WO2022048492A1 - 一种akr1c3检测方法、检测akr1c3的诊断试剂盒及其用途 - Google Patents

一种akr1c3检测方法、检测akr1c3的诊断试剂盒及其用途 Download PDF

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WO2022048492A1
WO2022048492A1 PCT/CN2021/114774 CN2021114774W WO2022048492A1 WO 2022048492 A1 WO2022048492 A1 WO 2022048492A1 CN 2021114774 W CN2021114774 W CN 2021114774W WO 2022048492 A1 WO2022048492 A1 WO 2022048492A1
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akr1c3
cancer
solution
antigen retrieval
formalin
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PCT/CN2021/114774
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English (en)
French (fr)
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谢燕彬
孟繁英
段建新
郝静
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深圳艾欣达伟医药科技有限公司
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Priority to CA3192258A priority Critical patent/CA3192258A1/en
Priority to US18/043,610 priority patent/US20240142453A1/en
Priority to BR112023003973A priority patent/BR112023003973A2/pt
Priority to CN202180031349.XA priority patent/CN115485560A/zh
Priority to JP2023514086A priority patent/JP2023540283A/ja
Priority to IL300833A priority patent/IL300833A/en
Priority to KR1020237011169A priority patent/KR20230058507A/ko
Priority to EP21863557.1A priority patent/EP4209785A1/en
Priority to AU2021337711A priority patent/AU2021337711A1/en
Publication of WO2022048492A1 publication Critical patent/WO2022048492A1/zh

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    • 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
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • 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
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • 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
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • 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
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • 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.)
    • G01N2333/90206—Oxidoreductases (1.) acting on the CH-CH group of donors (1.3)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2474/00—Immunochemical assays or immunoassays characterised by detection mode or means of detection
    • G01N2474/20—Immunohistochemistry assay

Definitions

  • the present invention relates to the technical field of cancer treatment, in particular to a method for detecting AKR1C3, a diagnostic kit for detecting AKR1C3 and uses thereof.
  • the conventional and effective detection method for specific enzymes or proteins in the pathological tissue of patients is to use immunohistochemical (IHC) staining methods.
  • IHC immunohistochemical
  • a separate IHC staining method needs to be developed to detect hepatocellular carcinoma, and another IHC staining method needs to be developed separately to detect prostate cancer. That is to say, the existing IHC staining detection method cannot achieve the staining detection of the expression level of AKR1C3 (aldehyde-ketone reductase 1C3) in a variety of cancer tissues corresponding to one IHC method.
  • AKR1C3 aldehyde-ketone reductase 1C3
  • the IHC detection method since further cancer treatment plans need to be determined according to the detection results of IHC, the IHC detection method must have stable staining results, that is, the IHC detection method used in large-scale commercial kits must have good sensitivity, precision and Consistent (different laboratories, different operators, different operation times), and can adapt to many different cancer tumor tissues.
  • the purpose of the present invention is to provide a method for detecting AKR1C3, a diagnostic kit for detecting AKR1C3 and the use thereof, which can be applied to the detection and staining of AKR1C3 expression levels in various cancer tumor tissues at the same time.
  • the results are stable with good sensitivity, precision and consistency.
  • one aspect of the present invention provides a method for detecting AKR1C3, using immunohistochemical staining to detect the expression level of AKR1C3 in isolated formalin-fixed paraffin-embedded human tissue sections, comprising the following steps:
  • the formalin-fixed paraffin-embedded human tissue sections after antigen retrieval are mixed with AKR1C3 monoclonal antibody solution with a concentration of 0.5-5.0 ⁇ g/ml and incubated for 25-700 minutes;
  • the formalin-fixed paraffin-embedded human tissue sections incubated with the primary antibody were mixed with a secondary antibody solution with a concentration of 0.5-5.0 ⁇ g/ml, and incubated for 25-700 minutes.
  • step a) antigen retrieval the pH value of the antigen retrieval solution is 2.0-9.0;
  • the pH value of the antigen retrieval solution is 6.0-9.0;
  • the pH value of the antigen retrieval solution is 6.0.
  • the antigen retrieval solution includes sodium citrate antigen retrieval solution or EDTA antigen retrieval solution.
  • step a) antigen retrieval the formalin-fixed paraffin-embedded human tissue section is heated at 92-102° C. for 18-25 minutes;
  • the formalin-fixed paraffin-embedded human tissue section is heated at 97° C. for 20 minutes.
  • step b) primary antibody incubation the concentration of the AKR1C3 monoclonal antibody solution is 1.0-3.0 ⁇ g/ml;
  • the concentration of the AKR1C3 monoclonal antibody solution is 1.2 ⁇ g/ml
  • step c) secondary antibody incubation the concentration of the secondary antibody solution is 1.0-3.0 ⁇ g/ml;
  • the concentration of the secondary antibody solution is 1.2 ⁇ g/ml.
  • both the AKR1C3 monoclonal antibody solution and the secondary antibody solution contain NaN 3 , H + ions, Cl - ions and tromethamine.
  • the AKR1C3 monoclonal antibody solution and the secondary antibody solution are obtained by diluting an antibody dilution buffer, and the antibody dilution buffer comprises the following components:
  • the antibody dilution buffer comprises the following components:
  • step b) primary antibody incubation in step b) primary antibody incubation, the incubation time of the formalin-fixed paraffin-embedded human tissue section after antigen retrieval and the AKR1C3 monoclonal antibody solution is 30-45 minutes ;
  • the incubation time between the formalin-fixed paraffin-embedded human tissue section after antigen retrieval and the AKR1C3 monoclonal antibody solution is 45 minutes.
  • step c) incubation of the secondary antibody in step c) incubation of the secondary antibody, the incubation time of the formalin-fixed paraffin-embedded human tissue section after incubation with the primary antibody and the secondary antibody solution is 30 to 45 minutes;
  • the incubation time of the formalin-fixed paraffin-embedded human tissue section after incubation with the primary antibody and the secondary antibody solution is 30 minutes.
  • the AKR1C3 monoclonal antibody is a mouse monoclonal antibody
  • step c) secondary antibody incubation the secondary antibody is goat anti-mouse antibody, rabbit anti-mouse antibody, horse anti-mouse antibody or donkey anti-mouse antibody.
  • step c) secondary antibody incubation it also includes:
  • Formalin-fixed paraffin-embedded human tissue sections were stained with hematoxylin, dehydrated and mounted after staining.
  • step a) antigen retrieval it further comprises:
  • the formalin-fixed paraffin-embedded human tissue sections were dewaxed with an organic solvent, and the dewaxed sections were washed sequentially with alcohols with different water contents, and finally washed with water;
  • the organic solvent is acetone, toluene or xylene; more preferably, the organic solvent is xylene;
  • the alcohol is ethanol or methanol; more preferably, the alcohol is ethanol;
  • the dewaxed sections are first washed with absolute ethanol, and then washed with ethanol with a volume fraction of 90-97%.
  • step a) antigen retrieval and step b) primary antibody incubation further comprising:
  • the blocking solution is the serum of an animal derived from AKR1C3 monoclonal antibody
  • the blocking solution is mouse serum.
  • the formalin-fixed paraffin-embedded human tissue sections are breast cancer tissue sections, colorectal cancer tissue sections, esophageal cancer tissue sections, gastric cancer tissue sections, hepatocellular carcinoma tissue sections, Non-small cell lung cancer tissue sections, prostate cancer tissue sections, renal cell carcinoma sections, peripheral T cell lymphoma sections or nodular NK/T cell lymphoma sections.
  • a diagnostic kit for detecting AKR1C3 comprising:
  • AKR1C3 monoclonal antibody solution with a concentration of 0.5 to 5.0 ⁇ g/ml
  • the pH value of the antigen retrieval solution is 2.0-9.0;
  • the pH value of the antigen retrieval solution is 6.0-9.0;
  • the pH value of the antigen retrieval solution is 6.0.
  • the antigen retrieval solution includes sodium citrate antigen retrieval solution or EDTA antigen retrieval solution.
  • the concentration of the AKR1C3 monoclonal antibody solution is 1.0-3.0 ⁇ g/ml
  • the concentration of the AKR1C3 monoclonal antibody solution is 1.2 ⁇ g/ml
  • the concentration of the secondary antibody solution is 1.0-3.0 ⁇ g/ml
  • the concentration of the secondary antibody solution is 1.2 ⁇ g/ml.
  • both the AKR1C3 monoclonal antibody solution and the secondary antibody solution contain NaN 3 , H + ions, Cl - ions and tromethamine.
  • the AKR1C3 monoclonal antibody solution and the secondary antibody solution are obtained by diluting an antibody dilution buffer, and the antibody dilution buffer comprises the following components:
  • the antibody dilution buffer comprises the following components:
  • the AKR1C3 monoclonal antibody is a mouse monoclonal antibody
  • the secondary antibody is a goat anti-mouse antibody, a rabbit anti-mouse antibody, a horse anti-mouse antibody or a donkey anti-mouse antibody.
  • the above-mentioned diagnostic kit for detecting AKR1C3 further comprises:
  • a blocking solution preferably, the blocking solution is the serum of an AKR1C3 monoclonal antibody-derived animal;
  • the blocking solution is mouse serum.
  • the above-mentioned diagnostic kit for detecting AKR1C3 further comprises:
  • another aspect of the present invention provides the use of the above diagnostic kit for detecting AKR1C3 in the preparation of a medicament for treating cancer, tumor or cell proliferative disease.
  • the above-mentioned use comprises the steps of:
  • An anticancer drug containing AKR1C3 activation is administered to a patient whose expression level of AKR1C3 is greater than or equal to a predetermined expression level.
  • the AKR1C3-activated anticancer drug satisfies, but is not limited to, at least one of the following definitions:
  • the cancer cell proliferation inhibitory effect of a certain compound detected is less than the cancer cell proliferation inhibitory effect of the compound detected in the absence of an AKR1C3 inhibitor;
  • the AKR1C3-activated anticancer drug is selected from compounds of the following structures:
  • Patent PCT/NZ2019/050030 the compound 5-nitrobenzenesulfonamide dibromide, bromomethanesulfonate and bis-methanesulfonate mustard (compounds 562-674) in the publication number WO2019190331, 3-methyl-5-nitro Benzenesulfonamide dibromide, bromomethanesulfonate and bis-methanesulfonate mustard (compounds 679-791), 3-trifluoromethyl-5-nitrobenzenesulfonamide dibromide, bromomethanesulfonate and bis- Mesylate mustard (compounds 913-1025), 3-ethynyl-5-nitrobenzenesulfonamide dibromide, bromomethanesulfonate and bis-methanesulfonate mustard (1030-1142) and 5-nitrobenzene Sulfonamide bis-methanesulfonate mustard mesylate (compounds 640.Ms, 641.
  • Patent PCT/IB2020/057285 the publication number is compounds Ex1 to Ex170 in WO2021005586A1; or
  • the AKR1C3-activated anticancer drug is selected from compounds of the following structures:
  • the cancer, tumor or cell proliferative disease comprises:
  • Lung cancer non-small cell lung cancer, liver cancer, pancreatic cancer, breast cancer, stomach cancer, bone cancer, esophageal cancer, breast cancer, prostate cancer, testicular cancer, colon cancer, ovarian cancer, bladder cancer, cervical cancer, hepatocellular cancer, melanin tumor, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, renal cell carcinoma, cystic adenocarcinoma, cystic carcinoma, medullary carcinoma, bronchial carcinoma, bone cell Carcinoma, epithelial carcinoma, cholangiocarcinoma, choriocarcinoma, embryonic carcinoma, seminoma, Wilms carcinoma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma , pineal tumor, hemoblastoma, vocal cord neuro
  • the cancer, tumor or cell proliferative disease includes: ovarian cancer, cervical cancer, pancreatic cancer, breast cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma carcinoma, peripheral T-cell lymphoma, or nodular NK/T-cell lymphoma.
  • Figure 1 is a photo of IHC staining of CRC samples after repairing with high pH antigen retrieval solution.
