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WO2025092877A1 - Procédé d'association de marqueurs biomédicaux multiples pour métastases du carcinome nasopharyngé - Google Patents

Procédé d'association de marqueurs biomédicaux multiples pour métastases du carcinome nasopharyngé Download PDF

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Publication number
WO2025092877A1
WO2025092877A1 PCT/CN2024/128778 CN2024128778W WO2025092877A1 WO 2025092877 A1 WO2025092877 A1 WO 2025092877A1 CN 2024128778 W CN2024128778 W CN 2024128778W WO 2025092877 A1 WO2025092877 A1 WO 2025092877A1
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Prior art keywords
markers
subject
sample
asphd2
agap9
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Inventor
Wei Dai
Zhaozheng Hou
Ka-Lok HO
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New Life Medicine Technology Co Ltd
University of Hong Kong HKU
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New Life Medicine Technology Co Ltd
University of Hong Kong HKU
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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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • 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
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/715—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons
    • G01N2333/7158—Assays involving receptors, cell surface antigens or cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90245—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates to a method for cancer diagnosis and treatment.
  • the present invention further relates to a group of biomedical markers associated with a high likelihood of responsiveness of a subject to a cancer therapy.
  • an assay for detecting, diagnosing, monitoring or prognosticating a medical condition, or for detecting, diagnosing, monitoring or prognosticating the responsiveness of a subject to a therapy against said medical condition is provided, as well as a corresponding method and a kit for classifying a subject and a medical decision support system.
  • stage IV patients Although the average 5-year relative survival rate of nasopharyngeal carcinoma has been increased to over 80%in the last decade, the condition for stage IV patients is still lower than 60%. Therefore, it is of great importance to identify the patients who are more likely to develop to stage IV (having metastasis or relapse) after receiving the standard radiation therapy. Applying precise treatment to these patients as early as possible may significantly improve their clinical outcomes.
  • molecular markers associated with tumor metastasis cannot be systematically identified using single molecular marker screening methods. There are currently no clinically available molecular markers, relevant kits or assays that can be used to predict metastasis after treatment for nasopharyngeal carcinoma.
  • the present disclosure relates to the field of cancer diagnosis and treatment, and particularly relates to a set of marker genes for the prognostic assessment of nasopharyngeal carcinoma (NPC) metastasis after radiation therapy.
  • NPC nasopharyngeal carcinoma
  • a list of genes was found to be significantly upregulated/downregulated in nasopharyngeal carcinoma metastasis samples compared to non-metastasis samples; the results of using said genes as a prognostic metastasis marker in nasopharyngeal carcinoma were interpreted objectively and with high accuracy.
  • the said gene set can be used to develop a RT-qPCR Kit for in vitro prognostic assessment of NPC metastasis.
  • This method provides the advantage of being able to provide predictive information at an early developmental stage of a disease, e.g. a cancer disease, in particular nasopharyngeal carcinoma metastases. Furthermore, it allows the assessment of a therapeutic resistance, such as a resistance to radiation therapy.
  • the methodology has successfully been used to identify stratifying genes between resistant and sensitive radiation therapy patients.
  • the present disclosure provides a biomedical marker or group of biomedical markers associated with a high likelihood of responsiveness of a subject to a cancer therapy, preferably radiation therapy, wherein said biomedical marker or group of biomedical markers comprises at least 1, 2, 3, 4, or all markers selected from CXCR6, ASPHD2, DDX39B and AGAP9.
  • an assay for detecting, diagnosing, graduating, monitoring or prognosticating a medical condition, or for detecting, diagnosing, monitoring or prognosticating the responsiveness of a subject to a therapy against said medical condition, in particular nasopharyngeal cancer,
  • the present disclosure relates to an assay for detecting, diagnosing, monitoring or prognosticating a medical condition, or for detecting, diagnosing, monitoring or prognosticating the responsiveness of a subject to a therapy against said medical condition, preferably cancer, more preferably nasopharyngeal carcinoma metastasis, comprising at least the steps of: (a) testing in a sample obtained from a subject for the expression of a marker or a group of biomedical markers; (b) testing in a control sample for the expression of the same marker, group of markers in (a) ; (c) determining the difference in expression of markers of steps (a) and (b) ; and (d) deciding on the presence or stage of a medical condition or the responsiveness of a subject to a therapy against said medical condition, preferably cancer, more preferably nasopharyngeal cancer, based on the results obtained in step (c) .
