有极强的侵袭性、转移性。目前在我国新诊断的实体瘤患者中，2/3的患者已经出现临床检查转移，而在经过了局部治疗(手术、放疗)以及全身化疗后，仍有1/2患者出现亚临床隐性转移。肿瘤转移指肿瘤细胞脱离原发生长部位，通过各种途径的转运，在机体内远离原发部位的器官/组织继续增殖生长，形成同样性质肿瘤(转移瘤)的过程。肿瘤转移的主要途径包括淋巴道转移、血道转移和种植性转移。
对于肿瘤转移，临床上可以定期通过影像学、肿瘤标记物、活检穿刺以及细胞学或分子角度，如循环肿瘤细胞(CTC)和循环肿瘤DNA(ctDNA)来发现。通过活检穿刺可以进一步确认肿瘤是否转移复发，但对患者有创伤，而且存在一定的手术风险。从肿瘤分子生物学角度探寻肿瘤复发转移风险是目前研究的热点，可在患者血液样品中的游离DNA(cfDNA)中发现肿瘤特异性基因组改变，该方法可作为活检的补充，用于实时分子监测治疗、检测复发和跟踪耐药性。目前可通过血液检测循环肿瘤细胞(CTC)和循环肿瘤DNA(ctDNA)来监测肿瘤的早期发生和转移复发，但从检测成本而言，连续监测相对昂贵，很多患者无法承受。通过预后分析，可以预知癌症复发或转移的风险高低。临床上可以基于癌症预后分析选取治疗方案，确定治疗后患者复查的频率，或决定是否采取其他方式预防癌症复发和转移等。因此，需要提供一种成本较低的能够有效预测癌症预后风险的方法。
发明内容
根据本申请的一方面，提供了检测CH25H基因表达水平的试剂在制备用于癌症预后预测的试剂盒中的一种用途。所述预测可以包括使用所述试剂盒检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平。所述预测还可以包括基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数。所述预测还可以包括将所述目标参数与一项或多项参考阈值进行比较以获取比较结果。所述预测还可以包括基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。
在一些实施例中，所述一项或多项参考阈值包括第一参考阈值。所述基于所述比较结果，确定癌症复发或转移的可能性可以包括响应于所述比较结果为所述目标参数小于所述第一参考阈值，确定所述目标受试者癌症复发或转移的可能性较高。
在一些实施例中，所述一项或多项参考阈值包括第二参考阈值。所述基于所述比较结果，预测癌症复发或转移的可能性可以包括：响应于所述比较结果为所述目标参数大于所述第二参考阈值，预测所述目标受试者癌症复发或转移的可能性较低。
在一些实施例中，所述CH25H基因的所述标准化表达水平可以是基于CH25H基因在目标受试者中的表达水平和内参基因在目标受试者中的表达水平确定的。
在一些实施例中，所述内参基因可以包括RPLO、GAPDH、ACTB、B2M、SDHA、HPRT1、ARBP、18sRNA、28sRNA中的一种或多种。
在一些实施例中，所述CH25H基因的所述标准化表达水平可以是基于所述CH25H基因的RNA转录物确定的。
在一些实施例中，所述检测所述生物样品中CH25H基因的标准化表达水平可以包括从所述生物样品中提取所述CH25H基因的RNA和所述内参基因的 RNA。所述检测所述生物样品中CH25H基因的标准化表达水平还可以包括对所述CH25H基因的RNA和所述内参基因的RNA分别进行反转录，获取所述CH25H基因的cDNA和所述内参基因的cDNA。所述检测所述生物样品中CH25H基因的标准化表达水平还可以包括针对所述CH25H基因的cDNA和所述内参基因的cDNA，进行PCR扩增反应。所述检测所述生物样品中CH25H基因的标准化表达水平可以包括根据PCR扩增反应中CH25H基因的cDNA的第一循环阈值与所述内参基因的cDNA的第二循环阈值，确定所述生物样品中CH25H基因的标准化表达水平。
在一些实施例中，所述根据PCR扩增反应中CH25H基因的cDNA的第一循环阈值与所述内参基因的cDNA的第二循环阈值，确定所述生物样品中CH25H基因的标准化表达水平可以包括确定所述第一循环阈值与第二循环阈值的差值。确定所述生物样品中CH25H基因的标准化表达水平还可以包括用所述第一循环阈值与第二循环阈值的所述差值表示所述生物样品中CH25H基因的标准化表达水平。
在一些实施例中，所述基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数可以包括：基于所述目标受试者的所述生物样品中所述CH25H基因的标准化表达水平和多个未患癌症的第一参考受试者的生物样品中所述CH25H基因的标准化表达水平而确定的。
在一些实施例中，所述一项或多项参考阈值可以是基于多个患有癌症的第二参考受试者的生物样品中所述CH25H基因的标准化表达水平而确定的。
在一些实施例中，所述检测CH25H基因表达水平的试剂可以包括CH25H基因的特异性探针。
在一些实施例中，所述CH25H基因的特异性探针的序列与SEQ ID NO.：1所示序列的相似度可以≥95％。
在一些实施例中，所述检测CH25H基因表达水平的试剂可以包括所述 CH25H基因的特异性引物。
在一些实施例中，所述CH25H基因的特异性引物序列与SEQ ID NO.：2所示序列的相似度可以≥95％；并且所述CH25H基因的特异性引物序列与SEQ ID NO.：3所示序列的相似度可以≥95％。
在一些实施例中，所述癌症可以包括以下中的一个或多个：黑色素瘤、肺癌、白血病、胃癌、卵巢癌、胰腺癌、乳腺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、宫颈癌或骨肉瘤。
在一些实施例中，所述癌症可以是非小细胞肺癌、肺鳞癌、肺腺癌或小细胞肺癌。
在一些实施例中，所述生物样品可以是外周血样品。
在一些实施例中，所述使用所述试剂盒检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平还可以包括从所述外周血样品中提取白细胞。所述使用所述试剂盒检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平还可以包括使用所述试剂盒检测所述白细胞中CH25H基因的标准化表达水平。
根据本申请的另一方面，提供了一种癌症预后预测装置。所述装置可以包括获取模块，被配置为获取测试数据，所述测试数据与目标受试者的生物样品中CH25H基因的表达水平相关。所述装置可以包括标准化表达水平确定模块，被配置为基于所述测试数据，确定所述生物样品中CH25H基因的标准化表达水平。所述装置还可以包括预后分析模块。预后分析模块可以被配置为基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数。预后分析模块可以被配置为将所述目标参数与一项或多项参考阈值进行比较以获取比较结果。预后分析模块还可以被配置为基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。
根据本申请的另一方面，提供了一种计算机可读存储介质。所述存储介质 存储计算机指令，当所述计算机指令被处理器执行时，实现一种癌症预后预测方法。所述癌症预后预测方法可以包括获取测试数据，所述测试数据与目标受试者的生物样品中CH25H基因的表达水平相关。所述癌症预后预测方法可以包括基于所述测试数据，确定所述生物样品中CH25H基因的标准化表达水平。所述癌症预后预测方法可以包括基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数。所述癌症预后预测方法还可以包括将所述目标参数与一项或多项参考阈值进行比较以获取比较结果。所述癌症预后预测方法还可以包括基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。
根据本申请的另一方面，提供了一种癌症预后预测方法。所述方法可以包括获取测试数据，所述测试数据与目标受试者的生物样品中CH25H基因的表达水平相关。所述方法可以包括基于所述测试数据，确定所述生物样品中CH25H基因的标准化表达水平。所述方法可以包括基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数。所述方法还可以包括将所述目标参数与一项或多项参考阈值进行比较以获取比较结果。所述方法还可以包括基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。
根据本申请的再一方面，提供了一种用于治疗癌症或预防癌症复发转移的方法。所述方法可以包括检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平。所述方法可以包括基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数。所述方法还可以包括将所述目标参数与一项或多项参考阈值进行比较。所述方法还可以包括响应于所述目标参数小于所述参考阈值，为受试者施用一种组合物，所述组合物含有能提高CH25H基因的表达水平或能提高CH25H蛋白活性的试剂。
在一些实施例中，所述试剂还包括干扰素、Toll样受体4激动剂中的至少一个。
附图说明
本申请将以示例性实施例的方式进一步说明，这些示例性实施例将通过附图进行详细描述。这些实施例并非限制性的，其中：
图1为根据本申请一些实施例所示的癌症预后风险预测系统100的应用场景示意图；
图2是根据本申请一些实施例所示的计算设备200的架构的示意图；
图3是根据本申请一些实施例所示的癌症预后风险预测系统的模块图；
图4是根据本申请一些实施例所示的一种预测癌症预后风险的方法；以及
图5是根据本申请一些实施例所示的一种检测生物样品中CH25H基因的标准化表达水平的流程图。
具体实施方式
为了更清楚地说明本申请实施例的技术方案，下面将对实施例描述中所需要使用的附图作简单的介绍。显而易见地，下面描述中的附图仅仅是本申请的一些示例或实施例，对于本领域的普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图将本申请应用于其它类似情景。除非从语言环境中显而易见或另做说明，图中相同标号代表相同结构或操作。
应当理解，本文使用的“系统”、“装置”、“单元”和/或“模组”是用于区分不同级别的不同组件、元件、部件、部分或装配的一种方法。然而，如果其他词语可实现相同的目的，则可通过其他表达来替换该词语。
如本申请和权利要求书中所示，除非上下文明确提示例外情形，“一”、“一个”、“一种”和/或“该”等词并非特指单数，也可包括复数。一般说来，术语“包括”与“包含”仅提示包括已明确标识的步骤和元素，而这些步骤和元素不构成一个排它性的罗列，方法或者设备也可能包含其它的步骤或元素。
本申请中使用了流程图用来说明根据本申请的实施例的系统所执行的操作。应当理解的是，前面或后面操作不一定按照顺序来精确地执行。相反，可以按照倒序或同时处理各个步骤。同时，也可以将其他操作添加到这些过程中，或从这些过程移除某一步或数步操作。
以下是对本申请中一些术语的定义。