  • Scanscope scans 0.4x, CRC sample: 335933-P;
  • picture a corresponds to Ab (antibody): 1:1000, primary antibody and secondary antibody Antibody incubation time is 20min and 20min respectively;
  • picture b corresponds to Ab: 1:2000, primary antibody and secondary antibody incubation time are 45min and 30min respectively;
  • picture c corresponds to Ab: 1:2000, primary antibody and secondary antibody incubation time are 60min respectively and 30min;
  • Figure 2 is a photo of IHC staining of CRC samples after repair with low pH antigen retrieval solution, Scanscope scans 0.4x, CRC sample: 335933-P; where a picture corresponds to Ab: 1: 1000, the incubation time of primary and secondary antibodies 30min and 30min respectively; picture b corresponds to Ab: 1:2000, the incubation time of primary antibody and secondary antibody is 45min and 30min respectively; picture c corresponds to Ab: 1:2000, the incubation time of primary antibody and secondary antibody is 60min and 30min respectively;
  • Figure 3 is a photo of the results of IHC staining of gastric cancer samples at a low pH value; in which the picture a corresponds to Ab: 1:1000, and the incubation time of primary antibody and secondary antibody is 30min and 30min respectively; picture b corresponds to Ab: 1:2000, a Antibody and secondary antibody incubation times were 45min and 30min respectively; Figure c corresponds to Ab: 1:2000, and the primary and secondary antibody incubation times were 45min and 45min respectively;
  • Figure 4 is a photo of the results of IHC staining of breast cancer samples at low pH value; in which the picture a corresponds to Ab: 1:1000, and the incubation time of primary antibody and secondary antibody is 30min and 30min respectively; picture b corresponds to Ab: 1:2000, The incubation time of primary antibody and secondary antibody is 45min and 30min respectively; picture c corresponds to Ab: 1:2000, and the incubation time of primary antibody and secondary antibody is 45min and 45min respectively;
  • Figure 5 shows the comparison photos of staining performance under different dilutions and incubation times of monoclonal antibodies.
  • Scanscope scans 4x; in which the picture a corresponds to Ab: 1:1000, the incubation time of primary antibody and secondary antibody 30min and 30min respectively; picture b corresponds to Ab: 1:2000, the incubation time of primary antibody and secondary antibody is 45min and 30min respectively; picture c corresponds to Ab: 1:2000, the incubation time of primary antibody and secondary antibody is 30min and 30min respectively;
  • Figure 6 Comparison of staining performance between 5 different normal colon tissues using the optimal staining protocol: low pH TRS, 97°C for 20 minutes; AKR1C3 diluted 1:2000, 30 minutes; HRP incubation time: 30 minutes, Scanscope scanning 2x; in which picture a corresponds to sample 390211-YN, picture b corresponds to sample 390650-YN, picture c corresponds to sample 391182-YN, picture d corresponds to sample 391761-YN, and picture e corresponds to sample 3919951-YN;
  • Figure 7 The photos of AKR1C3 staining in normal tissues using the optimal staining conditions; where a and b correspond to the low and high magnification of the normal tissue of the tonsil, respectively; c and d correspond to the low and high magnifications of the normal gastric tissue, respectively High-magnification staining photos; e and f images correspond to low- and high-power staining photos of normal colon tissue, respectively;
  • Figure 8 Photos of AKR1C3 staining in non-small cell lung cancer using the optimal protocol; where a and b correspond to the low- and high-magnification staining photos of sample 1 (F102582A22), respectively; c and d correspond to sample 2 (F134064A12), respectively ) low and high magnification staining photos;
  • Figure 9 Photos of AKR1C3 staining in gastric cancer using the optimal protocol; where a and b correspond to the low- and high-power staining photos of sample 1 (F180723A5), respectively, up arrow: tumor cells, down arrow: remaining normal stomach Mucosal glandular epithelium; panels c and d correspond to the low magnification and high magnification staining photos of sample 2 (F180684A3), respectively, down arrow: tumor cells, up arrow: remaining normal gastric mucosa glandular epithelium;
  • Figure 10 The photos of AKR1C3 staining in breast cancer using the optimal protocol; where a and b correspond to the low- and high-magnification staining photos of sample 1 (F162870A5), respectively; c and d correspond to sample 2 (F130368B3), respectively Low and high magnification stained photos;
  • FIG 11 The photos of AKR1C3 staining in hepatocellular carcinoma using the optimal protocol; where a and b correspond to the low- and high-magnification staining photos of sample 1 (DLV13050B3), respectively; c and d correspond to sample 2 (DLV13052B5), respectively Low and high magnification stained photos;
  • Figure 13 shows the normal colon tissue staining with both positive and negative components under the optimal protocol for AKR1C3 IHC determination, and the scan photos at different magnifications; in which the picture a corresponds to the negative quality control reagent (Scanscope scan 4x), and the b picture corresponds to AKR1C3 (Scanscope scan 10x) , picture c corresponds to AKR1C3 (Scanscope scan 4x), picture d corresponds to AKR1C3 (Scanscope scan 10x);
  • Figure 14 is a photograph of a tissue quality control sample. Normal colon tissue with both positive and negative components was used as a double positive and negative tissue quality control for each staining operation; picture a corresponds to the negative control reagent (Scanscope scan 4x) , b map corresponds to AKR1C3 staining (Scanscope scan 4x);
  • Figure 15 is a photograph of sample F151286A5 HCC, H-score 300 (3+: 100%). All tumor cells showed strong cytoplasmic/nuclear staining; meanwhile, normal hepatocytes (arrows) near cancer nests, as well as stromal cells and endothelial cells showed different intensities of staining as internal quality controls; where a panel corresponds to a lower magnification (Scanscope Scan 0.4x), image b corresponds to higher magnification (Scanscope scan 10x);
  • Figure 16 is a photo of sample F151725A1 EC, H-score 160 (0: 0%; 1+: 60%; 2+: 20%; 3+: 20%); tumor cells show different cytoplasm/nuclear staining with different intensities; Among them, picture a corresponds to a lower magnification (Scanscope scan 0.4x), and picture b corresponds to a higher magnification (Scanscope scan 10x);
  • Figure 17 is a photograph of sample F152459A4 GC, H-score 35 (0: 85%; 1+: 5%; 2+: 0%; 3+: 10%); tumor cells exhibit different cytoplasmic/nuclear staining of different intensities. Higher magnifications show smaller areas (upward arrows); endothelial cells show staining as an internal quality control (downward arrows); where a panel corresponds to lower magnification (Scanscope scan 0.4x) and b panel corresponds to higher magnification multiples (Scanscope scans 10x);
  • Figure 18 is a photo of sample F151653A1 CRC, H-score 120 (0: 30%; 1+: 30%; 2+: 30%; 3+: 10%); tumor cells show different cytoplasm/nuclear staining with different intensities; The tumor area is a rightward blue arrow, and the normal tissue is a leftward arrow; among them, image a corresponds to a lower magnification (Scanscope scan 0.4x), and image b corresponds to a higher magnification (Scanscope scan 10x);
  • Figure 19 is a photograph of sample F183410A4 PC, H-score 0 (0:100%); no tumor cells showed cytoplasmic/nuclear staining of AKR1C3; however, endothelial cells showed staining as an internal control (arrow); Low magnification (Scanscope scan 0.4x), panel b corresponds to higher magnification (Scanscope scan 10x).
  • Patient and “individual” are used interchangeably and refer to a mammal in need of cancer treatment.
  • the patient is a human.
  • the patient is a human diagnosed with cancer.
  • patient or “individual” may refer to non-human mammals, such as non-human primates, dogs, cats, rabbits, pigs, mice, for use in screening, characterizing and evaluating drugs and therapies or rats.
  • a “prodrug” refers to a compound that, after administration or administration, is metabolized or otherwise converted into a biologically active or more active compound (or drug) with respect to at least one property. Relative to the drug, the prodrug is chemically modified in such a way that it is less active or inactive relative to the drug, but the chemical modification results in the production of the corresponding drug through metabolism or other biological processes after administration of the prodrug.
  • Prodrugs may have altered metabolic stability or delivery characteristics, less side effects or less toxicity, or improved flavor relative to the active drug (see, e.g., reference Nogrady, 1985, Medicinal Chemistry A Biochemical Approach, Oxford University Press, New York, pp. 388-392, incorporated herein by reference). Prodrugs can be synthesized using reactants other than the corresponding drug.
  • Treatment of a condition or patient refers to taking steps to obtain beneficial or desired results (including clinical results).
  • beneficial or desired clinical outcomes include, but are not limited to, alleviation or amelioration of one or more symptoms of cancer; attenuation of disease extent; delay or slowing of disease progression; improvement, remission or stabilization of disease state; or other beneficial results.
  • treatment of cancer can result in a partial response or stabilize the disease.
  • Tumor cell refers to a tumor cell of any suitable species (eg, mammalian, eg, murine, canine, feline, equine, or human).
  • suitable species eg, mammalian, eg, murine, canine, feline, equine, or human.
  • the anticancer prodrugs of DNA alkylating agents targeting overexpression of AKR1C3 developed by the applicant of the present invention include: 1) DNA alkylating agents, corresponding to PCT application number PCT/US2016/021581, publication number WO2016/145092A, corresponding to Chinese application No.
  • Compounds in the form of prodrugs are reduced under the catalysis of AKR1C3 in the biochemical environment of cells to obtain cytotoxic toxins and play a role in killing cancer cells.
  • the Chinese name is (S)-1-(3-(3-N,N-dimethylaminocarbonyl)phenoxy-4-nitrophenyl)-1-ethyl-N,N'-bis (Ethylene) phosphoramidate, also known as the S configuration compound of OBI-3424, AST-3424, TH-2870), CAS number is 2097713-69-2, and its structure is as follows:
  • the above drugs are only effective for patients with AKR1C3 expression, so it is necessary to detect the expression level of AKR1C3 in patients. In practical applications, it is necessary to determine whether the patient's tissue sample reaches the predetermined expression level of AKR1C3 and then meets the conditions for giving the medicines of the above three patents (CN107530556A, CN108290911A, CN108136214A), which requires that the IHC detection method must have stable staining results. The inventors tried to use the IHC detection method disclosed in the prior art to detect the expression level of AKR1C3 in various cancer tumor tissues.
  • the IHC staining detection methods used in the kits do not have good sensitivity, precision and consistency (different laboratories, different operators, different operation time), and cannot be adapted to the IHC detection of many different cancer tumor tissues . If the staining results are unstable, the determination of AKR1C3 expression levels will be inaccurate, resulting in unsatisfactory cancer treatment effects.
  • the inventors tried to further improve the existing IHC detection methods, and provided a The AKR1C3 detection method can be applied to the detection of AKR1C3 expression levels in a variety of cancer tumor tissues at the same time, and the staining results are stable, with good sensitivity, precision and consistency.
  • One aspect of the present invention provides a method for detecting AKR1C3, using immunohistochemical staining to detect the expression level of AKR1C3 in isolated formalin-fixed paraffin-embedded (FFPE) human tissue sections, comprising the following steps:
  • the formalin-fixed paraffin-embedded human tissue sections after antigen retrieval are mixed with AKR1C3 monoclonal antibody solution with a concentration of 0.5-5.0 ⁇ g/ml and incubated for 25-700 minutes;
  • the formalin-fixed paraffin-embedded human tissue sections incubated with the primary antibody were mixed with a secondary antibody solution with a concentration of 0.5-5.0 ⁇ g/ml, and incubated for 25-700 minutes.
  • the assay sensitivity results show that the analytical sensitivity of the IHC method provided by the present invention to determine the expression level of AKR1C3 in various human cancer tissues has acceptable performance characteristics, which shows the expected staining pattern and localization of AKR1C3 in the detection sample, and is in the quality control. Appropriate performance within the standard limits for analytical sensitivity studies. Assay precision results showed 100% agreement between batch (day/operator, operator and instrument) and within-assay precision, which met the ⁇ 95% standard limit based on AKR1C3 expression in tumor cell cytoplasm and nucleus.
  • FFPE formalin-fixed paraffin-embedded
  • antigen retrieval is a necessary step in immunohistochemical staining before antibody labeling, because the fixation process of tissue usually causes protein cross-linking, which often occurs when formalin fixation is used due to its chemical nature , the antigenic epitopes need to be re-exposed to facilitate the binding of antibodies by antigen retrieval steps.
  • the antigens are re-exposed by the action of antigen retrieval solution and heat, the slices to be repaired are placed in the antigen retrieval solution, and then heated, ie, antigen retrieval is realized through the combined action of chemical and heat.