  • the disclosure relates to a method for classifying a subject comprising: (a) providing a subject's dataset comprising data on gene expression of a stratifying biomedical marker or group of said markers obtained by a method as defined herein above, or as defined in the list or group of biomedical markers described herein above or below; (b) accessing a database comprising database values for a stratifying biomedical marker or group of said markers as defined herein above, or as defined in the list or group of biomedical markers described herein above or below; and (c) calculating a subject's classification score based on the difference between database between the results of step (a) and (b) .
  • the disclosure relates to a medical decision support system comprising: an input for providing a subject dataset comprising data on gene expression of a stratifying biomedical marker or group of said markers obtained by a method as defined herein above, or as defined in the list or group of biomedical markers described herein above; a computer program product for enabling a processor to carry out the method for classifying a subject comprising as define above; and an output for outputting the subject classification score.
  • predictive value for a medical condition refers to a value allowing the assessment of a medical condition or the development of said medical condition in the future, e.g. within a defined time frame of 1 to 3 weeks, 1 month, 2 month, 3 month, 4 months, 5 months, 6 months, 1, 2, 3, 4, 5, 6, 7, 10 years or more years or any other period of time.
  • the term also includes all situations associated with said medical condition, e.g. treatment results, responsiveness to treatments, development of resistance etc.
  • the disclosure relates to a composition for in vivo or in vitro diagnosing, detecting, monitoring or prognosticating a disease, e.g. cancer, e.g., nasopharyngeal cancer, or for diagnosing, detecting, monitoring or prognosticating the likelihood of responsiveness of a subject to a cancer therapy.
  • a disease e.g. cancer, e.g., nasopharyngeal cancer
  • the therapy is against nasopharyngeal cancer.
  • the therapy is radiation therapy.
  • Such a composition may alternatively or additionally comprise an antibody against any of the above mentioned markers.
  • a nucleic acid affinity ligand or peptide affinity ligand is modified to function as an imaging contrast agent.
  • a method of identifying a subject for eligibility for a cancer disease therapy comprising: (a) testing in a sample obtained from subject for a parameter associated with a marker or group of markers as indicated herein above; (b) classifying the levels of tested parameters; and (c) identifying the individual as eligible to receive a cancer therapy wherein the subject's sample is classified as having an increased expression of one or more of the above mentioned markers.
  • the present disclosure relates to an assay for detecting, diagnosing, graduating, monitoring or prognosticating a medical condition, such as cancer, such as nasopharyngeal cancer, comprising at least the steps of: (a) testing in a sample obtained from a subject for the expression of a stratifying biomedical markers or group of said markers obtained according to the above described method; alternatively, the testing may be carried out with a marker or group of markers as defined herein above; (b) determining the difference in expression of markers of steps (a) and (b) ; and (c) deciding on the presence or stage of medical condition or the responsiveness of a subject to a therapy against said medical condition, based on the results obtained in step (b) .
  • the present disclosure relates to an assay for detecting, diagnosing, graduating, monitoring or prognosticating the responsiveness of a subject to a therapy against said medical condition, such as cancer, more preferably nasopharyngeal cancer, such as the responsiveness of a subject to a radiation therapy, comprising at least the steps of: (a) testing in a sample obtained from a subject for the expression of a stratifying biomedical markers or group of said markers obtained according to the above described method; (b) testing in a control sample for the expression of the same marker, group of markers as in (a) ; (c) determining the difference in expression of markers of steps (a) and (b) ; and (d) deciding on the presence or stage of medical condition or the responsiveness of a subject to a therapy against said medical condition, preferably cancer, such as nasopharyngeal cancer, based on the results obtained in step (c) .
  • asopharyngeal cancer such as the responsiveness of a subject to a
  • the expression may be tested by any suitable means known to the person skilled in the art, such as room temperature polymerase chain reaction (RT-PCR) , RNA sequencing, or gene expression detection on microarrays.
  • RT-PCR room temperature polymerase chain reaction
  • the present disclosure relates to a medical decision support system comprising: an input for providing a subject dataset comprising data on gene expression of a stratifying biomedical marker or group of said markers obtained according to the above described method; a computer program product for enabling a processor to carry out the method for classifying a subject as defined above, and an output for outputting the subject classification score.
  • the present disclosure relates to a medical decision support system that is a molecular oncology decision making workstation.
  • the decision-making workstation may be used for deciding on the initiation and/or continuation of a cancer therapy for a subject.
  • the decision-making workstation is used for deciding on the probability and likelihood of responsiveness to a radiation therapy.