如本申请中所使用的，“受试者”(也可称为“个体”、“对象”)为接受检测或测试的对象。在一些实施例中，受试者可以是脊椎动物。在一些实施例中，脊椎动物为哺乳动物。哺乳动物包括但不限于灵长类(包括人和非人灵长类)以及啮齿动物(例如，小鼠和大鼠)。在一些实施例中，哺乳动物可以是人。“目标受试者”指的是患有癌症并且还未接受治疗(例如，化疗)的受试者，或曾患有(或当前仍患有)癌症并且已接受治疗的受试者。
术语“胆固醇-25-羟化酶(cholesterol-25-hydroxylase，CH25H)”是一种大小为31.6kDa的多跨膜内质网(endoplasmic reticulum，ER)相关酶，其主要功能是以O 2为附加底物，NADPH为辅助因子来催化胆固醇产生25-羟基胆固醇(25-hydroxycholesterol，25HC)。25HC属于胆固醇众多氧化产物中的内源性氧固醇，是一种通过调节固醇响应元件结合蛋白(sterol regulatory element-binding protein，SREBP)和核受体控制甾醇生物合成的可溶性因子。25HC可在多方面发挥作用，例如，在抗病毒中发挥重要作用、在免疫中发挥重要作用等。
术语“引物”是指能够与核酸杂交并且通常通过提供游离3’-OH基团，允许互补核酸聚合的单链多核苷酸。
术语“聚合酶链式反应(PCR)”是一种用于放大扩增特定的DNA片段的分子生物学技术。
术语“实时荧光定量PCR(Quantitative Real-time PCR)”也可称为qPCR、RT-PCR。qPCR是一种在DNA扩增反应中，通过荧光化学物质检测每次聚合酶链式反应(PCR)循环后产物总量的方法，可通过内参或者外参法对待测样品中 的特定DNA序列进行定量分析。
本申请提出，可以基于受试者的生物样品中CH25H的标准化基因表达水平，预测受试者癌症发生转移或复发的风险。并且可以根据癌症发生转移或复发的风险，采取干预治疗或调整治疗后复查频率等措施。本发明有望改善癌症患者的生存率，在临床上具有重要的应用价值。在本申请中，当CH25H基因的目标参数小于第一参考阈值时，确定该目标受试者癌症复发或转移的可能性较高；当CH25H基因的目标参数大于第二参考阈值时，确定该目标受试者癌症复发或转移的可能性较低。
肿瘤细胞转移至其他器官需要一个适合肿瘤细胞生长的微环境。来自原发肿瘤的癌细胞可以通过发送肿瘤源性细胞外囊泡(Tumor-derived extracellular vesicles，TEV)来帮助准备这种微环境。这些囊泡含有一种分子混合物，可以“驯化”健康细胞，为癌细胞的种子和繁殖做准备，促进肿瘤的生长、复发和转移。但并非所有与这些囊泡接触的健康细胞都会被驯化，可通过一些方式阻止该驯化过程。CH25H产生的25-羟基胆固醇，能抑制正常细胞对囊泡的吸收，因此可抑制肿瘤的复发和转移。
图1为根据本申请一些实施例所示的癌症预后风险预测系统100的应用场景示意图。如图1所示，癌症预后风险预测系统100可以包括处理设备110、网络120和存储器130。在一些实施例中，存储器130可以存储目标受试者(例如，目标受试者140)的基础信息、疾病史、治疗方案等数据，还可以存储目标受试者140的基因表达信息，例如目标受试者的CH25H基因和内参基因的标准化表达水平以及目标参数等。存储器130还可以存储一项或多项参考阈值等数据。目标受试者的生物样品，例如生物样品145，可以保存在专门的储藏设备中以备进一步处理，例如进行RNA提取等。在一些实施例中，生物样品145可以是组织样品或体液样品。具体的，生物样品145可以是血液，例如外周血。处理设备110可以用于对相关信息进行处理、分析以生成预后预测结果。在一些实 施例中，处理设备110可以从存储器130中获取相关的信息和/或数据(例如，第一参考阈值、第二参考阈值、生物样品中CH25H基因的标准化表达水平等)，也可以直接获取工作人员或者其他设备仪器对目标受试者140的生物样品145进行处理得到的相关信息和/或数据。
处理设备110可以处理从存储器130处获得的数据和/或信息。例如，处理设备110可以能够确定目标受试者110的生物样品145中CH25H基因的目标参数的指令，将目标参数与一项或多项参考阈值进行比较的指令，根据比较结果进而获得癌症预后预测结果。在一些实施例中，处理设备110可以是一个单个的处理器或者一个处理器群组。该处理器群可以是集中式的或分布式的(例如，处理设备110可以是一个分布式的系统)。在一些实施例中，处理设备110可以是本地的或远程的。在一些实施例中，处理设备110可以通过网络120从存储器130处获取信息和/或数据。在一些实施例中，处理设备110可以在一个云平台上实现。仅仅举个例子，该云平台可以包括私有云、公共云、混合云、社区云、分布云、云之间、多重云等或上述举例的任意组合。处理设备110可包含中央处理器(CPU)、专用集成电路(ASIC)、专用指令处理器(ASIP)、图形处理器(GPU)、物理处理器(PPU)、数字信号处理器(DSP)、现场可编程门阵列(FPGA)、可编辑逻辑电路(PLD)、控制器、微控制器单元、精简指令集电脑(RISC)、微处理器等或以上任意组合。
网络120可以提供信息交换的渠道。在一些实施例中，处理设备110和存储器130之间可以通过网络120交换信息。例如，处理设备110可以通过网络120接收存储器130中的数据。在一些实施例中，目标受试者140和/或生物样品145的相关信息可以通过网络120传输给处理设备110和/存储器130。例如，目标受试者140的信息(如CH25H基因的标准化表达水平等)可以通过网络120传输给处理设备110。在一些实施例中，网络120可以是任意类型的有线或无线网络。例如，网络120可包括一缆线网络、有线网络、光纤网络、电信网 络、内部网络、网际网络、区域网络(LAN)、广域网络(WAN)、无线区域网络(WLAN)、都会区域网络(MAN)、公共电话交换网络(PSTN)、蓝牙网络、ZigBee网络、近场通讯(NFC)网络等或以上任意组合。
存储器130可以用于存储数据和/或指令集。在一些实施例中，存储器130可以存储从处理设备110获得的数据。在一些实施例中，存储器130可存储供处理设备110执行或使用的信息和/或指令，以执行本申请中描述的示例性方法。在一些实施例中，存储器130中可以存储基因表达数据。在一些实施例中，存储器130可以包括大容量存储器、可移动存储器、易失性读写存储器、只读存储器(ROM)等或其任意组合。在一些实施例中，存储器130可以在云平台上实现。仅作为示例，云平台可以包括私有云、公共云、混合云、社区云、分布式云、中间云、多云等或其任意组合。在一些实施例中，存储器130可以是处理设备110的一部分。
在一些实施例中，目标受试者140可以是曾患有癌症并已接受治疗的受试者。治疗可以包括但不限于肿瘤切除手术、化疗、局部放射治疗、生物免疫治疗、靶向治疗、药物治疗等。在一些实施例中，目标受试者110可以患有一种或多种癌症。在一些实施例中，癌症可以包括乳腺癌、三阴性乳房癌症、化生乳腺癌(MpBC)、白血病、胃癌、卵巢癌、前列腺癌、膀胱癌、直肠癌、肝癌、宫颈癌、肺鳞癌、肺腺癌、头颈部鳞状细胞癌(HNSCC)、人乳头瘤病毒(HPV)阳性HNSCC、HPV阴性/TP53突变HNSCC、转移性HNSCC、口咽HNSCC、非口咽HNSCC、黑色素瘤、管腔A乳腺癌、管腔B乳腺癌、HER2+乳房癌症、高微卫星不稳定性(MSI-H)结直肠癌、微卫星稳定结直肠癌(MSS)、非小细胞肺癌(NSCLC)、小细胞肺癌、脊索瘤或肾上腺皮质癌。癌可以是乳腺癌、结肠癌、肺癌、胰腺癌、前列腺、Merkel细胞、卵巢、肝脏、子宫内膜、膀胱、肾脏或癌症未知初级(CUP)。肉瘤可以是脂肪肉瘤、骨肉瘤、骨骼外粘液样软骨肉瘤或子宫肉瘤。在一些实施方案中，肉瘤包含α肺泡软组织肉瘤(ASPS)、 血管肉瘤、乳腺血管肉瘤、软骨肉瘤、脊索瘤、透明细胞肉瘤、增生性小圆细胞瘤(DSRCT)、上皮样细胞血管内皮瘤(EHE)、上皮样肉瘤、子宫内膜间质肉瘤(ESS)、尤文肉瘤、纤维瘤病、纤维肉瘤、巨细胞瘤、平滑肌肉瘤(LMS)、子宫LMS、脂肪肉瘤、恶性纤维组织细胞瘤(MFH/UPS)、恶性周围神经鞘瘤(MPNST)、骨肉瘤、血管周围上皮样细胞瘤(PEComa)、横纹肌肉瘤、孤立性纤维瘤(SFT)、滑膜肉瘤、纤维粘液样肉瘤、婴儿期纤维性错构瘤、遗传性平滑肌瘤病、血管平滑肌脂肪瘤、血管肌瘤、非典型梭形细胞病变(纤维组织细胞分化)、软骨母细胞瘤、树突状细胞肉瘤、颗粒细胞瘤、高级粘液样肉瘤、高度肌上皮癌、透明变性成纤维细胞肉瘤、炎症肌纤维母细胞肉瘤、交叉树突状细胞瘤、内膜肉瘤、平滑肌瘤、淋巴管肉瘤病、恶性血管瘤、恶性肌上皮瘤、黑素细胞肿瘤、间充质肿瘤、肠系膜上皮瘤、转移性组织细胞样肿瘤、肌上皮瘤、粘液样肉瘤、粘液样基质、神经鞘瘤、叶状体、横纹肌样细胞、圆形细胞、未另行规定的肉瘤(NOS)、肉瘤间皮瘤、神经鞘瘤、纺锤体和圆形细胞肉瘤、或梭形细胞间充质肿瘤。在一些实施例中，目标受试者140含有的癌症可以处在各个阶段(如早期、中期、晚期等)。
在一些实施例中，生物样品145可以用于反映目标受试者140中CH25H基因的相关信息。一些实施例中，该生物样品可以包括组织样品或体液样品。在一些实施例中，体液样品可以包括外周血、组织液、淋巴液或脑脊液样品的一种或几种的组合。在一些实施例中，体液样品可以包括哺乳动物的血液样品、组织液样品或淋巴液样品。在一些实施例中，哺乳动物可以为人。在一些实施例中，生物样品可以是外周血，例如静脉血。
在一些实施例中，目标受试者140和/或生物样品145的相关信息可以通过人工(如工作人员手动输入)或机器(如仪器设备等)传输给癌症预后风险预测系统100的一个或多个组件(如处理设备110、存储器130)。
图2是根据本申请一些实施例所示的计算设备200的架构的示意图。如 图2所示，计算设备200可以包括处理器210、存储器220、输入/输出接口230和通信端口240。在该计算设备200上可以实现处理设备110和/或存储器130。在一些实施例中，一种癌症预后预测装置可以在计算设备200中实现。例如，处理设备110可以在计算设备200上实现并且被配置为执行本申请中处理设备110的功能。例如，处理设备110可以检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平。又例如，处理设备110可以基于该生物样品中CH25H基因的该标准化表达水平，确定目标参数。再例如，处理设备110可以将该目标参数与一项或多项参考阈值进行比较以获取比较结果，并基于该比较结果，确定该目标受试者癌症复发或转移的可能性。
处理器210可以执行计算指令(程序代码)并执行本申请描述的处理设备110的功能。计算指令可以包括程序、对象、组件、数据结构、过程、模块和功能(功能指本申请中描述的特定功能)。例如，处理器210可以处理癌症预后风险预测系统100中预测癌症预后效果的指令。在一些实施例中，处理器210可以包括微控制器、微处理器、精简指令集计算机(RISC)、专用集成电路(ASIC)、应用特定指令集处理器(ASIP)、中央处理器(CPU)、图形处理单元(GPU)、物理处理单元(PPU)、微控制器单元、数字信号处理器(DSP)、现场可编程门阵列(FPGA)、高级RISC机(ARM)、可编程逻辑器件以及能够执行一个或多个功能的任何电路和处理器等，或其任意组合。仅为了说明，图2中只描述了一个处理器210，但需要注意的是本申请可以包括多个处理器。