  • step a) antigen retrieval the formalin-fixed paraffin-embedded human tissue section is heated at 92-102° C. for 18-25 minutes; Marin-fixed paraffin-embedded human tissue sections are heated at 97°C for 20 minutes, which can achieve the best results: good staining effect on various cancer tumor tissue sections.
  • the present invention does not limit the cooling temperature after antigen retrieval, it can be cooled to room temperature naturally, or cooled to a certain temperature (for example, 65° C.) before subsequent operations are performed.
  • the pH value of the antigen retrieval solution will affect the antigen retrieval effect, and in combination with the subsequent use of hematoxylin staining, in a preferred embodiment of the present invention, the pH value of the antigen retrieval solution is 2.0 to 9.0; more preferably Preferably, the pH value of the antigen retrieval solution is 6.0-9.0; further preferably, the pH value of the antigen retrieval solution is 6.0.
  • the present invention does not limit the components of the antigen retrieval solution, as long as the pH value of the antigen retrieval solution meets the requirements, it can be used in the present invention.
  • the antigen retrieval solution includes but is not limited to: Sodium Citrate Antigen Retrieval Solution, EDTA antigen Repair solution (EDTA Antigen Retrieval solution), etc.
  • the concentration of the AKR1C3 monoclonal antibody solution has a great influence on the operation of the primary antibody incubation in step b). It is determined through experiments that the AKR1C3 monoclonal antibody solution with a concentration of 1.0 to 3.0 ⁇ g/ml has a better effect, more preferably 1.2ug/ml ml concentration, the dilution factor of AKR1C3 monoclonal antibody solution at this time is 1:2000.
  • the AKR1C3 monoclonal antibody solution is obtained by diluting a diluent containing the following components:
  • Tris-HCl buffer tromethamine-hydrochloric acid buffer
  • the antibody dilution buffer comprises the following components:
  • Tris-HCl buffer containing 0.1% mass concentration of polyethylene glycol or Tween, and 0.015mol/L sodium azide.
  • the secondary antibody for immunohistochemical staining must be the antigen against the primary antibody species, for example: the primary antibody used to detect the B protein of animal A is animal C anti-animal A protein B antibody, and the secondary antibody should be animal D anti-animal C antibody, the present invention is to detect human AKR1C3 (protein), the primary antibody used is mouse anti-human AKR1C3 monoclonal antibody, that is, mouse AKR1C3 monoclonal antibody, then the secondary antibody is other animals (such as goat, rabbit, horse, etc.). , donkey) anti-mouse antibody.
  • the primary antibody used to detect the B protein of animal A is animal C anti-animal A protein B antibody
  • the secondary antibody should be animal D anti-animal C antibody
  • the present invention is to detect human AKR1C3 (protein)
  • the primary antibody used is mouse anti-human AKR1C3 monoclonal antibody, that is, mouse AKR1C3 monoclonal antibody
  • the secondary antibody is other animals (such
  • step c) secondary antibody incubation it also includes:
  • Formalin-fixed paraffin-embedded human tissue sections were stained with hematoxylin, and dehydrated and mounted for subsequent observation and scoring.
  • step a) antigen retrieval it further comprises:
  • the formalin-fixed paraffin-embedded human tissue sections were dewaxed with organic solvents.
  • the deparaffinized sections were washed sequentially with alcohols with different water contents, and finally washed with water; the dewaxing and rehydration operation was to replenish the dried samples. Moisture is convenient for various subsequent operations.
  • paraffin-embedded human tissue sections since paraffin-embedded is carried out, and the occurrence of paraffin in the subsequent staining process will have a serious impact, the paraffin must be washed out with the corresponding organic solvent.
  • organic solvents for eluting paraffin without loss of sections include acetone, xylene, toluene, etc., but xylene has better effect and less toxicity. After washing the paraffin, it is necessary to use an alcohol (methanol or ethanol) dissolved in water to wash away the residual organic solvent, and ethanol is usually used.
  • a gradient elution method is used, that is, the deparaffinized sections are first washed with absolute ethanol, then washed with ethanol with a volume fraction of 90-97% (eg, 95%), and finally washed with water.
  • step a) antigen retrieval and step b) primary antibody incubation further comprising:
  • Blocking of endogenous enzymes and antibodies in tissue is important to minimize background staining and reduce false positive staining. This is usually accomplished by incubating the sample with a specific buffer that blocks non-specific sites to which the primary or secondary antibody may also bind.
  • a specific buffer that blocks non-specific sites to which the primary or secondary antibody may also bind.
  • using the mouse serum matched with the mouse monoclonal antibody can mask all complex interfering substances, and the operation is convenient.
  • a method for detecting AKR1C3, using immunohistochemical staining to detect the expression level of AKR1C3 in isolated formalin-fixed paraffin-embedded (FFPE) human tissue sections comprising the following steps:
  • Formalin-fixed paraffin-embedded human tissue sections of appropriate thickness were dewaxed with organic solvents, and the dewaxed sections were washed with alcohol with different water contents, and finally washed with water;
  • the dewaxed and rehydrated formalin-fixed paraffin-embedded human tissue sections were heated at 90-115°C for 17-30 minutes for antigen retrieval;
  • the formalin-fixed paraffin-embedded human tissue sections after blocking non-specific antigens are mixed with AKR1C3 monoclonal antibody solution with a concentration of 0.5-5.0 ⁇ g/ml, and incubated for 25-700 minutes;
  • the formalin-fixed paraffin-embedded human tissue sections incubated with the primary antibody are mixed with a secondary antibody solution with a concentration of 0.5-5.0 ⁇ g/ml and incubated for 25-700 minutes;
  • the stained human tissue sections were observed, and the expression level of AKR1C3 in the human tissue sections was evaluated according to the observed staining degree.
  • step c) the secondary antibody incubation operation
  • a step of blocking non-specific antigens can be added again, using the serum of the secondary antibody animal as the blocking solution.
  • the use of the above conditions includes breast cancer, colorectal cancer, esophageal cancer, gastric cancer, hepatocellular carcinoma, non-small cell lung cancer, prostate cancer, renal cell carcinoma, peripheral T-cell lymphoma, nodular NK/T-cell lymphoma in Formalin-fixed paraffin-embedded human tissue sections of various cancers (tumors) in the IHC detection can achieve good staining results, which can prove that the IHC detection method of AKR1C3 provided above is suitable for various cancers. of tumor tissue.
  • a diagnostic kit for detecting AKR1C3 comprising:
  • AKR1C3 monoclonal antibody solution with a concentration of 0.5 to 5.0 ⁇ g/ml
  • the above-mentioned diagnostic kit for detecting AKR1C3 further comprises:
  • the blocking solution preferably, the blocking solution is the serum of an AKR1C3 monoclonal antibody-derived animal; more preferably, the blocking solution is mouse serum.
  • the above-mentioned diagnostic kit for detecting AKR1C3 further comprises:
  • the Negative Control Reagent is a commercially available product: FLEX Negative Control from DAKO Company, Mouse, (Link). "Link” means that the reagent is used in conjunction with the Dako Autostainer Link 48 automatic immunohistochemical staining system used in the following examples.
  • another aspect of the present invention provides the use of the above diagnostic kit for detecting AKR1C3 in the preparation of a medicament for treating cancer, tumor or cell proliferative disease.
  • the above-mentioned use comprises the steps of:
  • An AKR1C3-activated anticancer drug is administered to a patient whose expression level of AKR1C3 is greater than or equal to a predetermined expression level.
  • the expression level of AKR1C3 is different, so the expression levels of AKR1C3 in cancer or tumor tissue section samples suitable for the administration of AKR1C3-activated anticancer prodrugs are also different: some Cancers require high expression, and some cancers have moderate expression for drug administration.
  • the predetermined expression level of AKR1C3 can be expressed by the H score, and the pre-expression level of AKR1C3 corresponding to each cancer type can be obtained by statistical method.
  • AKR1C3-activated anticancer drugs include AKR1C3-activated anticancer prodrugs, that is, compounds in the form of prodrugs in the biochemical environment of cells are catalyzed by AKR1C3 to finally obtain cytotoxic toxins to play cancer cells. Poisoning effect.
  • an AKR1C3-activated anticancer drug satisfies, but is not limited to, at least one of the following conditions:
  • the compound disclosed in the publication number CN108136214A is a cleavage prodrug, and the final cleavage metabolized to play a role in the original drug is And drugs such as paclitaxel and camptothecin;
  • patent PCT/NZ2019/050030 publication number WO2019190331, corresponding to Chinese application number CN2019800234236
  • the compound disclosed in publication number CN111918864A is a cleavage prodrug, and its final cleavage metabolizes the original drug that plays a role It is a nitrogen mustard structure drug;
  • Aldehyde and ketone reductase 1C3 has the function of reducing certain specific structures of aldehyde and ketone compounds containing carbon-oxygen double bonds to corresponding alcohol compounds containing hydroxyl groups.
  • the aldehyde-ketone compound containing carbon-oxygen double bond is an anticancer drug activated by AKR1C3 ( Reduction-Prodrug), the corresponding alcohol compound containing hydroxyl group is the original drug (Drug).
  • the publication number is WO2021005586A1
  • the full text of this patent document is hereby incorporated into the text of this patent application.
  • AKR1C3-activated anticancer drugs/prodrugs are preferred, and these compounds are all AKR1C3-activated anticancer drugs/prodrugs:
  • AKR1C3-activated anticancer drugs of type A above; or
  • these compounds are the above-mentioned C-type AKR1C3-activated anticancer drugs.
  • the present invention used a total of 46 tissue samples from 9 markers, including 5 RCC (renal cell carcinoma) samples, 5 HCC (hepatocellular carcinoma) samples, 5 NSCLC (non-small cell lung cancer) samples, 5 GC (gastric cancer) samples, 5 PC (prostate cancer) samples, 5 EC (esophageal cancer) samples, 5 CRC (colorectal cancer) samples, 6 peripheral T-cell lymphoma samples, and 5 NK/T cells Lymphoma samples.
  • FFPE formalin-fixed paraffin-embedded
  • Dako Autostainer Link 48 automatic immunohistochemical staining system also known as tissue section stainer serial number: AS5085D1611, AS2370D1203
  • Aperio Scanscope XT digital slide scanning system (serial number: SS001403)
  • Reactivity evaluation includes the following aspects:
  • the AKR1C3 assay was evaluated on a semi-quantitative scale and the percentage of cellular staining was recorded for cytoplasmic and nuclear staining at the following four levels (0, 1+, 2+ and 3+).
  • the degree of staining ie the level of AKR1C3 enzyme expression, was scored using the H-score: % of nuclear-cytoplasmic stained tumor cells (the total value from 0 to 3+ should not exceed 100):
  • Tumor nucleus-cytoplasm 1+ (weak staining): a value between 0 and 100
  • Tumor nucleus-cytoplasm 2+ (moderate staining): a value between 0 and 100
  • Tumor nucleus-cytoplasm 3+ strong staining: a value between 0 and 100
  • % of total nuclear-cytoplasmic positive staining a value between 0 and 100
  • H-score (% Weak [1+] ⁇ 1) + (% Moderate [2+] ⁇ 2) + (% Strong [3+] ⁇ 3)
  • the total positive % score ⁇ 10% was defined as the agreement between pathologists for the same sample. However, if the pathologist scores the same case as 0 and 1%, it should be considered discordant.
  • the inventors determined that the conditions that need to be optimized include:
  • High pH antigen retrieval solutions have extremely strong background staining. However, if the pH is low, background staining is significantly reduced. A low pH antigen retrieval solution will be used for this validation (Scanscope scan 1x).
  • the finalized AKR1C3 detection method includes the following steps:
  • IHC staining was performed on normal tissue of tonsil, normal tissue of stomach, normal tissue of colon, non-small cell lung cancer, gastric cancer, breast cancer, hepatocellular carcinoma and colorectal cancer under the optimal staining conditions, as shown in Figure 7-12. Staining of various normal tissues and solid tumors showed the best signal-to-noise ratio as judged by staining of positive and negative tissue components and specific positive staining with respect to cellular localization and range of staining intensity.
  • Normal tissue Stromal cells and endothelial cells may show different intensity levels of staining.
  • Normal colon tissue with both positive and negative components will be used as a double positive and negative tissue control for each staining operation and will be used for the following validation operations as well as future in vivo studies.