  • the present disclosure relates to the use of one or more reagents in the preparation of a kit for identifying a subject at risk for a medical condition, such as nasopharyngeal carcinoma metastases after radiation therapy, wherein the reagents are used for detecting one or more markers selected from the group consisting of CXCR6, ASPHD2, DDX39B, and AGAP9.
  • the kit further comprises one or more immunomodulatory agents that are used for treating the subject.
  • the present disclosure relates to the use of one or more reagents in the preparation of a kit for detecting, diagnosing, graduating, monitoring or prognosticating the responsiveness of a subject to a therapy against said medical condition, such as cancer, more preferably nasopharyngeal cancer, such as the responsiveness of a subject to a radiation therapy, wherein the reagents are used for detecting one or more markers selected from the group consisting of CXCR6, ASPHD2, DDX39B and AGAP9.
  • the kit further comprises one or more immunomodulatory agents that are used for treating the subject.
  • the marker described in the above-said methods/assays/composition/kit preparation/medical decision support system comprises at least two, at least three or all of the four markers selected from the group consisting of CXCR6, ASPHD2, DDX39B and AGAP9. More preferably, the marker comprises CXCR6; or the marker comprises ASPHD2; or the marker comprises DDX39B; or the marker comprises AGAP9.
  • the at least two of the markers comprise CXCR6 and ASPHD2, or CXCR6 and DDX39B, or CXCR6 and AGAP9, or ASPHD2 and DDX39B, or ASPHD2 and AGAP9, or DDX39B and AGAP9. Additionally, in some embodiments, the at least three of the markers comprise CXCR6, ASPHD2 and DDX39B, or CXCR6, ASPHD2 and AGAP9, or ASPHD2, DDX39B and AGAP9.
  • FIG. 1 Marker Selection.
  • the 4 genes involved in the patent have significantly different expression levels in samples with and without metastasis after treatment in 29 samples in each of the 2 regions (totaling 58 cases) .
  • A. CXCR6-is down-regulated in metastasis cases.
  • B. ASPHD2 is down-regulated in metastasis cases.
  • C. DDX39B is up-regulated in metastasis cases.
  • D. AGAP9 is up-regulated in metastasis cases.
  • FIG. 3 Corresponding score-probability conversion graph.
  • FIG. 4 ROC curves of individual markers from q-PCR trials.
  • the AUC for individual markers is calculated according to the Cohort 2 q-PCR validation data (10 cases) .
  • FIG. 5A-FIG. 5F Amino acid sequence of the markers.
  • the terms “patient” or “subject” are used interchangeably and mean a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.
  • a human or non-human mammal such as a bovine, equine, canine, ovine, or feline.
  • the patient is a human.
  • Reduce or inhibit or decrease refer to the ability to cause an overall decrease of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or greater.
  • Reduce or inhibit can refer to the symptoms of the disorder being treated, the presence or size of metastases, or the size of the primary tumor.
  • the phrases "treating cancer” and “treatment of cancer” and “treatment of tumors” mean to decrease, reduce, or inhibit the replication of cancer cells; decrease, reduce or inhibit the spread (formation of metastases) of cancer; decrease tumor size; decrease the number of tumors (i.e. reduce tumor burden) ; lessen or reduce the number of cancerous cells in the body; prevent recurrence of cancer after surgical removal or other anti-cancer therapies; or ameliorate or alleviate the symptoms of the disease caused by the cancer.
  • progeny As used herein, the expressions “cell, ” “cell line, ” and “cell culture” are used interchangeably and all such designations include progeny. In particular, by progeny is also intended cell clones obtained by limit dilution of the cell lines of the invention. As certain modifications may occur in succeeding generations due to mutation or environmental influences, or clonal selection, such progeny may not be identical to the parent cell, but is still included within the scope of the term as used herein.
  • Treating” or “treatment” of a state, disorder or condition includes:
  • the benefit to a subject to be treated is either statistically significant or at least perceptible to the patient or to the physician.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams &Wilkins (A. R. Gennaro edit. 2005) .
  • the choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a “therapeutically effective amount” means the amount of a compound that, when administered to an animal for treating a state, disorder or condition, is sufficient to effect such treatment.
  • the “therapeutically effective amount” will vary depending on the compound, the disease and its severity and the age, weight, physical condition and responsiveness of the animal to be treated.
  • compositions of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of a compound described herein.
  • a “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result.
  • a therapeutically effective amount of an antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response in the individual.