存储器220可以存储从癌症预后风险预测系统100中任何组件获得的数据/信息。在一些实施例中，存储器220可以包括大容量存储器、可移动存储器、易失性读取和写入存储器和只读存储器(ROM)等，或其任意组合。示例性大容量存储器可以包括磁盘、光盘和固态驱动器等。可移动存储器可以包括闪存驱动器、软盘、光盘、存储卡、U盘、压缩盘和移动硬盘等。易失性读取和写入存储器可以包括随机存取存储器(RAM)。RAM可以包括动态RAM(DRAM)、 双倍速率同步动态RAM(DDRSDRAM)、静态RAM(SRAM)、晶闸管RAM(T-RAM)和零电容(Z-RAM)等。ROM可以包括掩模ROM(MROM)、可编程ROM(PROM)、可擦除可编程ROM(PEROM)、电可擦除可编程ROM(EEPROM)、光盘ROM(CD-ROM)和数字通用盘ROM等。
输入/输出接口230可以用于输入或输出信号、数据或信息。在一些实施例中，输入/输出接口230可以用于实现用户(例如，目标受试者110、癌症预后风险预测系统100的使用者等)与处理设备110的交互行为。在一些实施例中，用户可以通过输入/输出接口230输入肿瘤患者的特征信息。在一些实施例中，输入/输出接口230可以包括输入装置和输出装置。示例性输入装置可以包括键盘、鼠标、触摸屏和麦克风等，或其任意组合。示例性输出设备可以包括显示设备、扬声器、打印机、投影仪等，或其任意组合。示例性显示装置可以包括液晶显示器(LCD)、基于发光二极管(LED)的显示器、平板显示器、曲面显示器、电视设备、阴极射线管(CRT)等，或其任意组合。
通信端口240可以连接到网络120以便数据通信。连接可以是有线连接、无线连接或两者的组合。有线连接可以包括电缆、光缆或电话线等，或其任意组合。无线连接可以包括蓝牙、WiFi、WiMax、WLAN、ZigBee、移动网络(例如，3G、4G或5G等)等，或其任意组合。在一些实施例中，通信端口240可以是标准化端口，如RS232、RS485等。在一些实施例中，通信端口240可以是专门设计的端口。
图3是根据本申请一些实施例所示的癌症预后风险预测系统的模块图。如图3所示，该癌症预后风险预测系统可以包括获取模块310、标准化表达水平确定模块320、预后分析模块330。
获取模块310可以用于获取测试数据，该测试数据与目标受试者的生物样品中CH25H基因的表达水平相关。测试数据可以包括目标受试者的血液样品数据、CH25H基因的RNA数据(例如，浓度、碱基序列等)、CH25H基因的 cDNA数据(例如，浓度、碱基序列、CT值、2 ΔCT、ΔCT值、2 dΔCT等)、内参基因的RNA数据和cDNA数据(例如，浓度、碱基序列、CT值、2 ΔCT、ΔCT值、2 dΔCT等)等。
标准化表达水平模块320基于该测试数据，确定该生物样品中CH25H基因的标准化表达水平。该CH25H基因的该标准化表达水平是基于CH25H基因在目标受试者中的表达水平和内参基因在目标受试者中的表达水平确定的。该内参基因包括RPLO、GAPDH、ACTB、B2M、SDHA、HPRT1、ARBP、18sRNA、28sRNA等中的一种或多种。该CH25H基因的该标准化表达水平是基于该CH25H基因的RNA转录物确定的。在一些实施例中，为检测CH25H基因的标准化表达水平，可以从该生物样品中提取该CH25H基因的RNA和该内参基因的RNA。在一些实施例中，该生物样品可以是外周血。例如可以从外周血中提取白细胞，继而提取CH25H基因的RNA和该内参基因的RNA。可以对该CH25H基因的RNA和该内参基因的RNA分别进行反转录，获取该CH25H基因的cDNA和该内参基因的cDNA。进一步地，针对该CH25H基因的cDNA和该内参基因的cDNA，可以进行PCR扩增反应。根据PCR扩增反应中CH25H基因的cDNA的第一循环阈值与该内参基因的cDNA的第二循环阈值，可以确定该第一循环阈值与第二循环阈值的差值。可以用第一循环阈值与第二循环阈值的差值表示该生物样品中CH25H基因的标准化表达水平。
预后分析模块330可以基于该生物样品中CH25H基因的标准化表达水平，确定目标参数。该目标参数是基于目标受试者的生物样品中CH25H基因的标准化表达水平和多个未患癌症的第一参考受试者的生物样品中CH25H基因的标准化表达水平而确定的。
预后分析模块330还可以用于将目标参数与一项或多项参考阈值进行比较以获取比较结果。参考阈值可以包括第一参考阈值和第二参考阈值。该一项或多项参考阈值是基于多个患有癌症的第二参考受试者的生物样品中CH25H基因 的标准化表达水平而确定的。
预后分析模块33还可以用于基于该比较结果，预测该目标受试者癌症复发或转移的可能性。在一些实施例中，响应于该比较结果为该目标参数小于该第一参考阈值，确定该目标受试者癌症复发或转移的可能性较高。响应于该比较结果为该目标参数大于该第二参考阈值，预测该目标受试者癌症复发或转移的可能性较低。
应当理解，图3所示的系统及其模块可以利用各种方式来实现。例如，在一些实施例中，系统及其模块可以通过硬件、软件或者软件和硬件的结合来实现。其中，硬件部分可以利用专用逻辑来实现；软件部分则可以存储在存储器中，由适当的指令执行系统，例如微处理器或者专用设计硬件来执行。本领域技术人员可以理解上述的方法和系统可以使用计算机可执行指令和/或包含在处理器控制代码中来实现，例如在诸如磁盘、CD或DVD-ROM的载体介质、诸如只读存储器(固件)的可编程的存储器或者诸如光学或电子信号载体的数据载体上提供了这样的代码。本申请的系统及其模块不仅可以有诸如超大规模集成电路或门阵列、诸如逻辑芯片、晶体管等的半导体、或者诸如现场可编程门阵列、可编程逻辑设备等的可编程硬件设备的硬件电路实现，也可以用例如由各种类型的处理器所执行的软件实现，还可以由上述硬件电路和软件的结合(例如，固件)来实现。
需要注意的是，以上对于候选项显示、确定系统及其模块的描述，仅为描述方便，并不能把本申请限制在所举实施例范围之内。可以理解，对于本领域的技术人员来说，在了解该系统的原理后，可能在不背离这一原理的情况下，对各个模块进行任意组合，或者构成子系统与其他模块连接。例如，在一些实施例中，获取模块310、标准化表达水平确定模块320和预后分析模块330可以是一个系统中的不同模块，也可以是一个模块实现上述的两个或两个以上模块的功能。例如，获取模块310和标准化表达水平确定模块320也可以是一个模块，该模块 可以同时具有获取测试数据和确定该生物样品中CH25H基因的标准化表达水平的功能。又例如，各个模块可以共用一个存储模块，各个模块也可以分别具有各自的存储模块。诸如此类的变形，均在本申请的保护范围之内。
图4是根据本申请一些实施例所示的一种预测癌症预后风险的方法。在一些实施例中，流程400中的至少一部分步骤(例如步骤403-407)可以由计算设备(如图2所示的计算设备200，图1中的处理设备)完成。例如，流程400中的至少一部分步骤可以被实现为存储在存储器130、存储器220中的一个指令(例如，应用程序)。图1中的处理设备110，图2中的处理器210和/或模块可以执行该指令，并且在执行指令时，处理设备110、处理器210和/或模块可以被配置为执行流程400。以下所示过程的操作仅出于说明的目的。在一些实施例中，流程400可以利用未描述的一个或以上附加操作和/或未描述的一个或以上操作来完成。另外，图4所示和以下描述的过程的操作顺序并非旨在限制。
在步骤401中，可以使用该试剂盒检测该获取自目标受试者的生物样品中CH25H基因的标准化表达水平。
在一些实施例中，生物样品可以是外周血，例如静脉血。可通过采血方法来采集目标受试者的外周血。例如，采血方法可以包括负压采血的方法、皮肤采血的方法、还有静脉采血法。在一些实施例中，可以在目标受试者所在医院获取生物样品。在一些实施例中，该生物样品可存储于低温保存装置中，需要时可取出进行检测。关于目标受试者和生物样品的详细信息可以参考图1及其描述，在此不再赘述。一些实施例中，可以在目标受试者接受癌症治疗之后，为目标受试者预测癌症复发或转移的可能性。如果癌症复发或转移的可能性较高，可以为受试者提供干预治疗，或建议受试者提高复查频率。在一些实施例中，可以在目标受试者接受癌症治疗之前，为目标受试者预测癌症复发或转移的可能性，为临床上选择治疗方案提供一定的参考价值。
在一些实施例中，获取了目标受试者的生物样品后，可以使用该试剂盒检 测生物样品中CH25H基因的表达水平。可以基于该CH25H基因的RNA转录物确定CH25H基因的表达水平。在检测CH25H基因的表达水平时，可进行标准化，即使用内参基因的表达水平作为参照，来表征CH25H基因的标准化表达水平。通过测定CH25H的mRNA的含量可以确定CH25H基因的表达水平。如本文中所使用的，术语“内参基因”是生物体或者细胞中稳定表达的基因，表达量几乎不变，用来作为CH25H基因表达量的对照。
在一些实施例中，可以从生物样品(例如，外周血)中提取白细胞，例如，通过自然沉降法、加速红细胞沉降法等。再从白细胞中提取RNA，例如通过RNA提取试剂盒。在一些实施例中，可以直接从生物样品(例如，外周血)中提取RNA。关于提取RNA的详细内容，可以在本披露的其他部分中找到，例如在图5中的步骤501及其描述中。
受试者的生物样品中可以含有目标RNA，该目标RNA可以源自于CH25H基因。在一些实施例中，目标RNA可以由CH25H基因经转录得到。生物样品中还含有内参RNA。内参RNA可以是内参基因所转录的RNA。内参基因可以包括但不限于核糖体蛋白(ribosomal protein，RPLO)、3-磷酸甘油醛脱氢酶(GAPDH)、β-肌动蛋白(β-actin，ACTB)、18sRNA、28sRNA、β2微球蛋白(B2M)、琥珀酸脱氢酶亚单位A(SDHA)、次黄嘌呤磷酸核糖转移酶1(hypoxanthine phosphoribosyltransferase 1，HPRT1)、区结合蛋白(attachment region binding protein，ARBP)等中的一种或多种。例如，内参RNA可以由RPLO基因经转录得到。
在一些实施例中，可以通过试剂盒来检测CH25H基因的标准化表达水平。试剂盒可以包括一个或多个不同用途的试剂盒。例如，试剂盒可以包括RNA提取试剂盒、反转录试剂盒、表达水平检测试剂盒(例如qPCR试剂盒)。具体的，可以通过RNA提取试剂盒提取生物样品中的RNA含量(例如，CH25H和内参基因的RNA)。可以通过反转录试剂盒将RNA反转录成cDNA(例如，CH25H 和内参基因的cDNA)。可以通过qPCR试剂盒基于实时荧光定量PCR技术检测CH25H基因的标准化表达水平。qPCR试剂盒中包括检测CH25H基因表达水平的试剂，其包括CH25H基因的特异性探针和CH25H基因的特异性引物。关于检测生物样品中CH25H基因的标准化表达水平的详细内容，可以在本披露的其他部分中找到，例如在图2及其描述中。
通过qPCR，可以确定CH25H基因的标准化表达水平。QPCR的定量方式可以分为绝对定量和相对定量两种方式。绝对定量是通过目标基因的标准品建立的外标准曲线对样品中目标基因(即CH25H基因)的拷贝值的准确含量。相对定量是用于分析特定样品相对于参照样品(比如，未处理的对照组)某个基因表达量的变化。仅作为示例，本申请中可使用相对定量的方式来确定CH25H基因的标准化表达水平。用于相对定量的计算方法有标准曲线法和比较循环阈值(CT)法。标准曲线法为通过制作校准品(用作比较结果基础的样品)的标准曲线来获取相对定量结果。比较CT法为基于比较目标基因和内参基因的CT值获取CH25H基因的标准化表达水平。