  • a total of 46 tissue samples including 5 RCC, 5 HCC, 5 NSCLC, 5 GC, 5 PC, 5 EC, 5 CRC, 6 peripheral T-cell lymphoma tissue samples and 5 NK/ T-cell lymphoma tissue samples were stained with AKR1C3 antibody to assess the sensitivity of this IHC assay.
  • Between-assay precision includes:
  • a single run contains 4 serial slices for within-assay precision.
  • the AKR1C3 IHC assay was performed by 2 operators on 2 instruments on 3 non-consecutive days with 3 staining runs of 5 samples with different AKR1C3 expression with a precision showing reproducible results. Based on AKR1C3 expression in the tumor cell cytoplasm and nucleus as detailed above, 100% inter- and intra-procedural concordance was shown, which met the standard limit of ⁇ 95%.
  • Inter-pathologist concordance for AKR1C3 IHC detection between two pathologists showed 100% concordance (46/46) based on AKR1C3 expression in tumor cell cytoplasm and nuclei, consistent with ⁇ 90% of the standardizable limit.
  • the diagnostic kit (Kit) for AKR1C3 detection includes:
  • AKR1C3 monoclonal antibody solution containing NaN 3 , H + ions, Cl - ions and tromethamine;
  • the mouse AKR1C3 monoclonal antibody solution with a concentration of 1.2ug/ml and the secondary antibody solution with a concentration of 1.2ug/ml are both obtained by diluting the antibody diluent.
  • the antibody diluent includes:
  • Tris-HCl buffer tromethamine-hydrochloric acid buffer
  • Secondary antibodies were goat anti-mouse, rabbit anti-mouse, horse anti-mouse or donkey anti-mouse.
  • the negative control reagent solution was a commercially available product: FLEX Negative Control from DAKO, Mice, (Link).
  • Diagnostic kits for AKR1C3 detection which are used together with drugs as AKR1C3-activated anticancer prodrugs, are routinely screened for patients.
  • the use of this kit is convenient for medical staff to use a unified test kit standard operation (SOP) to test in different laboratories before deciding to administer drugs to patients, so that the AKR1C3 test results obtained by the same reagents and the same operation can be obtained. Matches the recommended test results for the specific cancer in the package insert of the AKR1C3-activated anticancer prodrug.
  • SOP test kit standard operation
  • the specific operating method of the kit is described in the instructions, that is, the specific operating conditions in the above-mentioned instructions.
  • the description also gives the score values of the IHC staining detection method using AKR1C3-activated anticancer prodrugs for different cancer (tumor) types.
  • a patient's gastric cancer tissue section obtained by using the above-mentioned kit to detect and score the score is 209, and according to statistics, the gastric cancer patient uses AKR1C3-activated anticancer prodrug IHC staining
  • the score of the detection method cannot be lower than 165, so the doctor can prescribe the use of AKR1C3-activated anticancer prodrugs for this patient.
  • a patient's esophageal cancer tissue section is scored with the above kit and the score is 105, and according to statistics, esophageal cancer patients are detected by IHC staining of AKR1C3-activated anticancer prodrugs.
  • the score of the method cannot be lower than 115, so the doctor cannot prescribe the use of AKR1C3-activated anticancer prodrugs for this patient.
  • Example 6 Use of AKR1C3 detection method and AKR1C3 detection diagnostic kit in the treatment of cancer, tumor or cell proliferative disease
  • the scoring value (for example, H score) obtained after using the AKR1C3 detection method established in Example 1 or the diagnostic kit for AKR1C3 detection in Example 5 to detect the isolated formalin-fixed paraffin-embedded human tissue section of a gastric cancer patient is: 209, which is greater than the predetermined score of 165;
  • AKR1C3-activated anticancer drugs were administered to this gastric cancer patient.
  • AKR1C3-activated anticancer drugs selected from the following structures may have the best therapeutic effect:

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Abstract

一种AKR1C3检测方法、检测AKR1C3的诊断试剂盒及其用途，其中，AKR1C3检测方法包括以下步骤：将福尔马林固定石蜡包埋人体组织切片依次使用有机溶剂、醇、水处理；在抗原修复液的存在下，将处理后的福尔马林固定石蜡包埋人体组织切片进行抗原修复；将抗原修复后的福尔马林固定石蜡包埋人体组织切片与封闭液共同孵育以封闭非特异性抗原；将封闭后的福尔马林固定石蜡包埋人体组织切片与一定浓度的AKR1C3单克隆抗体溶液混合后进行一抗孵育；将一抗孵育后的福尔马林固定石蜡包埋人体组织切片与一定浓度的二抗溶液混合后进行二抗孵育。该AKR1C3检测方法能同时适用于多种癌症肿瘤组织中AKR1C3表达水平的检测且染色结果稳定，具有较好的灵敏度、精密度和一致性。

Description

一种AKR1C3检测方法、检测AKR1C3的诊断试剂盒及其用途 技术领域
本发明涉及癌症治疗技术领域，尤其涉及一种AKR1C3检测方法、检测AKR1C3的诊断试剂盒及其用途。
背景技术
对于患者病理组织中特定酶或蛋白的常规有效检测方法是使用免疫组织化学(IHC)染色方法进行检测，然而现有的IHC方法往往只能针对单一的癌症肿瘤组织开发AKR1C3表达水平的染色方法，例如为了检测肝细胞癌需要单独开发一种IHC染色方法，为了检测前列腺癌又需要再单独开发另一种IHC染色方法。也就是现有的IHC染色检测方法无法做到一种IHC方法对应多种癌症组织中AKR1C3(醛酮还原酶1C3)表达水平的染色检测。
另外，由于需要根据IHC的检测结果确定进一步的癌症治疗方案，因此IHC检测方法必须是染色结果稳定，即作为大规模的商业试剂盒中使用的IHC检测方法必须具有较好的灵敏度、精密度和一致性(不同实验室、不同操作人员、不同操作时间)，并且能适应多种不同癌症肿瘤组织。
但是IHC检测方法的染色结果受到多种因素的影响(方捷迪,王晓星,张梦玲等.水质对免疫组化染色结果的影响[J].临床与实验病理学杂志,2019,35(04):111-113；刘海洋,王效军,张海宇等.微波加热修复和盐酸水解修复方法对大鼠脑组织免疫组化染色结果的影响[J].宁夏医科大学学报,2011(11):1115-1116；张威,梁英杰.不同的抗原修复方法和染色条件对P53蛋白免疫组织化学方法结果的影响[J].中国组织化学与细胞化学杂志,2002(2)；刘小燕,杨鉴,陈莹,等.不同的抗原修复液和修复方法对免疫组化染色结果的影响[J].广东医学院学报,2013(05):43-44；骆新兰,林兴滔,罗东兰等.pH9.0不同成分的抗原修复液对免疫组织化学染色结果的影响[J].中华病理学杂志,2012,41(003):192-194；蔡广玲，余光银，赵洋.抗原修复液pH值对淋巴组织免疫组化染色的影响[J].华夏医学,2005,18(4):501-502；杜娟,石雪迎,郑杰等.抗原修复液pH值及修复时间对免疫组化染色效果的影响[J].北京大学学报(医学版),2005,037(002):195-197；孟奎,周晓军.抗原修复技术在免疫组织化学中的作用[J].中国组织化学与细胞化学杂志,2001,10(001):109-111.)，影响的过程包括抗原修复过程、染色过程等，影响的因素和条件包括加热的温度、加热的时间、使用的水质、抗原修复液的组成、抗原修复液的pH值以及修复反应的时间等，当然不同的组织类型将极大的影响IHC染色检测的结果。这些影响因素就造成了现有技术中公开的IHC检测方法染色结果不稳定，即不具有较好的灵敏度、精密度和一致性，而且不能适应多种不同癌症类型的检测。因此，需要找到一种AKR1C3检测方法，能同时适用于多种癌症肿瘤组织中AKR1C3表达水平的检测且染色结果稳定。