  • a therapeutically effective amount is also one in which any toxic or detrimental effects of the compound are outweighed by the therapeutically beneficial effects.
  • a “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.
  • screen and “screening” and the like as used herein means to test a subject or patient to determine if they have a particular illness or disease, or a particular manifestation of an illness or disease. The term also means to test an agent to determine if it has a particular action or efficacy.
  • identification means to recognize a disease state or a clinical manifestation or severity of a disease state in a subject or patient.
  • the term also is used in relation to test agents and their ability to have a particular action or efficacy.
  • prediction means to tell in advance based upon special knowledge.
  • prevention refers to acting prior to overt disease onset, to prevent the disease from developing or minimize the extent of the disease or slow its course of development.
  • agent means a substance that produces or is capable of producing an effect and would include, but is not limited to, chemicals, pharmaceuticals, biologics, small organic molecules, antibodies, nucleic acids, peptides, and proteins.
  • the term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system, i.e., the degree of precision required for a particular purpose, such as a pharmaceutical formulation.
  • “about” can mean within 1 or more than 1 standard deviations, per the practice in the art.
  • “about” can mean a range of up to 20%, preferably up to 10%, more preferably up to 5%, and more preferably still up to 1%of a given value.
  • the term can mean within an order of magnitude, preferably within 5-fold, and more preferably within 2-fold, of a value.
  • the term “about” meaning within an acceptable error range for the particular value should be assumed.
  • the present invention also provides methods for stratifying subjects prior to treatment and/or monitoring subjects for their responses to treatment, e.g, administration of agents, both oral and topical, life-style alterations such as diet and exercise, and non-traditional treatment such as acupuncture. This is useful in both patient care as well as clinical trials.
  • the methods comprise obtaining the expression of at least one gene in at least one gene signature, or the corresponding protein level, in a subject prior to any treatment.
  • the methods comprise obtaining the expression of at least one gene in at least one gene signature (or the corresponding protein) in a normal subject that serves as a reference expression value. After a course of treatment at a particular time period that a person of skill in the art can determine, the measurement of expression of the markers profile or desired gene or genes (or corresponding proteins) is measured, and differential expression or protein level, when compared to the reference (e.g. prior to treatment levels, or control levels) would indicate that the subject has late-stage MDS, or relatively severe MDS.
  • the expression of at least one gene in at least one gene signature, or corresponding protein level would be measured before and after treatment. In an alternative embodiment, more than one gene from each signature, or corresponding protein would be measured.
  • the present disclosure also provides a method for determining target genes or proteins for drug development.
  • the disclosure also contemplates that the protein products of any of the genes in the gene signatures found for example in datasets and/or described in any of the Tables or Figures herein may have diagnostic value, as well as to serve as potential therapeutic targets for patient monitoring, stratification, or drug development.
  • a sample of biological tissue or bodily fluid from a subject with nasopharyngeal cancer is obtained.
  • the sample is tested for protein levels (for protein corollaries of any of the markers as described herein.
  • the protein sample can be obtained from any biological tissue.
  • biological tissues include, but are not limited to, biopsies, epidermal, whole blood, and plasma.
  • the protein sample can be obtained from any biological fluid.
  • fluids include, but are not limited to, plasma, saliva, and urine. Protein can be isolated and/or purified from the sample using any method known in the art, including but not limited to immunoaffinity chromatography.
  • preferred methods for detecting and measuring increase levels of the proteins in a protein sample include quantitative Western blot, immunoblot, quantitative mass spectrometry, enzyme-linked immunosorbent assays (ELISAs) , radioimmunoassays (RIA) , immunoradiometric assays (IRMA) , and immunoenzymatic assays (IEMA) and sandwich assays using monoclonal and polyclonal antibodies.
  • ELISAs enzyme-linked immunosorbent assays
  • RIA radioimmunoassays
  • IRMA immunoradiometric assays
  • IEMA immunoenzymatic assays
  • Antibodies are a method of detecting and measuring target or desired proteins in a sample. Such antibodies are available commercially or can be made by conventional methods known in the art. Such antibodies can be monoclonal or polyclonal and fragments thereof, and immunologic binding equivalents thereof.
  • the term “antibody” means both a homologous molecular entity as well as a mixture, such as a serum product made up of several homologous molecular entities.
  • such antibodies will immunoprecipitate the desired proteins from a solution as well as react with desired/target proteins on a Western blot, immunoblot, ELISA, and other assays listed above.