在一些实施例中，可以通过内参基因的CT值和CH25H基因的CT值的差值来确定CH25H基因的标准化表达水平。例如，可以用ΔCT表示CH25H基因的标准化表达水平。ΔCT可以通过以下公式(1)确定：
ΔCT＝CT 内参-CT CH25H         (1)
其中，CT 内参表示同一生物样品内的内参基因的CT值，CT CH25H表示生物样品内的CH25H基因的CT值。在一些实施例中，也可以用-ΔCT表示CH25H基因的标准化表达水平。
在一些实施例中，可以通过内参基因的CT值和CH25H基因的CT值的比值来确定CH25H基因的标准化表达水平。例如，可以用2 CT 内参/2 CT CH25H来表示。2 CT 内参/2 CT CH25H相当于
Figure PCTCN2020103579-appb-000001
因此，CH25H基因的标准化表达水平还可以表示为2 ΔCT。在一些实施例中，也可以用2 -ΔCT表示CH25H基因的标准 化表达水平。
在一些实施例中，除了使用qPCR技术检测CH25H基因的mRNA表达量的外，还可以通过检测CH25H基因的蛋白质表达量来表示CH25H基因的表达水平。例如，使用ELISA试剂盒检测CH25H基因表达的蛋白质含量。仅作为示例，可以使用夹心ELISA原理，将捕获抗体预包被在96孔微型ELISA板上，并将生物素偶联的抗体用作检测抗体。随后将该酶的标准品、测试样品和生物素偶联的检测抗体添加到孔中，进行孵育，然后洗涤。加入辣根过氧化物酶标记链霉亲和素，并用洗涤缓冲液洗去未结合的结合物。3，3′，5，5′-四甲基联苯胺(TMB)底物用于可视化HRP(辣根过氧化物酶)酶促反应。通过HRP催化TMB生成蓝色产物，加入酸性终止液后变成黄色。颜色的密度与从样品中捕获的CH25H的量成正比，从而可以确定出CH25H的蛋白含量。
在一些实施例中，可以多次重复进行步骤401以检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平的多个数值，并根据多个数值确定平均数值后，在后续步骤403-407中使用该平均数值来预测目标受试者癌症复发或转移的可能性。
在一些实施例中，可以将步骤401获取的和与目标受试者的生物样品中CH25H基因的表达水平相关的测试数据发送给处理器110和/或存储器130。测试数据可以包括对应于CH25H基因的第一循环阈值与对应于内参基因的第二循环阈值。处理器110可以基于第一循环阈值与第二循环阈值来确定标准化表达水平。
在步骤403中，可以基于该生物样品中CH25H基因的该标准化表达水平，确定目标参数。
目标参数可以用于与参考阈值进行比较，从而预测患者预后肿瘤转移或复发的风险。在一些实施例中，步骤403-407中的至少一部分可以由用户手动进行。在一些实施例中，步骤403-407中的至少一部分可以由处理设备110完成。 例如，步骤403可以由预后分析模块330完成。
在一些实施例中，处理设备110可以基于该目标受试者的该生物样品中该CH25H基因的标准化表达水平(下文中简称为“样品标准化表达水平”)和多个未患癌症的第一参考受试者的生物样品中该CH25H基因的标准化表达水平来确定该目标参数。第一参考受试者可以是未患有癌症的健康受试者(例如人)，可以用作对照组。检测第一参考受试者的生物样品(例如，外周血)中该CH25H基因的标准化表达水平的方法与步骤401中该的检测目标受试者的生物样品中的CH25H基因的标准化表达水平的方法类似，在此不再赘述。在一些实施例中，可以确定多个第一参考受试者的标准化表达水平的平均值作为对照标准化表达水平。在一些实施例中，可以将第一参考受试者按照特征信息(例如年龄、性别、身体健康状况等)进行分组。例如，一组第一参考受试者可以为年龄段在30-50之间。又例如，一组第一参考受试者可以是全为男性或女性，或男性和女性各占一半。再例如，一组第一参考受试者可以是有慢性病(例如，糖尿病)的受试者。在一些实施例中，可以为每组第一参考受试者，分别确定对照标准化表达水平的一个平均数值。
在一些实施例中，可以将上述对照标准化表达水平的一个或多个数值存储在存储设备(例如存储设备130)中。处理设备110可以从存储设备中获取对照标准化表达水平的一个或多个数值。例如，处理设备110可以基于目标受试者的特征信息(例如年龄、性别、身体健康状况等中的至少一个)选取对应的对照标准化表达水平的数值。处理设备110可以进一步基于样品标准化表达水平和对照标准化表达水平，来确定目标参数。
例如，目标参数可以基于以ΔCT形式表示的标准化表达水平确定。目标参数可以通过比较样品标准化表达水平与对照标准化表达水平而确定。在一些实施例中，目标参数可以用dΔCT表示。dΔCT可以通过以下公式(2)确定：
dΔCT＝ΔCT 样品-ΔCT 对照          (2)，
其中，ΔCT 样品表示目标受试者中的生物样品中标准化表达水平，ΔCT 对照表示对照组中的生物样品中标准化表达水平。
又例如，目标参数可以基于指数函数确定，例如将基于CT值的差值确定的标准化表达水平作为指数。在一些实施例中，目标参数可以为2 dΔCT。再例如，目标参数可以为e dΔCT。在一些实施例中，目标参数可以为2 -dΔCT。再例如，目标参数可以为e -dΔCT。
在一些实施例中，目标参数可以根据基于以2 ΔCT形式表示的标准化表达水平确定。例如，目标参数可以通过样品标准化表达水平与对照标准化表达水平的比值确定。例如，可以通过以下公式(3)确定：
目标参数＝2 ΔCT 样品/2 ΔCT 对照          (3)
其中，2 ΔCT 样品表示目标受试者的生物样品内的CH25H基因的标准化表达水平，2 ΔCT 对照表示第一参考受试者的生物样品内的CH25H基因的标准化表达水平。
在步骤405中，处理设备(例如，预后分析模块330)可以将该目标参数与一项或多项参考阈值进行比较以获取比较结果。
在一些实施例中，该参考阈值可以是基于多个患有癌症的第二参考受试者的生物样品中CH25H基因的标准化表达水平而确定的。第二参考受试者是指曾患有癌症并接受过治疗的受试者。并且，对于第二参考受试者，在接受治疗后的一定时间段内癌症是否复发转移的情况是已知的。该时间段可以是三年，五年等。检测第二参考受试者的生物样品中该CH25H基因的标准化表达水平的方法与步骤401中该的检测目标受试者的生物样品中CH25H基因的标准化表达水平的方法类似，在此不再赘述。可以通过放射性检查、肿瘤标记物等方式检查第二参考受试者肿瘤是否有转移或复发，将第二参考受试者分成肿瘤已转移组和肿瘤未转移组。在一些实施例中，可以确定已转移组和未转移组中每个第二参考受试者的对照参数。对照参数的确定方法与目标参数的确定方法类似，在此不再赘述。在确定每个第二参考受试者的对照参数后，可以基于两组中的对照参数确定 参考阈值。在一些实施例中，该参考阈值包括第一参考阈值和第二参考阈值。例如，可以将已转移组的对照参数的平均值作为第一参考阈值，将肿瘤未转移组的对照参数的平均值作为第二参考阈值。
具体的，可以根据步骤403确定两组第二参考受试者中的目标参数，从而确定第一和第二参考阈值。例如，若目标参数用2 dΔCT(或2 ΔCT 样品/2 ΔCT 对照)表示，第一参考阈值可以为0.48、0.5、0.52、0.54、0.6等，第二参考阈值可以为0.9、0.95、1、1.05等。例如，第一参考阈值可以是0.5，第二参考阈值可以是1。又例如，若目标参数用dΔCT表示，第一参考阈值可以是-1.2、-1、-0.95、-0.82、-0.7、-0.6等。第二参考阈值可以是0、0.2、0.39、0.46、0.6、0.8等。例如，第一参考阈值可以是-1，第二参考阈值可以是0。
在步骤407中，处理设备110可以基于该比较结果，确定该目标受试者癌症复发或转移的可能性。
在一些实施例中，响应于该比较结果为该目标参数小于该第一参考阈值，确定该目标受试者癌症复发或转移的可能性较高。在一些实施例中，响应与该比较结果为目标参数大于第三阈值，确定该目标受试者癌症复发或转移的可能性较高。例如，该癌症复发或转移的可能性较高可以指癌症复发或转移的可能性大于70％，75％，或80％等。
在一些实施例中，响应于该比较结果为该目标参数大于该第二参考阈值，预测该目标受试者癌症复发或转移的可能性较低。在一些实施例中，响应于该比较结果为该目标参数小于该第四参考阈值，预测该目标受试者癌症复发或转移的可能性较低。该癌症复发或转移的可能性较低对应的是癌症复发或转移的可能性小于40％，35％，或30％等。
在一些实施例中，可以通过机器学习模型来确定癌症复发或转移的可能性的数值。在一些实施例中，可以将目标患者的特征信息(例如，目标受试者的测试数据、性别、年龄、目标受试者曾经患有或现在患有的癌症的种类、目标受 试者患有的其他疾病等)输入到训练好的机器学习模型中，以获得肿瘤患者的预后预测结果。该预测结果可以表现为：肿瘤是否有复发或转移；复发或转移的可能性(例如，较高或较低、具体百分比数值等)。在一些实施例中，机器学习模型可以是监督学习模型。具体的，机器学习模型可以包括：支持向量机模型、决策树模型、神经网络模型、最近邻分类器等中的一种或几种的组合。例如，可以使用多个第二受试者的测试数据、多个第二受试者的特征信息以及是否有复发或转移的结果来训练机器学习模型。
应当注意的是，上述有关流程400的描述仅仅是为了示例和说明，而不限定本申请的适用范围。对于本领域技术人员来说，在本申请的指导下可以对流程400进行各种修正和改变。然而，这些修正和改变仍在本申请的范围之内。
图5是根据本申请一些实施例所示的一种检测生物样品中CH25H基因的标准化表达水平的流程图。在一些实施例中，流程500中的至少一部分步骤(例如，步骤507)可以由计算设备(如图2所示的计算设备200，图1中的处理设备)完成。例如，流程500中的至少一部分步骤可以被实现为存储在存储器130、存储器220中的一个指令(例如，应用程序)。图1中的处理设备110，图2中的处理器210和/或模块可以执行该指令，并且在执行指令时，处理设备110、处理器210和/或模块可以被配置为执行流程500。以下所示过程的操作仅出于说明的目的。在一些实施例中，流程500可以利用未描述的一个或以上附加操作和/或未描述的一个或以上操作来完成。另外，图5所示和以下描述的过程的操作顺序并非旨在限制。
在步骤501中，可以从生物样品中提取CH25H基因的RNA和内参基因的RNA。
提取RNA的方法可以包括异硫氰酸胍氯化铯超速离心法、盐酸胍-有机溶剂法、氯化锂-尿素法、热酚法、快速提取法、细胞质RNA提取法、酚-氯化锂法同时提取细胞RNA和DNA、一步快速热酚抽提法。在一些实施例中，通过 提取RNA的方法可以在生物样品(例如，外周血)中提取RNA。在一些实施例中，提取目标RNA和内参RNA的步骤至少可以包括高温变性、萃取、沉淀、洗涤和溶解。在一些实施例中，目标RNA和内参RNA可以直接从生物样品(例如，外周血)中提取。不需要先从生物样品中提取白细胞，再从白细胞中提取目标RNA和内参RNA。在一些实施例中，可以先从生物样品中提取白细胞，例如，通过自然沉降法、加速红细胞沉降法等。再从白细胞中提取目标RNA和内参RNA。在一些实施例中，提取目标RNA和内参RNA的试剂盒可以为如表1所示的试剂盒。需要注意的是，表1中的试剂仅作为示例，本领域内技术人员可以对试剂的种类和用量进行变化和修改。