发明内容
有鉴于此，本发明的目的是提供一种AKR1C3检测方法、检测AKR1C3的诊断试剂 盒及其用途，该AKR1C3检测方法和试剂盒能同时适用于多种癌症肿瘤组织中AKR1C3表达水平的检测且染色结果稳定，具有较好的灵敏度、精密度和一致性。
基于上述目的，本发明的一个方面提供了一种AKR1C3检测方法，使用免疫组织化学染色法检测离体的福尔马林固定石蜡包埋人体组织切片中AKR1C3的表达水平，包括以下步骤：
a)抗原修复
在抗原修复液的存在下，将福尔马林固定石蜡包埋人体组织切片于90～115℃加热17～30分钟进行抗原修复；
b)一抗孵育
将抗原修复后的福尔马林固定石蜡包埋人体组织切片与浓度为0.5～5.0μg/ml的AKR1C3单克隆抗体溶液混合后孵育25～700分钟；
c)二抗孵育
将一抗孵育后的福尔马林固定石蜡包埋人体组织切片与浓度为0.5～5.0μg/ml的二抗溶液混合后孵育25～700分钟。
在本发明的优选的实施方案中，在步骤a)抗原修复中，所述抗原修复液的pH值为2.0～9.0；
更优选地，所述抗原修复液的pH值为6.0～9.0；
进一步优选地，所述抗原修复液的pH值为6.0。
在本发明的优选的实施方案中，在步骤a)抗原修复中，所述抗原修复液包括柠檬酸钠抗原修复液或EDTA抗原修复液。
在本发明的优选的实施方案中，在步骤a)抗原修复中，所述福尔马林固定石蜡包埋人体组织切片于92～102℃加热18～25分钟；
更优选地，所述福尔马林固定石蜡包埋人体组织切片于97℃加热20分钟。
在本发明的优选的实施方案中，在步骤b)一抗孵育中，所述AKR1C3单克隆抗体溶液的浓度为1.0～3.0μg/ml；
更优选地，所述AKR1C3单克隆抗体溶液的浓度为1.2μg/ml；
和/或，在步骤c)二抗孵育中，所述二抗溶液的浓度为1.0～3.0μg/ml；
更优选地，所述二抗溶液的浓度为1.2μg/ml。
在本发明的优选的实施方案中，所述AKR1C3单克隆抗体溶液和所述二抗溶液均含有NaN 3、H +离子、Cl -离子和氨丁三醇。
在本发明的优选的实施方案中，所述AKR1C3单克隆抗体溶液和所述二抗溶液由抗体稀释缓冲液稀释得到，所述抗体稀释缓冲液包含以下成分：
0.02～0.08mol/L的Tris-HCl缓冲液，
含有0.05～0.15％质量浓度的聚乙二醇或吐温，和
0.010～0.020mol/L叠氮化钠；
更优选地，所述抗体稀释缓冲液包含以下成分：
0.05mol/L的Tris-HCl缓冲液，
含有0.1％质量浓度的聚乙二醇或吐温，和
0.015mol/L叠氮化钠。
在本发明的优选的实施方案中，在步骤b)一抗孵育中，所述抗原修复后的福尔马林固定石蜡包埋人体组织切片与AKR1C3单克隆抗体溶液的孵育时间为30～45分钟；
更优选地，所述抗原修复后的福尔马林固定石蜡包埋人体组织切片与AKR1C3单克隆抗体溶液的孵育时间为45分钟。
在本发明的优选的实施方案中，在步骤c)二抗孵育中，所述一抗孵育后的福尔马林固定石蜡包埋人体组织切片与二抗溶液的孵育时间为30～45分钟；
更优选地，所述一抗孵育后的福尔马林固定石蜡包埋人体组织切片与二抗溶液的孵育时间为30分钟。
在本发明的优选的实施方案中，在步骤b)一抗孵育中，所述AKR1C3单克隆抗体为小鼠单克隆抗体；
和/或，在步骤c)二抗孵育中，所述二抗为山羊抗小鼠抗体、兔抗小鼠抗体、马抗小鼠抗体或驴抗小鼠抗体。
在本发明的优选的实施方案中，在步骤c)二抗孵育之后，还包括：
d)染色封固
使用苏木精对福尔马林固定石蜡包埋人体组织切片进行染色，染色后脱水封片。
在本发明的优选的实施方案中，在步骤a)抗原修复之前，还包括：
a1)脱蜡复水
将福尔马林固定石蜡包埋人体组织切片使用有机溶剂脱蜡，脱蜡后的切片经过不同水含量的醇依次洗涤，最后使用水洗涤；
优选地，所述有机溶剂为丙酮、甲苯或二甲苯；更优选地，所述有机溶剂为二甲苯；
和/或，优选地，所述醇为乙醇或甲醇；更优选地，所述醇为乙醇；
和/或，优选地，所述脱蜡后的切片先经过无水乙醇洗涤，再经过体积分数为90～97％的乙醇洗涤。
在本发明的优选的实施方案中，在步骤a)抗原修复和步骤b)一抗孵育之间，还包括：
b1)封闭非特异性抗原
将抗原修复后的福尔马林固定石蜡包埋人体组织切片与封闭液共同孵育以封闭非特异性抗原；
优选地，所述封闭液为AKR1C3单克隆抗体来源动物的血清；
更优选地，所述封闭液为小鼠血清。
在本发明的优选的实施方案中，所述福尔马林固定石蜡包埋人体组织切片为乳腺癌组织切片、结直肠癌组织切片、食管癌组织切片、胃癌组织切片、肝细胞癌组织切片、非小细胞肺癌组织切片、前列腺癌组织切片、肾细胞癌切片、外周性T细胞淋巴瘤切片或结节性NK/T细胞淋巴瘤切片。
基于相同的发明构思，本发明的另一个方面提供了一种检测AKR1C3的诊断试剂盒，包括：
抗原修复液；
浓度为0.5～5.0μg/ml的AKR1C3单克隆抗体溶液；
浓度为0.5～5.0μg/ml的二抗溶液。
在本发明的优选的实施方案中，所述抗原修复液的pH值为2.0～9.0；
更优选地，所述抗原修复液的pH值为6.0～9.0；
进一步优选地，所述抗原修复液的pH值为6.0。
在本发明的优选的实施方案中，所述抗原修复液包括柠檬酸钠抗原修复液或EDTA抗原修复液。
在本发明的优选的实施方案中，所述AKR1C3单克隆抗体溶液的浓度为1.0～3.0μg/ml；
更优选地，所述AKR1C3单克隆抗体溶液的浓度为1.2μg/ml；
和/或，所述二抗溶液的浓度为1.0～3.0μg/ml；
更优选地，所述二抗溶液的浓度为1.2μg/ml。
在本发明的优选的实施方案中，所述AKR1C3单克隆抗体溶液和所述二抗溶液均含有NaN 3、H +离子、Cl -离子和氨丁三醇。
在本发明的优选的实施方案中，所述AKR1C3单克隆抗体溶液和所述二抗溶液由抗体稀释缓冲液稀释得到，所述抗体稀释缓冲液包含以下成分：
0.02～0.08mol/L的Tris-HCl缓冲液，
含有0.05～0.15％质量浓度的聚乙二醇或吐温，和
0.010～0.020mol/L叠氮化钠；
更优选地，所述抗体稀释缓冲液包含以下成分：
0.05mol/L的Tris-HCl缓冲液，
含有0.1％质量浓度的聚乙二醇或吐温，和
0.015mol/L叠氮化钠。
在本发明的优选的实施方案中，所述AKR1C3单克隆抗体为小鼠单克隆抗体；
和/或，所述二抗为山羊抗小鼠抗体、兔抗小鼠抗体、马抗小鼠抗体或驴抗小鼠抗体。
在本发明的优选的实施方案中，上述检测AKR1C3的诊断试剂盒还包括：
封闭液，优选地，所述封闭液为AKR1C3单克隆抗体来源动物的血清；
更优选地，所述封闭液为小鼠血清。
在本发明的优选的实施方案中，上述检测AKR1C3的诊断试剂盒还包括：
阴性质控试剂溶液；以及
说明书。
基于相同的发明构思，本发明的另一个方面提供了上述检测AKR1C3的诊断试剂盒在制备治疗癌症、肿瘤或细胞增生性疾病的药物中的用途。
在本发明的优选的实施方案中，上述用途包括以下步骤：
使用上述检测AKR1C3的诊断试剂盒获得患者离体的福尔马林固定石蜡包埋人体组织切片中AKR1C3的表达水平；
对AKR1C3的表达水平大于或等于预定表达水平的患者施加含有AKR1C3活化的抗癌药物。
在本发明的优选的实施方案中，所述AKR1C3活化的抗癌药物满足但不限于以下定义中的至少一项：
A.在存在AKR1C3抑制剂的环境下，检测得到的某化合物的癌细胞增殖抑制作用小于不存在AKR1C3抑制剂的环境下检测得到的该化合物的癌细胞增殖抑制作用；
B.某化合物对AKR1C3酶不同表达水平的癌细胞的增殖抑制作用具有显著差异，且对高表达AKR1C3酶的癌细胞增殖抑制作用远大于低表达AKR1C3酶的癌细胞增殖抑制作用；
C.当某些含有碳氧双键的醛酮化合物对AKR1C3酶不同表达水平的癌细胞增殖抑制作用具有显著差异时，且对高表达AKR1C3酶的癌细胞增殖抑制作用远大于低表达AKR1C3酶的癌细胞增殖抑制作用，且对应的含有羟基的醇类化合物对于AKR1C3酶不同表达水平的癌细胞增殖抑制作用差距不大或近似，则该含有碳氧双键的醛酮化合物为AKR1C3活化的抗癌药物，对应的含有羟基的醇类化合物为原体药物。
在本发明的优选的实施方案中，所述AKR1C3活化的抗癌药物选自以下结构的化合物：
Figure PCTCN2021114774-appb-000001
Figure PCTCN2021114774-appb-000002
Figure PCTCN2021114774-appb-000003
Figure PCTCN2021114774-appb-000004
Figure PCTCN2021114774-appb-000005
或者
专利PCT/NZ2019/050030，公开号WO2019190331中的化合物5-硝基苯磺酰胺二溴、溴甲磺酸盐和双甲磺酸盐芥(化合物562-674)，3-甲基-5-硝基苯磺酰胺二溴、溴甲磺酸盐和双甲磺酸盐芥(化合物679-791)，3-三氟甲基-5-硝基苯磺酰胺二溴、溴甲磺酸盐和双甲磺酸盐芥(化合物913-1025)，3-乙炔基-5-硝基苯磺酰胺二溴、溴甲磺酸盐和双甲磺酸盐芥(1030-1142)和5-硝基苯磺酰胺双甲磺酸盐芥的甲磺酸盐(化合物640.Ms、641.Ms、642.Ms、643.Ms、644.Ms、757.Ms、758.Ms、991.Ms、992.Ms、1108.Ms和1109.Ms)；或者
Figure PCTCN2021114774-appb-000006
Figure PCTCN2021114774-appb-000007
或其药学上可接受的盐或其异构体；或者
专利PCT/IB2020/057285，公开号为WO2021005586A1中的化合物Ex1～Ex170；或者
Figure PCTCN2021114774-appb-000008
或其药学上可接受的盐或其异构体；
优选地，所述AKR1C3活化的抗癌药物选自以下结构的化合物：
Figure PCTCN2021114774-appb-000009
Figure PCTCN2021114774-appb-000010
或者
Figure PCTCN2021114774-appb-000011
或者
Figure PCTCN2021114774-appb-000012
或其药学上可接受的盐或其异构体。
在本发明的优选的实施方案中，所述癌症、肿瘤或细胞增生性疾病包括：
肺癌、非小细胞肺癌、肝癌、胰腺癌、乳腺癌、胃癌、骨癌、食管癌、乳房癌、前列腺癌、睾丸癌、结肠癌、卵巢癌、膀胧癌、宫颈癌、肝细胞癌、黑色素瘤、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、肾细胞癌、囊性腺癌、囊性癌、髓状癌、支气管癌、骨细胞癌、上皮癌、胆管癌、绒毛膜癌、胚癌、精原细胞癌、维尔姆斯癌、胶质细胞癌、星形细胞瘤、成神经管细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、成血细胞瘤、声带神经瘤、脑膜瘤、成神经细胞瘤、成视神经细胞瘤、成视网膜细胞瘤、神经纤维瘤、纤维肉瘤、成纤维细胞瘤、纤维瘤、纤维腺瘤、纤维软骨瘤、纤维囊瘤、纤维粘液瘤、纤维骨瘤、纤维粘液肉瘤、纤维乳头状瘤、粘液肉瘤、粘液囊瘤、粘液软骨瘤、粘液软骨肉瘤、粘液软骨纤维肉瘤、粘液腺瘤、成粘液细胞瘤、脂肉瘤、脂肪瘤、脂肪腺瘤、成脂细胞瘤、脂肪软骨瘤、脂肪纤维瘤、脂肪血管瘤、粘液脂瘤、软骨肉瘤、软骨瘤、软骨肌瘤、脊索瘤、绒毛膜腺瘤、绒毛上皮瘤、成绒毛膜细胞瘤、骨肉瘤、成骨细胞瘤、骨软骨纤维瘤、骨软骨肉瘤、骨软骨瘤、骨囊瘤、骨牙质瘤、骨纤维瘤、骨纤维肉瘤、血管肉瘤、血管瘤、血管脂肪瘤、血管软骨瘤、成血管细胞瘤、血管角质瘤、血管神经胶质瘤、血管内皮瘤、血管纤维瘤、血管肌瘤、血管脂肪瘤、血管淋巴管瘤、血管脂肪平滑肌瘤、血管肌脂瘤、血管肌神经瘤、血管粘液瘤、血管网状内皮瘤、淋巴管肉瘤、淋巴肉芽瘤、淋巴管瘤、淋巴瘤、淋巴粘液瘤、淋巴肉瘤、淋巴管纤维瘤、淋巴细胞瘤、淋巴上皮瘤、成淋巴细胞瘤、外周性T细胞淋巴瘤、结节性NK/T细胞淋巴瘤、内皮瘤、成 内皮细胞瘤、滑膜瘤、滑膜肉瘤、间皮瘤、结缔组织瘤、尤因瘤、平滑肌瘤、平滑肌肉瘤、成平滑肌瘤、平滑肌纤维瘤、横纹肌瘤、横纹肌肉瘤、横纹肌粘液瘤、急性淋巴白血病、急性骨髓性白血病、慢性病细胞、红细胞增多症、淋巴瘤、子宫内膜癌、胶质瘤、结直肠癌、甲状腺癌、尿路上皮癌或多发性骨髓瘤；