  • Antibodies for use in these assays can be labeled covalently or non-covalently with an agent that provides a detectable signal. Any label and conjugation method known in the art can be used. Labels, include but are not limited to, enzymes, fluorescent agents, radiolabels, substrates, inhibitors, cofactors, magnetic particles, and chemiluminescent agents. A number of fluorescent materials are known and can be utilized as detectable labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. A particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • Any desired targets or binding partner (s) can also be labeled with a radioactive element or with an enzyme.
  • the radioactive label can be detected by any of the currently available counting procedures.
  • the preferred isotope may be selected from 3H, 14C, 32P, 35S, 36Cl, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re.
  • Enzyme labels are likewise useful, and can be detected by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques.
  • the enzyme is conjugated to the selected particle by reaction with bridging molecules such as carbodiimides, diisocyanates, glutaraldehyde and the like.
  • the enzymes which can be used in these procedures are known and can be utilized.
  • the enzymes can be are peroxidase, ⁇ -glucuronidase, ⁇ -D-glucosidase, ⁇ -D-galactosidase, urease, glucose oxidase plus peroxidase and alkaline phosphatase.
  • U.S. Patent Nos. 3,654,090; 3,850,752; and 4,016,043 are referred to by way of example for their disclosure of alternate labeling material and methods.
  • sample refers to a sample of biological fluid, tissue, or cells, in a healthy and/or pathological state obtained from a subject.
  • samples include, but are not limited to, blood, bronchial lavage fluid, sputum, saliva, urine, amniotic fluid, lymph fluid, tissue or fine needle biopsy samples, peritoneal fluid, cerebrospinal fluid, and includes supernatant from cell lysates, lysed cells, cellular extracts, and nuclear extracts.
  • the whole blood sample is further processed into serum or plasma samples.
  • the sample includes blood spotting tests.
  • all of the assays disclosed herein can be in kit form for use by a health care provider and/or a diagnostic laboratory.
  • the present disclosure provides for a kit comprising one or more probes and/or antibodies for detecting expression levels of one or more markers as described herein.
  • kits may include probes for one or more of the genes from one or more signatures, as described herein, reagents for isolating and purifying nucleic acids from biological tissue or bodily fluid, reagents for performing assays on the isolated and purified nucleic acid, instructions for use, and reference values or the means for obtaining reference values in a control sample for the included genes.
  • a preferred kit for patient classification with regard to disease activity and clinical manifestations would include probes for at least one gene from each of the signatures described herein.
  • the kit would include reagents for testing for markers, for example.
  • reagents for testing for markers for example.
  • Such a kit could include antibodies that recognize the peptide of interest, reagents for isolating and/or purifying protein from a biological tissue or bodily fluid, reagents for performing assays on the isolated and purified protein, instructions for use, and reference values or the means for obtaining reference values for the quantity or level of peptides in a control sample.
  • a preferred kit for monitoring or use to disease activity would include probes from at least one gene from each of the markers signatures described herein.
  • Such a kit could include antibodies that recognize the peptide of interest, reagents for isolating and/or purifying protein from a biological tissue or bodily fluid, reagents for performing assays on the isolated and purified protein, instructions for use, and reference values or the means for obtaining reference values for the quantity or level of peptides in a control sample.
  • the kit for diagnosing or prognosing nasopharyngeal carcinoma would include probes for at least one gene from each of the determinative signatures, such as any combination of markers as described herein, or corresponding protein.
  • kits would have the probes attached to a solid state.
  • Another embodiment would have the probes in a microarray format wherein nucleic acid probes for one or more of the genes from one or more of the gene signatures would be in an ordered arrangement on a surface or substrate.
  • kits suitable for use by a medical specialist may be prepared to determine the presence or amount of a desired gene or protein activity, expression or signature gene amplification in suspected cancer cells or biopsy or tumor samples.
  • One class of such kits will contain at least the labeled target or its binding partner, for instance an antibody specific thereto, and directions, of course, depending upon the method selected, e.g., "competitive, " "sandwich, " “DASP” and the like.
  • the kits may also contain peripheral reagents such as buffers, stabilizers, etc.
  • the kits comprise one or more PCR primers described herein.
  • test kit may be prepared for the determination and quantitation of a desired target or protein in cells or a cellular or biopsy sample, comprising:
  • the diagnostic test kit may comprise:
  • test kit may be prepared and used for the purposes stated above, and comprises:
  • an assay system for screening potential drugs effective to modulate the activity or expression of the target or gene signature may be prepared and is provided.