表1.RNA提取试剂盒
名称 规格 主要成分
Buffer A 40ml Tris、NaCl、SDS
Buffer B 15ml 氯仿
Buffer C 15ml 糖原、醋酸钠
Buffer E 30ml 无水乙醇
Buffer F 30ml 无水乙醇
Buffer G 15ml H 2O
T1离心管 50个 聚丙烯
T2离心管 100个 聚丙烯
在步骤503中，可以对CH25H基因的RNA和内参基因的RNA分别进行反转录，获取该CH25H基因的cDNA和该内参基因的cDNA。
反转录可以是以目标基因和内参基因分别转录成的RNA为模板，通过反转录酶，合成互补的单链DNA(cDNA)。
在一些实施例中，目标RNA反转录使用的特异性反转录引物可以包括：与SEQ ID NO.：4所示序列的相似度≥70％的核苷酸，与SEQ ID NO.：4所示序列的相似度≥75％的核苷酸，与SEQ ID NO.：4所示序列的相似度≥80％的核苷酸，与SEQ ID NO.：4所示序列的相似度≥85％的核苷酸，与SEQ ID NO.：4所示序列的相似度≥90％的核苷酸，与SEQ ID NO.：4所示序列的相似度≥95％的核苷酸。 在一些实施例中，该内参RNA反转录使用的特异性反转录引物可以包括：与SEQ ID NO.：5所示序列的相似度≥70％的核苷酸，与SEQ ID NO.：5所示序列的相似度≥75％的核苷酸，与SEQ ID NO.：5所示序列的相似度≥80％的核苷酸，与SEQ ID NO.：5所示序列的相似度≥85％的核苷酸，与SEQ ID NO.：5所示序列的相似度≥90％的核苷酸，与SEQ ID NO.：5所示序列的相似度≥95％的核苷酸。
可以通过表2中的反转录试剂盒对CH25H基因和内参基因进行反转录。需要注意的是，表2中的反转录试剂盒仅作为示例，本领域内技术人员可以对试剂的种类和用量进行变化和修改。
表2.反转录试剂盒
名称 规格
5×RT Buffer 4μL
引物终浓度 200nM
dNTP 1mM
super RT 200U
RNA 6μL
补DEPC水至 20μL
在步骤505中，可以针对CH25H基因的cDNA和内参基因的cDNA，进行PCR扩增反应。
将目标cDNA进行的PCR扩增反应为实时荧光定量PCR。PCR扩增的基本原理是：以单链DNA(cDNA)为模板，4种dNTP为底物，在模板3’末端有引物存在的情况下，用酶进行互补链的延伸，多次反复的循环能使微量的模板DNA得到极大程度的扩增。在微量离心管中，加入与待扩增的DNA片段两端已知序列分别互补的两个引物、适量的缓冲液、微量的DNA膜板、四种dNTP溶液、耐热Taq DNA聚合酶、Mg 2+等。反应时先将上述溶液加热，使模板DNA在高温下变性，双链解开为单链状态；然后降低溶液温度，使合成引物在低温下与其靶序列配对，形成部分双链，称为退火；再将温度升至合适温度，在TaqDNA聚合酶的催化下，以dNTP为原料，引物沿5’→3’方向延伸，形成新的DNA 片段，该片段又可作为下一轮反应的模板，如此重复改变温度，由高温变性、低温复性和适温延伸组成一个周期，反复循环，使目的基因得以迅速扩增。
在一些实施例中，该检测CH25H基因表达水平的试剂包括该CH25H基因的特异性引物。在一些实施例中，该CH25H基因的特异性引物序列与SEQ ID NO.：2所示序列的相似度大于或等于70％、75％、80％、85％、90％或95％；或者该CH25H基因的特异性引物序列与SEQ ID NO.：3所示序列的相似度大于或等于70％、75％、80％、85％、90％或95％。CH25H基因的特异性引物序列可以包括正向引物序列和反向引物序列，正向引物序列对应于SEQ ID NO.：2，反向引物序列对应于SEQ ID NO.：3。
将该内参cDNA进行的PCR扩增反应为实时荧光定量PCR(Q-PCR)。在一些实施例中，内参cDNA进行扩增使用的qPCR特异性引物可以包括：与SEQ ID NO.：6所示序列的相似度≥70％的核苷酸，与SEQ ID NO.：6所示序列的相似度≥75％的核苷酸，与SEQ IDNO.：6所示序列的相似度≥80％的核苷酸，与SEQ ID NO.：6所示序列的相似度≥85％的核苷酸，与SEQ ID NO.：6所示序列的相似度≥90％的核苷酸，与SEQ ID NO.：6所示序列的相似度≥95％的核苷酸；或与SEQ ID NO.：7所示序列的相似度≥70％的核苷酸，与SEQ ID NO.：7所示序列的相似度≥75％的核苷酸，与SEQ ID NO.：7所示序列的相似度≥80％的核苷酸，与SEQ ID NO.：7所示序列的相似度≥85％的核苷酸，与SEQ ID NO.：7所示序列的相似度≥90％的核苷酸，与SEQ IDNO.：7所示序列的相似度≥95％的核苷酸。内参基因的特异性引物序列可以包括正向引物序列和反向引物序列，正向引物序列对应于SEQ ID NO.：6，反向引物序列对应于SEQ ID NO.：7。
在一些实施例中，该检测CH25H基因表达水平的试剂包括CH25H基因的特异性探针。目标cDNA进行扩增使用的探针可以包括：与SEQ ID NO.：1所示序列的相似度≥70％的核苷酸，与SEQ ID NO.：1所示序列的相似度≥75％的核苷酸，与SEQ ID NO.：1所示序列的相似度≥80％的核苷酸，与SEQ ID NO.： 1所示序列的相似度≥85％的核苷酸，与SEQ IDNO.：1所示序列的相似度≥90％的核苷酸，与SEQ ID NO.：1所示序列的相似度≥95％的核苷酸等。在一些实施例中，内参cDNA进行扩增使用的探针可以包括：与SEQ ID NO.：8所示序列的相似度≥70％的核苷酸，与SEQ ID NO.：8所示序列的相似度≥75％的核苷酸，与SEQ ID NO.：8所示序列的相似度≥80％的核苷酸，与SEQ ID NO.：8所示序列的相似度≥85％的核苷酸，与SEQ ID NO.：8所示序列的相似度≥90％的核苷酸，与SEQ ID NO.：8所示序列的相似度≥95％的核苷酸。
可以通过表3中的qPCR试剂盒对CH25H基因和内参基因进行qPCR。需要注意的是，表3中的qPCR试剂盒仅作为示例，本领域内技术人员可以对试剂的种类和用量进行变化和修改。
表3.qPCR试剂盒
名称 规格
10×PCR Buffer 稀释为1×
dNTP 0.2mM
模板 2uL
各引物 200-400nM
各探针 100-400nM
热启动Taq酶 1U
MgCL 2 2-5mM
总体积 20μL
在步骤507中，可以根据PCR扩增反应中CH25H基因的cDNA的第一循环阈值与该内参基因的cDNA的第二循环阈值，确定该生物样品中CH25H基因的标准化表达水平。
在一些实施例中，目标cDNA的数量和内参cDNA的数量可以表征为目标cDNA的循环阈值(Cycle threshold，CT)(也称为第一循环阈值)和内参cDNA的CT值(也称为第二循环阈值)。在实时荧光定量PCR技术中，CT值是指每个反应管内的荧光信号达到设定的阈值时所经历的循环阈值。
该根据PCR扩增反应中CH25H基因的cDNA的第一循环阈值(可表示 为CT CH25H)与该内参基因的cDNA的第二循环阈值(可表示为CT 内参)，确定该生物样品中CH25H基因的标准化表达水平包括确定该第一循环阈值与第二循环阈值的差值(可表示为ΔCT)，用该第一循环阈值与第二循环阈值的该差值表示该生物样品中CH25H基因的标准化表达水平(例如，ΔCT或2 ΔCT)。
根据本申请的一方面，提供了一种检测CH25H基因表达水平的试剂在制备用于癌症预后预测的试剂盒中的一种用途。预测可以包括使用该试剂盒检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平；基于该生物样品中CH25H基因的该标准化表达水平，确定目标参数；将该目标参数与一项或多项参考阈值进行比较以获取比较结果；基于该比较结果，确定该目标受试者癌症复发或转移的可能性。
根据本申请的另一方面，提供了一种癌症预后预测装置。该装置可以包括获取模块，用于被配置为获取测试数据，该测试数据与目标受试者的生物样品中CH25H基因的表达水平相关；标准化表达水平确定模块，用被配置为于基于该测试数据，确定该生物样品中CH25H基因的标准化表达水平；预后分析模块，被配置为基于该生物样品中该CH25H基因的该标准化表达水平，确定目标参数；将该目标参数与一项或多项参考阈值进行比较以获取比较结果；以及基于该比较结果，确定该目标受试者癌症复发或转移的可能性。
根据本申请的另一方面，提供了一种计算机可读存储介质。该存储介质存储计算机指令，当该计算机指令被处理器执行时，实现一种癌症预后预测方法，其特征在于，该癌症预后预测方法包括：获取测试数据，该测试数据与目标受试者的生物样品中CH25H基因的表达水平相关；基于该测试数据，确定该生物样品中CH25H基因的标准化表达水平；基于该生物样品中该CH25H基因的该标准化表达水平，确定目标参数；将该目标参数与一项或多项参考阈值进行比较以获取比较结果；以及基于该比较结果，确定该目标受试者癌症复发或转移的可能性。
根据本申请的另一方面，提供了一种癌症预后预测方法。该方法可以包括：获取测试数据，该测试数据与目标受试者的生物样品中CH25H基因的表达水平相关检测该生物样品中CH25H基因的标准表达水平；基于该测试数据，确定该生物样品中CH25H基因的标准化表达水平；基于该生物样品中该CH25H基因的该标准化表达水平，确定目标参数；将该目标参数与一项或多项参考阈值进行比较以获取比较结果；以及基于该比较结果，确定该目标受试者癌症复发或转移的可能性。
根据本申请的另一方面，提供了一种方法，用于治疗癌症或预防癌症复发转移。该方法可以包括：检测获取自目标受试者的该生物样品中CH25H基因的标准化表达水平；基于该生物样品中该CH25H基因的该标准化表达水平，确定目标参数；将该目标参数与一项或多项标准参考阈值进行比较；响应于该目标参数小于该参考标准阈值，为受试者施用一种组合物，该组合物含有能提高CH25H基因的表达水平或能提高CH25H蛋白活性的试剂。
在一些实施例中，当预测出癌症复发或转移的可能性较高时，可以为目标受试者提供干预治疗，还可以提高复查频率。
在一些实施例中，当预测出癌症复发或转移的可能性较高时，可为目标受试者提供药物治疗，例如为目标受试者施用一种组合物。该组合物含有能提高CH25H基因的表达水平或能提高CH25H蛋白活性的试剂。在一些实施例中，该试剂可以包括利血平。在一些实施例中，该试剂还可以包括干扰素或Toll样受体4(TLR4)激动剂等。TLR4激动剂可以包括但不限于脂多糖(LPS)、类脂A及其衍生物。在一些实施例中，该组合物可以单独用药或与其他抗癌药物(例如，单克隆抗体、抗肿瘤抗生素等)一起使用。
应当注意的是，上述有关流程500的描述仅仅是为了示例和说明，而不限定本申请的适用范围。对于本领域技术人员来说，在本申请的指导下可以对流程500进行各种修正和改变。然而，这些修正和改变仍在本申请的范围之内。
下述实施例中的实验方法，如无特殊说明，均为常规方法。下述实施例中所用的试验材料，如无特殊说明，均为自常规生化试剂公司购买得到的。以下实施例中的定量试验，均设置三次重复实验，结果取平均值。