优选地，所述癌症、肿瘤或细胞增生性疾病包括：卵巢癌、宫颈癌、胰腺癌、乳腺癌、结直肠癌、食管癌、胃癌、肝细胞癌、非小细胞肺癌、前列腺癌、肾细胞癌、外周性T细胞淋巴瘤或结节性NK/T细胞淋巴瘤。
附图说明
图1为对CRC样本采用高pH值抗原修复液进行修复后的IHC染色照片，Scanscope扫描0.4x，CRC样本：335933-P；其中a图对应Ab(抗体)：1：1000，一抗和二抗孵育时间分别为20min和20min；b图对应Ab：1：2000，一抗和二抗孵育时间分别为45min和30min；c图对应Ab：1：2000，一抗和二抗孵育时间分别为60min和30min；
图2为对CRC样本采用低pH值抗原修复液进行修复后的IHC染色照片，Scanscope扫描0.4x，CRC样本：335933-P；其中a图对应Ab：1：1000，一抗和二抗孵育时间分别为30min和30min；b图对应Ab：1：2000，一抗和二抗孵育时间分别为45min和30min；c图对应Ab：1：2000，一抗和二抗孵育时间分别为60min和30min；
图3为胃癌样本在低pH值下进行IHC染色的结果照片；其中a图对应Ab：1：1000，一抗和二抗孵育时间分别为30min和30min；b图对应Ab：1：2000，一抗和二抗孵育时间分别为45min和30min；c图对应Ab：1：2000，一抗和二抗孵育时间分别为45min和45min；
图4为乳腺癌样本在低pH值下进行IHC染色的结果照片；其中a图对应Ab：1：1000，一抗和二抗孵育时间分别为30min和30min；b图对应Ab：1：2000，一抗和二抗孵育时间分别为45min和30min；c图对应Ab：1：2000，一抗和二抗孵育时间分别为45min和45min；
图5为不同单克隆抗体稀释和孵育时间下的染色性能比较照片，以正常结肠组织：391761-YN为例，Scanscope扫描4x；其中a图对应Ab：1：1000，一抗和二抗孵育时间分别为30min和30min；b图对应Ab：1：2000，一抗和二抗孵育时间分别为45min和30min；c图对应Ab：1：2000，一抗和二抗孵育时间分别为30min和30min；
图6为使用最佳染色方案比较5种不同正常结肠组织间一致性的染色性能：低pH值TRS，97℃20分钟；AKR1C3稀释1:2000，30分钟；HRP孵育时间：30分钟，Scanscope扫描2x；其中a图对应样本390211-YN，b图对应样本390650-YN，c图对应样本391182-YN，d图对应样本391761-YN，e图对应样本3919951-YN；
图7在正常组织中使用最佳染色条件进行的AKR1C3染色照片；其中a图和b图分别对应扁桃体正常组织的低倍和高倍染色照片；c图和d图分别对应胃正常组织的低倍和高倍染色照片；e图和f图分别对应结肠正常组织的低倍和高倍染色照片；
图8在非小细胞肺癌中使用最佳方案进行的AKR1C3染色照片；其中a图和b图分别对应样本1(F102582A22)的低倍和高倍染色照片；c图和d图分别对应样本2(F134064A12)的低倍和高倍染色照片；
图9在胃癌中使用最佳方案进行的AKR1C3染色照片；其中a图和b图分别对应样本1(F180723A5)的低倍和高倍染色照片，向上箭头：肿瘤细胞，向下箭头：剩余的正常胃粘膜腺上皮；c图和d图分别对应样本2(F180684A3)的低倍和高倍染色照片，向下箭头：肿瘤细胞，向上箭头：剩余的正常胃粘膜腺上皮；
图10在乳腺癌中使用最佳方案进行的AKR1C3染色照片；其中a图和b图分别对应样本1(F162870A5)的低倍和高倍染色照片；c图和d图分别对应样本2(F130368B3)的低倍和高倍染色照片；
图11在肝细胞癌中使用最佳方案进行的AKR1C3染色照片；其中a图和b图分别对应样本1(DLV13050B3)的低倍和高倍染色照片；c图和d图分别对应样本2(DLV13052B5)的低倍和高倍染色照片；
图12在结直肠癌中使用最佳方案进行的AKR1C3染色照片；其中a图和b图分别对应样本(335933-P)的低倍和高倍染色照片；
图13为AKR1C3 IHC测定最佳方案下同时具有阳性和阴性成分的正常结肠组织染色，不同倍数扫描照片；其中a图对应阴性质控试剂(Scanscope扫描4x)，b图对应AKR1C3(Scanscope扫描10x)，c图对应AKR1C3(Scanscope扫描4x)，d图对应AKR1C3(Scanscope扫描10x)；
图14为组织质控样本照片，将同时具有阳性和阴性成分的正常结肠组织用作双重阳性和阴性组织质控，并用于每次染色操作；其中a图对应阴性质控试剂(Scanscope扫描4x)，b图对应AKR1C3染色(Scanscope扫描4x)；；
图15为样本F151286A5 HCC的照片，H-评分300(3+：100％)。所有肿瘤细胞均呈现强烈的细胞质/细胞核染色；同时，癌巢附近的正常肝细胞(箭头)以及基质细胞和内皮细胞显示不同强度的染色作为内部质控；其中a图对应较低放大倍数(Scanscope扫描0.4x)，b图对应较高放大倍数(Scanscope扫描10x)；
图16为样本F151725A1 EC的照片，H-评分160(0：0％；1+：60％；2+：20％；3+：20％)；肿瘤细胞呈现不同强度的不同细胞质/细胞核染色；其中a图对应较低放大倍数(Scanscope扫描0.4x)，b图对应较高放大倍数(Scanscope扫描10x)；
图17为样本F152459A4 GC的照片，H-评分35(0：85％；1+：5％；2+：0％；3+：10％)；肿瘤细胞呈现不同强度的不同细胞质/细胞核染色。放大倍数越高，显示的面积(向上箭头)越小；内皮细胞显示染色作为内部质控(向下箭头)；其中a图对应较低放大倍数(Scanscope扫描0.4x)，b图对应较高放大倍数(Scanscope扫描10x)；
图18为样本F151653A1 CRC的照片，H-评分120(0：30％；1+：30％；2+：30％；3+：10％)；肿瘤细胞呈现不同强度的不同细胞质/细胞核染色；肿瘤区域为向右蓝色箭头，正常组织为向左箭头；其中a图对应较低放大倍数(Scanscope扫描0.4x)，b图对应较高放大倍数(Scanscope扫描10x)；
图19为样本F183410A4 PC的照片，H-评分0(0：100％)；无肿瘤细胞出现AKR1C3的细胞质/细胞核染色；然而，内皮细胞显示染色作为内部质控(箭头)；其中a图对应较低放大倍数(Scanscope扫描0.4x)，b图对应较高放大倍数(Scanscope扫描10x)。
具体实施方式
需要说明的是，除非另外定义，本说明书一个或多个实施例使用的技术术语或者科学术语应当为本公开所属领域内具有一般技能的人士所理解的通常意义。
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药材原料、试剂材料等，如无特殊说明，均为市售购买产品。
“患者”及“个体”可互换使用，是指需要癌症治疗的哺乳动物。通常，患者是人类。通常，患者是诊断患有癌症的人类。在某些实施例中，“患者”或“个体”可指用于筛选、表征及评估药物及疗法的非人类哺乳动物，例如非人类灵长类动物、狗、猫、兔、猪、小鼠或大鼠。
“前药”是指投与或施用之后经新陈代谢或以其他方式转化为关于至少一种性质的生物学活性或活性更高的化合物(或药物)的化合物。相对于药物，前药以使其相对于药物活性较低或无活性的方式化学修饰，但化学修饰使得在前药投与之后通过代谢或其他生物过程产生相应药物。前药可相对于活性药物具有改变的代谢稳定性或输送特征、较少副作用或较低毒性或经改良的风味(参见(例如)参考文献Nogrady，1985，Medicinal Chemistry A Biochemical Approach，0xford University Press，New York，第388页至392页，其以引用式并入本文中)。前药可使用除相应药物以外的反应物来合成。
病况或患者的“治疗”是指采取步骤以获得有益或期望结果(包括临床结果)。出于本发明的目的，有益或期望临床结果包括(但不限于)一或多种癌症症状的缓和或改善；疾病程度的减弱；疾病进展的延迟或减缓；疾病状态的改善、缓解或稳定；或其他有益结果。在一些情形下，癌症的治疗可使得部分反应或稳定疾病。
“肿瘤细胞”是指任何适当物种(例如，哺乳动物，例如鼠类、犬、猫、马或人类)的肿瘤细胞。
本发明的申请人开发的以过表达AKR1C3为靶标的DNA烷化剂抗癌前药包括：1)DNA烷化剂，对应PCT申请号PCT/US2016/021581，公开号WO2016/145092A，对应中国申请号2016800150788，公开号CN107530556A；2)(R)-及(S)-1-(3-(3-N,N-二甲基胺基羰基)苯氧基-4-硝苯基)-1-乙基-N,N’-双(伸乙基)胺基磷酸酯、组合物及其使用及制备方法，对应PCT申请号PCT/US2016/062114，公开号WO2017087428A1，对应中国申请号2016800446081，公开号CN108290911A中的S构型化合物；3)硝基苄基衍生物抗癌试剂，对应PCT申请号PCT/US2016/025665，公开号WO2016/161342，对应中国申请号2016800200132，公开号CN108136214A。前药形式的化合物在细胞中的生化环境中通过AKR1C3的催化下发生还原，得到具有细胞毒性的毒素而发挥癌细胞毒杀作用。
特别的，中文名为(S)-1-(3-(3-N,N-二甲氨基羰基)苯氧基-4-硝基苯基)-1-乙基-N,N'-双(亚乙基)氨基磷酸酯，也称为OBI-3424、AST-3424、TH-2870的S构型化合物)，CAS号为2097713-69-2，其结构如下：
Figure PCTCN2021114774-appb-000013
已分别在美国和中国进行一期临床试验。
上述药物只对AKR1C3有表达的患者有效，因此有必要对患者的AKR1C3表达水平进行检测。在实际应用中，需要判断患者的组织样本是否达到预定的AKR1C3表达水平进而满足给予上述三个专利(CN107530556A、CN108290911A、CN108136214A)药物的条件，这就要求IHC检测方法必须是染色结果稳定。发明人尝试将现有技术中公开的IHC检测方法用于多种癌症肿瘤组织中AKR1C3表达水平的检测，研究发现，现有技术中公开的IHC检测方法并不能满足实际需求，这是因为现有的IHC检测方法都是某个医院实验室或科研院所实验室针对某个特定的组织就某个酶或蛋白开发一个特定的IHC检测某个酶或蛋白的方法，而不能作为大规模的商业试剂盒中使用的IHC染色检测方法，这些方法不具有较好的灵敏度、精密度和一致性(不同实验室、不同操作人员、不同操作时间)，而且不能适应多种不同癌症肿瘤组织的IHC检测。如果染色结果不稳定，那么AKR1C3表达水平的判断就会不准确，进而造成癌症治疗效果不理想。
为了解决现有技术中IHC检测方法不能适应多种癌症肿瘤组织中AKR1C3表达水平的检测且染色结果不稳定的问题，发明人尝试对现有的IHC检测方法进行进一步的改进，并提供了一种AKR1C3检测方法，该检测方法能同时适用于多种癌症肿瘤组织中AKR1C3表达水平的检测且染色结果稳定，具有较好的灵敏度、精密度和一致性。
本发明的一个方面提供了一种AKR1C3检测方法，使用免疫组织化学染色法检测离体的福尔马林固定石蜡包埋(FFPE)人体组织切片中AKR1C3的表达水平，包括以下步骤：
a)抗原修复
在抗原修复液的存在下，将福尔马林固定石蜡包埋人体组织切片于90～115℃加热17～30分钟进行抗原修复；
b)一抗孵育
将抗原修复后的福尔马林固定石蜡包埋人体组织切片与浓度为0.5～5.0μg/ml的AKR1C3单克隆抗体溶液混合后孵育25～700分钟；
c)二抗孵育
将一抗孵育后的福尔马林固定石蜡包埋人体组织切片与浓度为0.5～5.0μg/ml的二抗溶液混合后孵育25～700分钟。