  • the target may be introduced into a test system, and the prospective drug may also be introduced into the resulting cell culture, and the culture thereafter examined to observe any changes in the target activity of the cells, or in the proliferation or division of the cells, due either to the addition of the prospective drug alone, or due to the effect of added quantities of the known target.
  • target can include any of the following: any of the genes (including any single or combinations) of the markers as described herein, any corresponding protein of these genes; alone or in combination with one or more hematologic cancer markers.
  • nasopharyngeal carcinoma patients from 2 regions/cohorts from hospitals in southern China (cohort 1: Guangzhou; cohort 2: Hong Kong) . 29 from each for marker selection, 71 from cohort 1 for estimating the scoring vector, and 46 from cohort 2 for accuracy validation.
  • NPC patients were randomly selected from the database who had 5 years of clinical follow-up after receiving their first radiation therapy for NPC.
  • the control group corresponds to the patients who do not develop metastasis in 5 years after radiation therapy.
  • the proposed test kit contains the forward and reverse primers of 4 marker genes and 1 housekeeping gene.
  • Green qPCR ReadyMix can be included if the user does not have similar reagents.
  • the developed toolkit and scoring vector led to 79.24%AUC in RNA-seq data, and 93.75%AUC in q-PCR. Such accuracy can already provide a valuable reference in clinical practice.
  • X refers to the four marker genes.
  • Treatment methods for nasopharyngeal cancer after diagnosis with the present method If a patient has a higher risk of metastasis, the physician may take a more aggressive approach to treatment, such as adding induction chemotherapy, extending the course of induction chemotherapy, changing from a two-drug regimen (e.g., gemcitabine + cisplatin, GP) to a three-drug regimen (docetaxel + cisplatin + fluorouracil, TPF) , concurrent chemotherapy, or adjuvant chemotherapy, etc.
  • a two-drug regimen e.g., gemcitabine + cisplatin, GP
  • TPF trifluorouracil
  • the quality of the raw reads was examined using FastQC (v0.11.8) .
  • RSeQC1 v2.6.4
  • Picard v2.17.4
  • All samples had a mapping rate of least 95%.
  • STAR v2.7.5c 2 was utilized to align the reads onto the reference genome using default parameters.
  • the reference genome was integrated from the human genome (hg38) and the EBV genome (NC_007065) .
  • the genome annotation files (GTF) for human genome and EBV genome were downloaded from GENCODE3 (v27) and GeneBank respectively.
  • HTSeq4 v0.9.1) was utilized for quantifying read counts at the gene-level in intersection-nonempty mode.
  • Library normalization and differential gene expression analysis were performed using DESeq25 (v1.30.1) .
  • VST Variance-stabilizing transformation
  • RNA sequencing results of the samples were obtained to calculate the read count of genes.
  • the read count is normalized using the median of ratios method in DEseq2 before further analysis.
  • Nasopharyngeal carcinoma patients were analyzed for differential expression of Over 29,000 genes, using the R toolkit DESeq2. Marker genes were identified by Wald test and corrected for batch effect of relapse condition.

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Abstract

Procédé de diagnostic et de traitement du cancer. LA présente invention permet plus particulièrement d'identifier les associations entre les marqueurs biomédicaux qui présentent une valeur diagnostique, pronostique ou prédictive pour un état pathologique, en particulier les métastases du cancer nasopharyngé (NPC) après une thérapie anticancéreuse. Le marqueur biomédical ou le groupe de marqueurs biomédicaux associé à une forte probabilité de réactivité d'un sujet à une thérapie anticancéreuse, de préférence une radiothérapie, ledit marqueur biomédical ou groupe de marqueurs biomédicaux comportant au moins 1, 2, 3, 4, ou tous les marqueurs choisis parmi CXCR6, ASPHD2, DDX39B et AGAP9. En outre, la présente invention concerne un dosage pour détecter, diagnostiquer, graduer, surveiller ou pronostiquer une condition médicale, ou pour détecter, diagnostiquer, surveiller ou pronostiquer la réactivité d'un sujet à une thérapie contre ledit état pathologique, en particulier le cancer nasopharyngé, ainsi qu'un procédé correspondant pour classer un sujet et un système d'aide à la décision médicale.
PCT/CN2024/128778 2023-11-01 2024-10-31 Procédé d'association de marqueurs biomédicaux multiples pour métastases du carcinome nasopharyngé Pending WO2025092877A1 (fr)

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