实施例1、提取该目标RNA和内参RNA。
1.1将血液样品置于离心管(自备)中，1200rpm常温离心20分钟。
1.2取上层全部血浆于离心管(自备)中，抽取其中的1mL血浆于T1离心管中，剩余的血浆2100rpm常温离心20分钟。
1.3抽取上层血浆于-80℃保存或直接提取ctDNA，沉淀为血小板。
1.4用步骤1.2中的1mL血浆依次悬浮血小板。
1.5沸水孵育5分钟，冰上冰浴5分钟。
1.6短暂离心，保持含RNA的离心管置于冰上，捣碎，加入600ul Buffer A，涡旋震荡混匀，加入200ul Buffer B，涡旋震荡混匀，15000rpm4℃离心20分钟。
1.7保持含RNA的离心管置于冰上，抽取上清置于T2离心管中(分装两管，每管约500ul)，加入50ul Buffer C和1mL无水乙醇，上下颠倒至混匀。置于-80℃，沉淀2小时。
1.8 15000rpm 4℃离心10分钟，弃上清。
1.9加入1mL Buffer E，涡旋震荡，使沉淀悬浮，15000rpm 4℃离心10分钟，弃上清。
1.10加入1mL Buffer F，涡旋震荡，使沉淀悬浮，15000rpm 4℃离心10分钟，弃上清。
1.11短暂离心(离心机升到最大转速停止)，吸弃残余液体，将离心管置于室温数分钟以彻底晾干。
1.12向每个离心管中加入10ul Buffer G，立即置于冰上，轻弹，短暂离心，保持RNA置于冰上。提取好的RNA应尽快进行反转录，防止RNA降解。
实施例2、将该目标RNA和内参RNA分别反转录为目标cDNA和内参cDNA。
该反转录体系如下：
5×RT Buffer 4μL
引物终浓度 200nM
dNTP 1mM
super RT 200U
RNA 6μL
补DEPC水至20μL。
2.1取样品RNA 11.5μL，super RT反转录酶1μL，反转录引物1μL，dNTP混合物2μL，缓冲液4μL，RNA酶抑制剂0.5μL，加入无菌离心管中，混匀。其中，目标RNA的反转录引物为：ccacattgtctgctcccaca(SEQ ID NO.：4)，内参RNA的反转录引物为：atgtcgaagaagcccaaaga(SEQ ID NO.：5)。
2.2启动PCR仪程序：42℃保温1个小时，70℃孵育10分钟；反应结束后，短暂离心，置于10℃冷却10分钟。
2.3反转录产物可直接用于后续PCR扩增反应。
实施例3、将该目标cDNA和内参cDNA进行PCR扩增反应。
PCR扩增体系如下：
10×PCR Buffer 稀释为1×
dNTP 0.2mM
模板 2uL
各引物 200-400nM
各探针 100-400nM
热启动Taq酶 1U
MgCL2 2-5mM
总体积 20uL。
取缓冲液18μL，0.2μM的PCR特异引物和0.2μM的探针的混合物，反转录样品cDNA 1μL，Tac酶1μL，加入无菌离心管中，混匀。其中，目标基因CH25H的特异性引物为：aaggtgcaccaccagaactc(SEQ ID NO.：2，正向引物)，atgtcgaagaagcccaaaga(SEQ ID NO.：3，反向引物)。内参基因RPLO的特异性引物为：gcgacctggaagtccaacta(SEQ ID NO.：6，正向引物)，ccacattgtctgctcccaca(SEQ ID NO.：7，反向引物)。目标基因CH25H的特异性探针的序列为：caacgcagtatatgagc(SEQ ID NO.：1)，内参基因RPLO的特异性探针的序列为：cttaagatcatccaactattg(SEQ ID NO.：8)。
PCR扩增反应的条件如下：
表4.PCR扩增反应的条件
Figure PCTCN2020103579-appb-000002
实施例4、确定第一参考受试者的目标RNA和内参RNA的表达水平。
选取健康人的血液样品11例，进行上述实施例1到3的步骤。PCR扩增反应之后，可以手动调节扩增曲线中的基线(baseline)的起始点、终止点和荧光阈值(threshold)。扩增曲线指的是以循环数为横坐标，以反应过程中实时荧光强度为纵坐标所做的曲线。基线指的是在PCR扩增反应的最初数个循环里，荧光强度变化不大而接近直线的一条线。荧光阈值是PCR前3-15个循环荧光强度标准差的10倍，荧光阈值设定在PCR扩增的指数期。CT值表示每个PCR反应管内荧光信号到达设定的阈值时所经历的循环数。根据调节的阈值和基线，确定健康人的血液样品中的CH25H基因和内参基因RPLO的CT值并取平均值，将健康人的CH25H基因和内参基因的CT值的平均值作为对照。
部分第一参考受试者的CH25H基因和内参基因的CT值如下表5所示：
表5.第一参考受试者的标准化表达水平
编号 CT CH25H CT RPLO ΔCT 2 ΔCT
1 19.52 6.48 -13.04 0.000118732
2 19.56 6.85 -12.71 0.000149248
3 21.39 7.79 -13.6 8.05364E-05
4 19.85 8.21 -11.64 0.000313336
5 21.6 7.77 -13.83 6.86681E-05
6 19.15 7.85 -11.3 0.000396608
7 19.94 7.81 -12.13 0.000223103
8 17.62 6.54 -11.08 0.000461942
9 19.88 6.92 -12.96 0.000125502
10 19.25 7.63 -11.62 0.00031771
11 20.88 9.63 -11.25 0.000410594
平均值 19.87 7.58 -12.29 0.000199683
将第一参考受试者的血液样品中的CH25H基因和内参基因RPLO的CT值取平均值，作为对照。从该表中，可知第一参考受试者的标准化表达水平。仅作为示例，标准化表达水平可使用ΔCT或2 ΔCT来表示。ΔCT为-12.29，2 ΔCT为0.000199683。
实施例5、确定样品中的目标RNA和内参RNA的表达水平。
选取20例患有不同种类的肺癌并已接受过治疗的患者(即，第二参考受试者)，经过标准手术治疗后，采取10ml血液样品，进行上述实施例1-3的步骤，其中实施例3与实施例4中的生物样品同时进行反应。在手动调节基线和 阈值之后，得到癌症患者样品的CH25H基因和内参基因，并计算标准化表达水平(例如，以ΔCT或2 ΔCT表示)和对照参数(例如，以dΔCT、2 dΔCT或2 ΔCT 样 品/2 ΔCT 对照表示)。数值如下表6所示：
表6.对照参数示意表
编号 CT CH25H CT RPLO ΔCT dΔCT 2 dΔCT 2 ΔCT 样品/2 ΔCT 对照
1 24.21 12.31 -11.9 0.39 1.310393404 1.31039419
2 24.19 11.3 -12.89 -0.6 0.659753955 0.659754351
3 35 18.05 -16.95 -4.66 0.039554894 0.039554917
4 35 12.01 -22.99 -10.7 0.000601145 0.000601145
5 22.07 11.93 -10.14 2.15 4.438277888 4.43828055
6 35 11.98 -23.02 -10.73 0.000588773 0.000588774
7 35 12.01 -22.99 -10.7 0.000601145 0.000601145
8 35 9.13 -25.87 -13.58 8.16606E-05 8.16607E-05
9 22.06 10.23 -11.83 0.46 1.375541818 1.375542643
10 21.57 13.55 -8.02 4.27 19.29292524 19.29293681
11 24.33 11.09 -13.24 -0.95 0.517632462 0.517632772
12 24.44 11.33 -13.11 -0.82 0.566441943 0.566442282
13 22.02 13.43 -8.59 3.7 12.99603834 12.99604614
14 35 14.12 -20.88 -8.59 0.002595089 0.002595091
15 18.35 13.26 -5.09 7.2 147.0333894 147.0334776
16 22.59 12.02 -10.57 1.72 3.294364069 3.294366045
17 35 14.01 -20.99 -8.7 0.002404579 0.00240458
18 35 13.97 -21.03 -8.74 0.002338826 0.002338827
19 35 15.67 -19.33 -7.04 0.007598867 0.007598871
20 21.16 9.68 -11.48 0.81 1.753211443 1.753212494
根据上述表格，通过CH25H的目标参数dΔCT、2 dΔCT和2 ΔCT 样品/2 ΔCT 对照的数值，当dΔCT的数值大于0时，表示第二参考受试者中CH25H基因的表达量相对于健康人该基因的表达量高；反之，则低。当2 dΔCT大于1时，第二参考受试者中CH25H基因的表达量相对于健康人该基因的表达量高；反之，则低。当2 ΔCT 样品/2 ΔCT 对照大于1时，第二参考受试者中CH25H基因的表达量相对于健康人该基因的表达量高；反之，则低。
由于2 ΔCT 样品/2 ΔCT 对照还可以表示为
Figure PCTCN2020103579-appb-000003
即2 dΔCT。因此，2 ΔCT 样 品/2 ΔCT 对照和2 dΔCT表达方式不同，但结果相同。
实施例6、患者转移情况与目标参数和参考阈值的比较结果的关系。
随访患者，看癌症是否有转移。以下表7中是20个患者癌症是否转移与CH25H相对表达量的关系：
表7.患者随访结果
Figure PCTCN2020103579-appb-000004
Figure PCTCN2020103579-appb-000005
从此表中可知，相对于未发生转移的肺癌患者，发生转移的患者白细胞上CH25H基因mRNA表达明显下降，并且具有显著性差异(p＜0.01％)，CH25H mRNA表达可作为肺癌转移与否的良好生物标记物。例如，当CH25H的目标参数以dΔCT表示时，第一参考阈值为-1，当dΔCT小于-1时，CH25H基因的表达量较低，癌症转移的可能性较高；第二参考阈值为0，当dΔCT大于0时，CH25H基因的表达量较高，癌症转移的可能性较低。又例如，当CH25H的目标参数以2 dΔCT表示时，第一参考阈值为0.5，当2 dΔCT小于0.5时，CH25H基因的 表达量较低，癌症转移的可能性较高；第二参考阈值为1，当2 dΔCT大于1时，CH25H基因的表达量较高，癌症转移的可能性较低。再例如，当CH25H的目标参数以2 ΔCT 样品/2 ΔCT 对照表示时，第一参考阈值为0.5，当2 ΔCT 样品/2 ΔCT 对照小于0.5时，CH25H基因的表达量较低，癌症转移的可能性较高；第二参考阈值为1，当2 ΔCT 样品/2 ΔCT 对照大于1时，CH25H基因的表达量较高，癌症转移的可能性较低。