测定灵敏度结果显示，本发明提供的IHC法测定多种人体癌症组织中AKR1C3表达水平的分析灵敏度具有可接受的性能特征，其显示了检测样本中AKR1C3的预期染色模式和定位，并在质控上具有适当的性能，其符合分析灵敏度研究的标准限度。测定精密度结果显示，批间(日间/操作间、操作员间和仪器间)和批内精密度100％一致，这符合基于肿瘤细胞细胞质和细胞核中AKR1C3表达的≥95％标准限度。测定一致性结果显示，基于AKR1C3在肿瘤细胞细胞质和细胞核中的表达，病理学家间的一致性为100％，且符合≥90％的标准限度。总体而言，该测定显示了可接受的结果，证明了使用包含上述关键步骤的IHC方法来进行多种人体癌症组织的福尔马林固定石蜡包埋样本切片中AKR1C3表达水平的测定是可行的。
在本发明中，样品制备，使用福尔马林和石蜡对组织样本进行处理后得到福尔马林固定石蜡包埋(FFPE)样本，然后进行切片，福尔马林固定石蜡包埋人体组织切片的厚度一般为4mm。
在本发明中，抗原修复是免疫组织化学染色法在进行抗体标记前的必要步骤，因为组织的固定过程通常会引起蛋白交联，这在使用福尔马林固定时因其化学属性而经常发生，需要通过抗原修复步骤使得抗原表位被重新暴露以便于抗体的结合。本发明利用抗原修复液和热的作用将这些抗原重新暴露出来，将待修复的切片置于抗原修复液中，然后进行加热，即通过化学-热的联合作用来实现抗原修复。常用的操作是将切片放入抗原修复液中，连同容器放入高压锅中加热，保持预定的温度一段时间后取出，自然降温。在本发明的优选的实施方案中，在步骤a)抗原修复中，所述福尔马林固定石蜡包埋人体组织切片于92～102℃加热18～25分钟；更优选地，所述福尔马林固定石蜡包埋人体组织切片于97℃加热20分钟，这样可以取得最好的效果：对各种癌症肿瘤组织切片染色效果好。本发明并不对抗原修复后的冷却温度作限定，可自然冷却至室温，也可冷却至一定温度(例如65℃)再进行后续的操作。
另外，抗原修复液的pH值会影响抗原修复效果，并结合后续使用的是苏木精染色，在本发明的优选的实施方案中，所述抗原修复液的pH值为2.0～9.0；更优选地，所述抗原修复液的pH值为6.0～9.0；进一步优选地，所述抗原修复液的pH值为6.0。本发明并不对抗原修复液的成分进行限制，只要抗原修复液的pH值满足要求均可用于本发明，抗原修复液包括但不限于：柠檬酸钠抗原修复液(Citrate Antigen Retrieval Solution)、EDTA抗原修复液(EDTA Antigen Retrieval solution)等。
AKR1C3单克隆抗体溶液的浓度对于步骤b)一抗孵育操作具有较大的影响，通过实验确定1.0～3.0μg/ml浓度的AKR1C3单克隆抗体溶液具有较好的效果，更优选的为1.2ug/ml浓度，此时AKR1C3单克隆抗体溶液的稀释倍数为1:2000。
为了取得更好的效果，其中所述AKR1C3单克隆抗体溶液由包含以下成分的稀释液稀释得到：
0.02～0.08mol/L的Tris-HCl缓冲液(氨丁三醇-盐酸缓冲液)，含有0.05～0.15％质量浓度的聚乙二醇或吐温，和0.010～0.020mol/L叠氮化钠；
更优选地，所述抗体稀释缓冲液包含以下成分：
0.05mol/L的Tris-HCl缓冲液，含有0.1％质量浓度的聚乙二醇或吐温，和0.015mol/L叠氮化钠。
免疫组织化学染色法的二抗必须是抗一抗物种的抗原，比如说：检测A动物的B蛋白所用的一抗为动物C抗动物A的B蛋白抗体，二抗就应该是动物D抗动物C抗体，本发明是为了检测人类的AKR1C3(蛋白)，其所用的一抗为小鼠抗人的AKR1C3单克隆抗体即鼠AKR1C3单克隆抗体，那么二抗就是其他动物(如山羊、兔、马、驴)抗小鼠抗体。
在本发明的优选的实施方案中，在步骤c)二抗孵育之后，还包括：
d)染色封固
使用苏木精对福尔马林固定石蜡包埋人体组织切片进行染色，染色后脱水封片以便于后续观察评分。
在本发明的优选的实施方案中，在步骤a)抗原修复之前，还包括：
a1)脱蜡复水
将福尔马林固定石蜡包埋人体组织切片使用有机溶剂脱蜡，脱蜡后的切片经过不同水 含量的醇依次洗涤，最后使用水洗涤；脱蜡复水操作是为了将干化的样本补充水分，便于后续的各种操作。
对于福尔马林固定石蜡包埋人体组织切片而言，由于是进行了石蜡包埋，而后续的染色过程中出现石蜡会产生严重影响，因此必须使用相应的有机溶剂将石蜡洗脱干净。常用的洗脱石蜡但又不会损失切片的有机溶剂包括丙酮、二甲苯、甲苯等，但二甲苯效果较好，毒性较小。洗净石蜡后，需要使用溶于水的醇(甲醇或乙醇)来洗净残留的有机溶剂，通常使用乙醇。为了到达较好的洗涤效果，使用梯度洗脱法，即脱蜡后的切片先经过无水乙醇洗涤，再经过体积分数为90-97％(例如95％)的乙醇洗涤，最后使用水洗涤。
在本发明的优选的实施方案中，在步骤a)抗原修复和步骤b)一抗孵育之间，还包括：
b1)封闭非特异性抗原
将抗原修复后的福尔马林固定石蜡包埋人体组织切片与封闭液共同孵育以封闭非特异性抗原；
组织中内源性酶和抗体的封闭对于背景染色的最小化以及降低假阳性染色十分重要。这通常是通过用可封闭一抗或二抗也可能结合的非特异性位点的特定缓冲液对样品进行孵育而实现的。封闭非特异性抗原操作中使用的试剂比较多，其主要的目的是使得样本中的其他蛋白、生物素、内源性酶以及IHC检测体系中的其他物质(干扰物质)不会干扰IHC检测中的被测的AKR1C3的检测结果。在本发明中使用与小鼠单克隆抗体相匹配的小鼠血清可以将复杂的干扰物质都掩蔽，操作方便。
在本发明的优选的实施方案中，一种AKR1C3检测方法，使用免疫组织化学染色法检测离体的福尔马林固定石蜡包埋(FFPE)人体组织切片中AKR1C3的表达水平，包括以下步骤：
a1)脱蜡复水
将适当厚度的福尔马林固定石蜡包埋人体组织切片使用有机溶剂脱蜡，脱蜡后的切片经过不同水含量的醇洗涤，最后使用水洗涤；
a)抗原修复
在抗原修复液存在下，将脱蜡复水操作后的福尔马林固定石蜡包埋人体组织切片于90～115℃加热17～30分钟进行抗原修复；
b1)封闭非特异性抗原
将抗原修复后的福尔马林固定石蜡包埋人体组织切片与封闭液共同孵育以封闭非特异性抗原；
b)一抗孵育
将封闭非特异性抗原后的福尔马林固定石蜡包埋人体组织切片与浓度为0.5～5.0μg/ml的AKR1C3单克隆抗体溶液混合后孵育25～700分钟；
c)二抗孵育
将一抗孵育后的福尔马林固定石蜡包埋人体组织切片与浓度为0.5～5.0μg/ml的二抗溶液混合后孵育25～700分钟；
d)染色封固
使用苏木精对福尔马林固定石蜡包埋人体组织切片进行染色，染色后脱水封片；
e)观察评分
观察染色后的人体组织切片，根据观察到的染色程度来评价人体组织切片中AKR1C3的表达水平的高低。
当然，在步骤c)二抗孵育操作前可以再增加一次封闭非特异性抗原步骤，使用二抗动物的血清作为封闭液。
使用上述条件对包括乳腺癌、结直肠癌、食管癌、胃癌、肝细胞癌、非小细胞肺癌、前列腺癌、肾细胞癌、外周性T细胞淋巴瘤、结节性NK/T细胞淋巴瘤在内的多种癌(肿瘤)的福尔马林固定石蜡包埋人体组织切片进行IHC检测，均能取得较好的染色效果，由此可以证明上述提供的AKR1C3的IHC检测方法是适应多种癌症肿瘤组织的。
基于相同的发明构思，本发明的另一个方面提供了一种检测AKR1C3的诊断试剂盒，包括：
抗原修复液；
浓度为0.5～5.0μg/ml的AKR1C3单克隆抗体溶液；
浓度为0.5～5.0μg/ml的二抗溶液。
在本发明的优选的实施方案中，上述检测AKR1C3的诊断试剂盒还包括：
封闭液，优选地，所述封闭液为AKR1C3单克隆抗体来源动物的血清；更优选地，所述封闭液为小鼠血清。
在本发明的优选的实施方案中，上述检测AKR1C3的诊断试剂盒还包括：
阴性质控试剂溶液；以及
记载相关操作方法的说明书。
阴性质控试剂溶液的加入是为了更好的保证检测的结果，但不是必须的，可以添加或不添加：添加后结果中由于有了阴性质控试剂的存在，便于进行对照来检验染色检测操作是否正常。优选地，阴性质控试剂(NCR)为市售的产品：DAKO公司的FLEX阴性质控，小鼠，(Link)。“Link”是指该试剂与下面实施例使用的Dako Autostainer Link 48全自动免疫组化染色系统配合使用。
基于相同的发明构思，本发明的另一个方面提供了上述检测AKR1C3的诊断试剂盒在制备治疗癌症、肿瘤或细胞增生性疾病的药物中的用途。
在本发明的优选的实施方案中，上述用途包括以下步骤：
使用上述检测AKR1C3的诊断试剂盒获得患者离体的福尔马林固定石蜡包埋人体组织切片中AKR1C3的表达水平；
对AKR1C3的表达水平大于或等于预定表达水平的患者施加AKR1C3活化的抗癌药物。
在本发明中，对于不同的癌症或肿瘤，AKR1C3的表达水平的高低是不同的，因此适合施用AKR1C3活化的抗癌前药的药物的癌症或肿瘤组织切片样本的AKR1C3表达水平也对应不同：有些癌症需要高表达，有些癌症中等表达就可以施用药物。该AKR1C3预定表达水平可用H评分表示，并可用统计学方法得到每种癌症类型对应的AKR1C3预表达水平。
在本发明中，AKR1C3活化的抗癌药物，当然包括AKR1C3活化的抗癌前药，即前药形式的化合物在细胞中的生化环境中通过AKR1C3的催化最终得到具有细胞毒性的毒 素而发挥癌细胞毒杀作用。
广义而言，AKR1C3活化的抗癌药物满足但不限于以下条件中的至少一项：
A.在存在AKR1C3抑制剂(如上述三个专利中公开的TH-3021，Flanagan等人，Bioorganic and Medicinal Chemistry(2014)第962-977页中的化合物36即
Figure PCTCN2021114774-appb-000014
)的环境下，检测得到的某化合物的癌细胞增殖抑制作用小于不存在AKR1C3抑制剂(如上述三个专利中公开的TH-3021)的环境下检测得到的该化合物的癌细胞增殖抑制作用，当癌细胞增殖抑制作用使用IC 50进行量化时，则如果某个化合物对某个癌细胞系在存在AKR1C3抑制剂时检测得到的IC 50大于不存在AKR1C3抑制剂时检测得到的IC 50，则可以判定该化合物为AKR1C3活化的抗癌药物(裂解前药Lysis-Prodrug)。具体的如以下专利文献：
PCT/US2016/021581，公开号WO2016145092A1，对应中国申请号2016800150788，公开号CN107530556A；
PCT/US2016/062114，公开号WO2017087428，对应中国申请号2016800446081，公开号CN108290911A；
PCT/US2016/025665，公开号WO2016161342，对应中国申请号2016800200132，公开号CN108136214A；以及
PCT/NZ2019/050030，公开号WO2019190331，对于中国申请号CN2019800234236，公开号CN111918864A中所公开的化合物，在此将以上专利文献的全文引入到本专利申请文本中。
其中，专利PCT/US2016/021581，公开号WO2016145092A1，对应中国申请号2016800150788，公开号CN107530556A；PCT/US2016/062114，公开号WO2017087428，对应中国申请号2016800446081，公开号CN108290911A；PCT/US2016/025665，公开号WO2016161342，对应中国申请号2016800200132，公开号CN108136214A中所公开的化合物为裂解前药，其最终裂解代谢出的发挥作用的原体药物为
Figure PCTCN2021114774-appb-000015
以及紫杉醇、喜树碱等药物；专利PCT/NZ2019/050030，公开号WO2019190331，对应中国申请号CN2019800234236，公开号CN111918864A中公开的化合物为裂解前药，其最终裂解代谢出的发挥作用的原体药物为氮芥结构药物；
B.某化合物对AKR1C3酶不同表达水平的癌细胞的增殖抑制作用具有显著差异，且对高表达AKR1C3酶的癌细胞增殖抑制作用远大于低表达AKR1C3酶的癌细胞增殖抑制作用，当癌细胞增殖抑制作用使用IC 50进行量化时，则如果某个化合物高表达AKR1C3酶的癌细胞的IC 50值小于低表达AKR1C3的癌细胞的IC 50值，则可以判定该化合物。具体的如专利PCT/CN2020/120281，公开号WO2021068952A1中所公开的化合物。在此将此专利文献的全文引入到本专利申请文本中。