本申请所披露的一种癌症预后预测的方法，可能带来的有益效果包括但不限于：(1)CH25H基因可作为癌症预后预测的标记物，能够实时、无创监测肿瘤术后复发转移；(2)通过CH25H基因的目标参数与参考阈值相比较，能够准确预测出肿瘤复发转移的可能性高低，对选择患者的治疗方案可起到一定帮助；(3)若复发转移的可能性较高，可在手术、放疗或化疗后加大复查频率，并在手术、放疗或化疗前后进行辅助性治疗。
需要说明的是，不同实施例可能产生的有益效果不同，在不同的实施例里，可能产生的有益效果可以是以上任意一种或几种的组合，也可以是其他任何可能获得的有益效果。
本领域的技术人员应当理解，以上实施例仅为说明本发明，而不对本发明构成限制。凡在本发明的精神和原则内所作的任何修改、等同替换和变动等，均应包含在本发明的保护范围之内。

Claims (82)

  1. 检测CH25H基因表达水平的试剂在制备用于癌症预后预测的试剂盒中的一种用途，其特征在于，所述预测包括：
    使用所述试剂盒检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平；
    基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数；
    将所述目标参数与一项或多项参考阈值进行比较以获取比较结果；以及
    基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。
  2. 如权利要求1所述的用途，其特征在于，所述一项或多项参考阈值包括第一参考阈值，所述基于所述比较结果，确定癌症复发或转移的可能性包括：
    响应于所述比较结果为所述目标参数小于所述第一参考阈值，确定所述目标受试者癌症复发或转移的可能性较高。
  3. 如权利要求1或2所述的用途，其特征在于，所述一项或多项参考阈值包括第二参考阈值，所述基于所述比较结果，预测癌症复发或转移的可能性包括：
    响应于所述比较结果为所述目标参数大于所述第二参考阈值，预测所述目标受试者癌症复发或转移的可能性较低。
  4. 如权利要求1-3中任一项所述的用途，其特征在于，所述CH25H基因的所述标准化表达水平是基于CH25H基因在目标受试者中的表达水平和内参基因在目标受试者中的表达水平确定的。
  5. 如权利要求4所述的用途，其特征在于，所述内参基因包括RPLO、GAPDH、ACTB、B2M、SDHA、HPRT1、ARBP、18sRNA、28sRNA中的一种或多种。
  6. 如权利要求4所述的用途，其特征在于，所述CH25H基因的所述标准化表达水平是基于所述CH25H基因的RNA转录物确定的。
  7. 如权利要求6所述的用途，其特征在于，所述检测所述生物样品中CH25H基因的标准化表达水平包括：
    从所述生物样品中提取所述CH25H基因的RNA和所述内参基因的RNA；
    对所述CH25H基因的RNA和所述内参基因的RNA分别进行反转录，获取所述CH25H基因的cDNA和所述内参基因的cDNA；
    针对所述CH25H基因的cDNA和所述内参基因的cDNA，进行PCR扩增反应；以及
    根据PCR扩增反应中CH25H基因的cDNA的第一循环阈值与所述内参基因的cDNA的第二循环阈值，确定所述生物样品中CH25H基因的标准化表达水平。
  8. 如权利要求7所述的用途，其特征在于，所述根据PCR扩增反应中CH25H基因的cDNA的第一循环阈值与所述内参基因的cDNA的第二循环阈值，确定所述生物样品中CH25H基因的标准化表达水平包括：
    确定所述第一循环阈值与第二循环阈值的差值；以及
    用所述第一循环阈值与第二循环阈值的所述差值表示所述生物样品中CH25H基因的标准化表达水平。
  9. 如权利要求1-8中任一项所述的用途，其特征在于，所述基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数包括：
    基于所述目标受试者的所述生物样品中所述CH25H基因的标准化表达水平和多个未患癌症的第一参考受试者的生物样品中所述CH25H基因的标准化表达水平而确定的。
  10. 如权利要求1-9中任一项所述的用途，其特征在于，所述一项或多项参考阈值是基于多个患有癌症的第二参考受试者的生物样品中所述CH25H基因的标准化表达水平而确定的。
  11. 如权利要求1-10中任一项所述的用途，其特征在于，所述检测CH25H基因表达水平的试剂包括CH25H基因的特异性探针。
  12. 如权利要求11所述的用途，其特征在于，所述CH25H基因的特异性探针的序列与SEQ ID NO.：1所示序列的相似度≥95％。
  13. 如权利要求1-12中任一项所述的用途，其特征在于，所述检测CH25H基因表达水平的试剂包括所述CH25H基因的特异性引物。
  14. 如权利要求13所述的用途，其特征在于，所述CH25H基因的特异性引物序列与SEQ ID NO.：2所示序列的相似度≥95％；并且所述CH25H基因的特异性引物序列与SEQ ID NO.：3所示序列的相似度≥95％。
  15. 如权利要求1-14中任一项所述的用途，其特征在于，所述癌症包括以下中的一个或多个：黑色素瘤、肺癌、白血病、胃癌、卵巢癌、胰腺癌、乳腺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、宫颈癌或骨肉瘤。
  16. 如权利要求1-15中任一项所述的用途，其特征在于，所述癌症是非小细胞肺癌、肺鳞癌、肺腺癌或小细胞肺癌。
  17. 如权利要求1-16中任一项所述的用途，其特征在于，所述生物样品是外周血样品。
  18. 如权利要求1-17中任一项所述的用途，其特征在于，所述生物样品包括外周血中的白细胞。
  19. 一种癌症预后预测装置，其特征在于，所述装置包括：
    获取模块，被配置为获取测试数据，所述测试数据与目标受试者的生物样品中CH25H基因的表达水平相关；
    标准化表达水平确定模块，被配置为基于所述测试数据，确定所述生物样品中CH25H基因的标准化表达水平；以及
    预后分析模块，被配置为
    基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数；
    将所述目标参数与一项或多项参考阈值进行比较以获取比较结果；以及
    基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。
  20. 如权利要求19所述的装置，其特征在于，所述一项或多项参考阈值包括第一参考阈值，为了基于所述比较结果，确定癌症复发或转移的可能性，所述预后分析模块还被配置为：
    响应于所述比较结果为所述目标参数小于所述第一参考阈值，确定所述目标受试者癌症复发或转移的可能性较高。
  21. 如权利要求19或20所述的装置，其特征在于，所述一项或多项参考阈值包括第二参考阈值，为了基于所述比较结果，预测癌症复发或转移的可能性，所述预后分析模块还被配置为：
    响应于所述比较结果为所述目标参数大于所述第二参考阈值，预测所述目 标受试者癌症复发或转移的可能性较低。
  22. 如权利要求19-21中任一项所述的装置，其特征在于，所述CH25H基因的所述标准化表达水平是基于CH25H基因在目标受试者中的表达水平和内参基因在目标受试者中的表达水平确定的。
  23. 如权利要求22所述的装置，其特征在于，所述内参基因包括RPLO、GAPDH、ACTB、B2M、SDHA、HPRT1、ARBP、18sRNA、28sRNA中的一种或多种。
  24. 如权利要求22所述的装置，其特征在于，所述CH25H基因的所述标准化表达水平是基于所述CH25H基因的RNA转录物确定的。
  25. 如权利要求24所述的装置，其特征在于，所述测试数据包括在针对CH25H基因的cDNA和所述内参基因的cDNA进行的PCR扩增反应中CH25H基因的cDNA的第一循环阈值与所述内参基因的cDNA的第二循环阈值，其中所述CH25H基因的cDNA和所述内参基因的cDNA是通过基于从所述生物样品中提取所述CH25H基因的RNA和所述内参基因的RNA并进行反转录而得到的。
  26. 如权利要求25所述的装置，其特征在于，为了确定所述生物样品中CH25H基因的标准化表达水平，所述标准化表达水平确定模块被配置为：
    基于所述第一循环阈值与所述第二循环阈值，确定所述生物样品中CH25H基因的标准化表达水平。
  27. 如权利要求19-26中任一项所述的装置，其特征在于，为了基于所述生 物样品中CH25H基因的所述标准化表达水平，确定目标参数，所述预后分析模块被配置为基于所述目标受试者的所述生物样品中所述CH25H基因的标准化表达水平和多个未患癌症的第一参考受试者的生物样品中所述CH25H基因的标准化表达水平，确定所述目标参数。
  28. 如权利要求19-27中任一项所述的装置，其特征在于，所述一项或多项参考阈值是基于多个患有癌症的第二参考受试者的生物样品中所述CH25H基因的标准化表达水平而确定的。
  29. 如权利要求19-28中任一项所述的装置，其特征在于，所述测试数据是使用检测CH25H基因表达水平的试剂得到的，所述检测CH25H基因表达水平的试剂包括CH25H基因的特异性探针。
  30. 如权利要求29所述的装置，其特征在于，所述CH25H基因的特异性探针的序列与SEQ ID NO.：1所示序列的相似度≥95％。
  31. 如权利要求28所述的装置，其特征在于，所述检测CH25H基因表达水平的试剂还包括所述CH25H基因的特异性引物。
  32. 如权利要求31所述的装置，其特征在于，所述CH25H基因的特异性引物序列与SEQ ID NO.：2所示序列的相似度≥95％；并且所述CH25H基因的特异性引物序列与SEQ ID NO.：3所示序列的相似度≥95％。
  33. 如权利要求19-32中任一项所述的装置，其特征在于，所述癌症包括以下中的一个或多个：黑色素瘤、肺癌、白血病、胃癌、卵巢癌、胰腺癌、乳腺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、宫颈癌或骨肉瘤。
  34. 如权利要求19-33中任一项所述的装置，其特征在于，所述癌症是非小细胞肺癌、肺鳞癌、肺腺癌或小细胞肺癌。
  35. 如权利要求19-34中任一项所述的装置，其特征在于，所述生物样品是外周血样品。
  36. 如权利要求19-35中任一项所述的装置，其特征在于，所述生物样品包括外周血中的白细胞。
  37. 一种计算机可读存储介质，其特征在于，所述存储介质存储计算机指令，当所述计算机指令被处理器执行时，实现一种癌症预后预测方法，其特征在于，所述癌症预后预测方法包括：
    获取测试数据，所述测试数据与目标受试者的生物样品中CH25H基因的表达水平相关；
    基于所述测试数据，确定所述生物样品中CH25H基因的标准化表达水平；
    基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数；
    将所述目标参数与一项或多项参考阈值进行比较以获取比较结果；以及
    基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。
  38. 如权利要求37所述的计算机可读存储介质，其特征在于，所述一项或多项参考阈值包括第一参考阈值，所述基于所述比较结果，确定癌症复发或转移的可能性包括：
    响应于所述比较结果为所述目标参数小于所述第一参考阈值，确定所述目标受试者癌症复发或转移的可能性较高。
  39. 如权利要求37或38所述的计算机可读存储介质，其特征在于，所述一 项或多项参考阈值包括第二参考阈值，所述基于所述比较结果，预测癌症复发或转移的可能性包括：
    响应于所述比较结果为所述目标参数大于所述第二参考阈值，预测所述目标受试者癌症复发或转移的可能性较低。
  40. 如权利要求37-39中任一项所述的计算机可读存储介质，其特征在于，所述CH25H基因的所述标准化表达水平是基于CH25H基因在目标受试者中的表达水平和内参基因在目标受试者中的表达水平确定的。
  41. 如权利要求40所述的计算机可读存储介质，其特征在于，所述内参基因包括RPLO、GAPDH、ACTB、B2M、SDHA、HPRT1、ARBP、18sRNA、28sRNA中的一种或多种。
  42. 如权利要求40所述的计算机可读存储介质，其特征在于，所述CH25H基因的所述标准化表达水平是基于所述CH25H基因的RNA转录物确定的。
  43. 如权利要求42所述的计算机可读存储介质，其特征在于，所述测试数据包括在针对CH25H基因的cDNA和所述内参基因的cDNA进行的PCR扩增反应中CH25H基因的cDNA的第一循环阈值与所述内参基因的cDNA的第二循环阈值，其中所述CH25H基因的cDNA和所述内参基因的cDNA是通过基于从所述生物样品中提取所述CH25H基因的RNA和所述内参基因的RNA并进行反转录而得到的。
  44. 如权利要求43所述的计算机可读存储介质，其特征在于，所述基于所述测试数据，确定所述生物样品中CH25H基因的标准化表达水平包括：
    基于所述第一循环阈值与所述第二循环阈值，确定所述生物样品中CH25H 基因的标准化表达水平。
  45. 如权利要求44所述的计算机可读存储介质，其特征在于，所述根据第一循环阈值与所述第二循环阈值，确定所述生物样品中CH25H基因的标准化表达水平包括
    确定所述第一循环阈值与第二循环阈值的差值；以及
    用所述第一循环阈值与第二循环阈值的所述差值表示所述生物样品中CH25H基因的标准化表达水平。
  46. 如权利要求37-45中任一项所述的计算机可读存储介质，其特征在于，所述基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数包括：
    基于所述目标受试者的所述生物样品中所述CH25H基因的标准化表达水平和多个未患癌症的第一参考受试者的生物样品中所述CH25H基因的标准化表达水平而确定目标参数。
  47. 如权利要求37-46中任一项所述的计算机可读存储介质，其特征在于，所述一项或多项参考阈值是基于多个患有癌症的第二参考受试者的生物样品中所述CH25H基因的标准化表达水平而确定的。
  48. 如权利要求37-47中任一项所述的计算机可读存储介质，其特征在于，所述测试数据是使用检测CH25H基因表达水平的试剂得到的，所述检测CH25H基因表达水平的试剂包括CH25H基因的特异性探针。
  49. 如权利要求48所述的计算机可读存储介质，其特征在于，所述CH25H基因的特异性探针的序列与SEQ ID NO.：1所示序列的相似度≥95％。
  50. 如权利要求48所述的计算机可读存储介质，其特征在于，所述检测CH25H基因表达水平的试剂还包括所述CH25H基因的特异性引物。
  51. 如权利要求50所述的计算机可读存储介质，其特征在于，所述CH25H基因的特异性引物序列与SEQ ID NO.：2所示序列的相似度≥95％；并且所述CH25H基因的特异性引物序列与SEQ ID NO.：3所示序列的相似度≥95％。
  52. 如权利要求37-51中任一项所述的计算机可读存储介质，其特征在于，所述癌症包括以下中的一个或多个：黑色素瘤、肺癌、白血病、胃癌、卵巢癌、胰腺癌、乳腺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、宫颈癌或骨肉瘤。
  53. 如权利要求37-52中任一项所述的计算机可读存储介质，其特征在于，所述癌症是非小细胞肺癌、肺鳞癌、肺腺癌或小细胞肺癌。
  54. 如权利要求37-53中任一项所述的计算机可读存储介质，其特征在于，所述生物样品是外周血样品。
  55. 如权利要求37-54中任一项所述的计算机可读存储介质，其特征在于，所述生物样品包括外周血中的白细胞。
  56. 一种癌症预后预测方法，其特征在于，所述方法包括：
    获取测试数据，所述测试数据与目标受试者的生物样品中CH25H基因的表达水平相关；
    基于所述测试数据，确定所述生物样品中CH25H基因的标准化表达水平；
    基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数；
    将所述目标参数与一项或多项参考阈值进行比较以获取比较结果；以及
    基于所述比较结果，确定所述目标受试者癌症复发或转移的可能性。
  57. 如权利要求56所述的方法，其特征在于，所述一项或多项参考阈值包括第一参考阈值，所述基于所述比较结果，确定癌症复发或转移的可能性包括：
    响应于所述比较结果为所述目标参数小于所述第一参考阈值，确定所述目标受试者癌症复发或转移的可能性较高。
  58. 如权利要求56或57所述的方法，其特征在于，所述一项或多项参考阈值包括第二参考阈值，所述基于所述比较结果，预测癌症复发或转移的可能性包括：
    响应于所述比较结果为所述目标参数大于所述第二参考阈值，预测所述目标受试者癌症复发或转移的可能性较低。
  59. 如权利要求56-58中任一项所述的方法，其特征在于，所述CH25H基因的所述标准化表达水平是基于CH25H基因在目标受试者中的表达水平和内参基因在目标受试者中的表达水平确定的。
  60. 如权利要求59所述的方法，其特征在于，所述内参基因包括RPLO、GAPDH、ACTB、B2M、SDHA、HPRT1、ARBP、18sRNA、28sRNA、β-actin中的一种或多种。
  61. 如权利要求59所述的方法，其特征在于，所述CH25H基因的所述标准化表达水平是基于所述CH25H基因的RNA转录物确定的。
  62. 如权利要求61所述的方法，其特征在于，
    所述测试数据包括在针对CH25H基因的cDNA和所述内参基因的cDNA 进行的PCR扩增反应中CH25H基因的cDNA的第一循环阈值与所述内参基因的cDNA的第二循环阈值，其中所述CH25H基因的cDNA和所述内参基因的cDNA是通过基于从所述生物样品中提取所述CH25H基因的RNA和所述内参基因的RNA并进行反转录而得到的。
  63. 如权利要求62所述的方法，其特征在于，所述确定所述生物样品中CH25H基因的标准化表达水平包括
    基于所述第一循环阈值与所述第二循环阈值，确定所述生物样品中CH25H基因的标准化表达水平。
  64. 如权利要求56-63中任一项所述的方法，其特征在于，所述目标参数是基于所述目标受试者的所述生物样品中所述CH25H基因的标准化表达水平和多个未患癌症的第一参考受试者的生物样品中所述CH25H基因的标准化表达水平，确定所述目标参数。
  65. 如权利要求56-64中任一项所述的方法，其特征在于，所述一项或多项参考阈值是基于多个患有癌症的第二参考受试者的生物样品中所述CH25H基因的标准化表达水平而确定的。
  66. 如权利要求56-65中任一项所述的方法，其特征在于，所述检测所述生物样品中所述CH25H基因的标准化表达水平包括使用所述CH25H基因的特异性探针。
  67. 如权利要求66所述的方法，其特征在于，所述CH25H基因的特异性探针的序列与SEQ ID NO.：1所示序列的相似度≥95％。
  68. 如权利要求56-67中任一项所述的方法，其特征在于，所述检测所述生物样品中所述CH25H基因的标准化表达水平包括使用所述CH25H基因的特异性引物。
  69. 如权利要求68所述的方法，其特征在于，所述CH25H基因的特异性引物序列与SEQ ID NO.：2所示序列的相似度≥95％；并且所述CH25H基因的特异性引物序列与SEQ ID NO.：3所示序列的相似度≥95％。
  70. 如权利要求56-69中任一项所述的方法，其特征在于，所述癌症包括以下中的一个或多个：黑色素瘤、肺癌、白血病、胃癌、卵巢癌、胰腺癌、乳腺癌、前列腺癌、膀胱癌、结肠癌、直肠癌、肝癌、宫颈癌或骨肉瘤。
  71. 如权利要求56-70中任一项所述的方法，其特征在于，所述癌症是非小细胞肺癌、肺鳞癌、肺腺癌或小细胞肺癌。
  72. 如权利要求56-71中任一项所述的方法，其特征在于，所述生物样品是外周血样品。
  73. 如权利要求56-72中任一项所述的方法，其特征在于，所述生物样品包括外周血中的白细胞。
  74. 一种方法，用于治疗癌症或预防癌症复发转移，包括：
    检测获取自目标受试者的生物样品中CH25H基因的标准化表达水平；
    基于所述生物样品中CH25H基因的所述标准化表达水平，确定目标参数；
    将所述目标参数与一项或多项参考阈值进行比较；以及
    响应于所述目标参数小于所述参考阈值，为受试者施用一种组合物，所述组 合物含有能提高CH25H基因的表达水平或能提高CH25H蛋白活性的试剂。
  75. 如权利要求74所述的方法，其特征在于，所述CH25H基因的所述标准化表达水平是基于CH25H基因在目标受试者中的表达水平和内参基因在目标受试者中的表达水平确定的。
  76. 如权利要求74或75所述的方法，其特征在于，所述测试数据包括在针对CH25H基因的cDNA和所述内参基因的cDNA进行的PCR扩增反应中CH25H基因的cDNA的第一循环阈值与所述内参基因的cDNA的第二循环阈值，其中所述CH25H基因的cDNA和所述内参基因的cDNA是通过基于从所述生物样品中提取所述CH25H基因的RNA和所述内参基因的RNA并进行反转录而得到的。
  77. 如权利要求76所述的方法，其特征在于，所述基于所述测试数据，确定所述生物样品中CH25H基因的标准化表达水平包括：
    基于所述第一循环阈值与所述第二循环阈值，确定所述生物样品中CH25H基因的标准化表达水平。
  78. 如权利要求74-77中任一项所述的方法，其特征在于，所述癌症是非小细胞肺癌、肺鳞癌、肺腺癌或小细胞肺癌。
  79. 如权利要求74-78中任一项所述的方法，其特征在于，所述生物样品是外周血样品。
  80. 如权利要求74-79中任一项所述的方法，其特征在于，所述生物样品包括外周血中的白细胞。
  81. 如权利要求74-80中任一项所述的方法，其特征在于，所述试剂包括利血平。
  82. 如权利要求74-81中任一项所述的方法，其特征在于，所述试剂还包括干扰素、Toll样受体4激动剂中的至少一个。
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