C.醛酮还原酶1C3(AKR1C3)具有将某些特定结构的含有碳氧双键的醛酮化合物还原为对应的含有羟基的醇类化合物的功能。当某些含有碳氧双键的醛酮化合物对AKR1C3酶不同表达水平的癌细胞增殖抑制作用具有显著差异时，且对高表达AKR1C3酶的癌细胞增殖抑制作用远大于低表达AKR1C3酶的癌细胞增殖抑制作用，且对应的含有羟基的 醇类化合物对于AKR1C3酶不同表达水平的癌细胞增殖抑制作用差距不大或近似，则该含有碳氧双键的醛酮化合物为AKR1C3活化的抗癌药物(还原前药Reduction-Prodrug)，对应的含有羟基的醇类化合物为原体药物(Drug)。具体的如专利PCT/IB2020/057285，公开号为WO2021005586A1中所公开的化合物，在此将此专利文献的全文引入到本专利申请文本中。
上述专利申请公开的通式化合物、具体化合物均属于AKR1C3活化的抗癌药物/前药，在此将上述申请引用到本申请说明书中。
进一步的，优选以下结构的化合物，这些化合物均为AKR1C3活化的抗癌药物/前药：
Figure PCTCN2021114774-appb-000016
Figure PCTCN2021114774-appb-000017
Figure PCTCN2021114774-appb-000018
Figure PCTCN2021114774-appb-000019
Figure PCTCN2021114774-appb-000020
这些化合物是上述A类型的AKR1C3活化的抗癌药物；或者
Figure PCTCN2021114774-appb-000021
Figure PCTCN2021114774-appb-000022
Figure PCTCN2021114774-appb-000023
或其药学上可接受的盐或其异构体，这些化合物是上述B类型的AKR1C3活化的抗癌药物；或者
Figure PCTCN2021114774-appb-000024
或其药学上可接受的盐或其异构体，这些化合物是上述C类型的AKR1C3活化的抗癌药物。
本发明使用了来自9个标志物的共46个组织样本，包括5个RCC(肾细胞癌)样本、5个HCC(肝细胞癌)样本、5个NSCLC(非小细胞肺癌)样本、5个GC(胃癌)样本、5个PC(前列腺癌)样本、5个EC(食道癌)样本、5个CRC(结直肠癌)样本、6个外周性T细胞淋巴瘤样本和5个NK/T细胞淋巴瘤样本。
对于每次染色操作，将同时具有阳性和阴性成分的正常结肠组织用作双重阳性和阴性组织质控。
上述所有样本均为福尔马林固定石蜡包埋(FFPE)人体组织切片，且切成4μm厚的组织切片置于带正电荷的载玻片上，并在环境温度下保存直至染色。
实验生化试剂及化学试剂见下表1。
表1：实验生化试剂及化学试剂
Figure PCTCN2021114774-appb-000025
仪器设备
Ventana Benchmark Ultra全自动免疫组化仪(也称为组织切片染色机，序列号：311434、316829)
Dako Autostainer Link 48全自动免疫组化染色系统(也称为组织切片染色机序列号：AS5085D1611、AS2370D1203)
DAKO PT Link全自动免疫组化预处理系统(序列号：MY1716P184、PT3543Y1310)
Sakura Tissue-Tek DRS载玻片染色机(序列号：49310219-0409)
Sakura载玻片加热器(序列号：14881799-0409)
Sakura切片机(序列号：1429-1407)
NIKON显微镜(序列号：940776)
HANNA pH计(序列号：08678109)
带正电荷的显微镜载玻片
Aperio Scanscope XT数字切片扫描系统(序列号：SS001403)
下面结合具体的实施例对本发明提供的技术方案做进一步的描述。下述实施例仅用于对本发明进行说明，并不会对本发明的保护范围进行限制。
以下实施例中涉及到的染色结果评价标准为：
(1)染色说明
由AKR1C3抗体标记的细胞显示细胞质和/或细胞核染色。
(2)一般评分指南
任何染色或染色缺失的解释均通过形态学研究使用适当的质控来完成，并由合格的病理学家(GH和KZ)进行评价，以确定AKR1C3染色在肿瘤样本中的定位、分布和强度。
反应性评价包括以下几个方面：
染色的细胞定位
染色强度
亚细胞定位
关注区域中细胞染色的百分比
AKR1C3测定在半定量的尺度上进行评价，并记录细胞质和细胞核染色在以下四个水平(0、1+、2+和3+)的细胞染色百分比。
(3)肿瘤样本评分标准
使用H-评分：细胞核-细胞质染色肿瘤细胞的％(0到3+的总数值不应超过100)来进行染色程度即AKR1C3酶表达水平高低的评分：
0(未染色)：0到100之间的值
肿瘤细胞核-细胞质1+(弱染色)：0到100之间的值
肿瘤细胞核-细胞质2+(中等染色)：0到100之间的值
肿瘤细胞核-细胞质3+(强染色)：0到100之间的值
细胞核-细胞质阳性染色的总数％：0到100之间的值
将根据每个强度的肿瘤比例评分来计算总的H评分。最终H评分计算如下：H-评分＝(％弱[1+]×1)+(％中等[2+]×2)+(％强[3+]×3)
在本验证过程中，没有为临床解释指定特定的临界评分来确定AKR1C3阳性和阴性状态，这可能在临床试验完成后确定。
将总阳性％评分±10％定义为相同样本在病理学家之间的一致性。然而，如果病理学家将同一病例评分为0和1％，则应视为不一致。
实施例1 AKR1C3检测方法的建立
本实施例中，首先对商业购买的Dako Autostainer Link 48平台和Ventana Benchmark Ultra平台提供的现有方法进行预验证，预验证结果表明，这两个平台提供的现有方法都不适用于AKR1C3的IHC方法。因此，需要对Ventana Benchmark Ultra和Dako Autostainer Link 48进行条件优化。
发明人根据IHC的原理并结合预验证的染色结果，确定需要优化的条件包括：
优化Ab浓度和孵育时间
优化抗原修复条件
优化检测系统的条件
1.1优化Ab浓度
预验证结果表明，AKR1C3 1:100和1:500具有极强的背景染色。1:4000的染色过弱。然后，在下一步中主要关注AKR1C3 1:1000和1:2000，尽管两者也均显示背景染色。
1.2.优化抗原修复条件
使用高pH值(pH 9.0)抗原修复液进行染色实验，结果如表2所示：
表2高pH值抗原修复液进行染色实验结果
Figure PCTCN2021114774-appb-000026
使用低pH值(pH 6.0)抗原修复液进行染色实验，结果如表3所示：
表3低pH值抗原修复液进行染色实验结果
Figure PCTCN2021114774-appb-000027
高pH值抗原修复液有极强的背景染色。然而，如果低pH值，背景染色明显减少。低pH值抗原修复液将用于本验证(Scanscope扫描1x)。
1.3更多样本中进行最佳测试
在包括正常组织和各种实体肿瘤在内的更多样本中进行最佳测试，使用抗原修复低pH值来确定最佳Ab浓度和孵育时间。
表4胃癌样本在低pH值下进行IHC染色的结果
Figure PCTCN2021114774-appb-000028
由图3a-3c的染色结果可以看出，图3a中出现背景/非特异性染色，图3b中适当染色，图3c中出现背景/非特异性染色。
表5乳腺癌样本在低pH值下进行IHC染色的结果
Figure PCTCN2021114774-appb-000029
由图4a-4c的染色结果可以看出，图4a中出现背景/非特异性染色，图4b中适当染色，图4c中出现背景/非特异性染色。
由此可以确定，在以下条件下显示最佳染色性能：低pH值抗原修复液，97℃20分钟；AKR1C3稀释1:2000，孵育时间：45分钟；HRP(辣根过氧化物酶)孵育时间：30分钟，根据阳性和阴性组织成分的染色以及关于细胞定位和染色强度范围的特异性阳性染色来判断，产生最佳信噪比。然后，该最佳染色方案将用于该验证(Scanscope扫描1x)。
1.4 QC(质量控制)确认
使用上述最佳染色方案对5个正常结肠组织进行预验证，以测试染色性能，并确认其是否是研究的合适QC质控。
表6正常结肠组织在最佳染色方案下的染色结果
Figure PCTCN2021114774-appb-000030
由图5a-5c的染色结果可以看出，图5a中出现背景/非特异性染色，图5b中适当染色，图5c中出现弱特异性染色。采用最佳染色方案，所有5个正常结肠组织均表现出一致和最佳的染色性能(低pH值抗原修复液，97℃20分钟；AKR1C3稀释1:2000，孵育时间：45分钟；HRP孵育时间：30分钟)，通过阳性和阴性组织成分的染色以及关于细胞定位和染色强度范围的特异性阳性染色来判断，产生最佳信噪比，如图6所示。然后，同时将具有阳性和阴性成分的正常结肠组织用作双重阳性和阴性组织质控，并用于研究的每次染色操作。
根据上述试验结果确定了最佳染色条件，如表7所示。
表7最佳染色条件
低pH值TRS 97℃ 20min
AKR1C3稀释 1:2000(1.2ug/ml)
一抗孵育时间 45min
二抗/HRP孵育时间 30min
即最终确定的AKR1C3检测方法包括以下步骤：
抗原修复步骤，在pH 6.0的抗原修复液存在下，将福尔马林固定石蜡包埋人体组织切片于97℃加热20分钟进行抗原修复；
一抗孵育步骤，将福尔马林固定石蜡包埋人体组织切片与1.2ug/ml浓度的AKR1C3单克隆抗体溶液和阴性质控试剂溶液混合后孵育45分钟；阴性质控试剂溶液为市售的产品：DAKO公司的FLEX阴性质控，小鼠，(Link)；
二抗孵育步骤，将福尔马林固定石蜡包埋人体组织切片与1.2ug/ml浓度的二抗溶液混合后孵育30分钟。
使用最佳染色条件下对扁桃体正常组织、胃正常组织、结肠正常组织、非小细胞肺癌、胃癌、乳腺癌、肝细胞癌和结直肠癌进行了IHC染色，如图7-12所示，各种正常组织和实体肿瘤的染色显示出最佳的信噪比，该数值通过阳性和阴性组织成分的染色以及关于细胞定位和染色强度范围的特异性阳性染色来判断。正常组织：基质细胞和内皮细胞可能显示不同强度水平的染色。
1.5 QC测定
同时具有阳性和阴性成分的正常结肠组织将用作每次染色操作的双重阳性和阴性组织质控，并将用于以下验证操作以及未来的活体研究。
如图13所示，使用上述的最优方案对具有阳性和阴性成分的正常结肠组织进行染色实验，同时扫描不同倍数的照片，可以明显发现，染色后阳性和阴性区分明显，染色效果好，便于后续由病理专家评分来判断AKR1C3表达水平的高低。
实施例2分析方法的灵敏度
2.1试验方法
总共46个组织样本，包括5个RCC、5个HCC、5个NSCLC、5个GC、5个PC、5个EC、5个CRC、6个外周性T细胞淋巴瘤组织样本和5个NK/T细胞淋巴瘤组织样本，用AKR1C3抗体进行染色，以评估该IHC测定的灵敏度。
用作双重阳性和阴性组织质控的正常结肠组织和每个样本的阴性质控试剂(NRC)包含在每次染色操作中，首先由QBEJ病理学家对其进行评价，并且必须显示出生物标志物预期的可接受染色。
标准限度：
(1)用NRC染色的样本必须显示0特异性染色和<1+强度背景染色。
(2)用AKR1C3抗体染色的样本必须显示<1+非特异性背景染色强度。
(3)用AKR1C3抗体染色的细胞必须显示适当的细胞定位。
2.2试验结果
将46个组织样本的灵敏度评估结果总结在表8中。
首先评估质控的所有结果，包括每次操作的阳性和阴性质控以及每个样本的阴性质控试剂，并显示生物标志物预期的可接受染色。
表8分析灵敏度研究的汇总结果
Figure PCTCN2021114774-appb-000031
Figure PCTCN2021114774-appb-000032
所有质控载玻片和肿瘤样本的分析灵敏度操作结果表明，AKR1C3抗体显示AKR1C3在正常结肠组织质控和肿瘤样本中的预期染色和定位，符合实施例3中提到的标准限度。
实施例3分析方法的精密度
3.1试验方法
使用上述分析灵敏度研究的5个具有不同AKR1C3表达的样本，由2名操作员在2台仪器上非连续3天进行3次染色操作，以评估批间(日间/操作间、操作员间、仪器间)精密度和批内精密度。精密度研究设计见下表9。
批间精密度包括：
(1)日间/批间精密度，要求：
染色以非连续3天为周期进行。
每个样本1张NRC片，1张AKR1C3片
(2)操作员间和仪器间精密度，要求：
在3次染色操作中利用了两个染色平台，并涉及两个操作员。
(3)批内精密度
一次操作包含4个系列切片，用于批内精密度。
每个样本1张NRC片，3张AKR1C3片
表9精密度研究实验设计
Figure PCTCN2021114774-appb-000033
Figure PCTCN2021114774-appb-000034
用作双重阳性和阴性组织质控的正常结肠组织和每个样本的阴性质控试剂(NRC)包含在每次染色操作中，首先由病理学家进行评价，并且必须显示出生物标志物预期的可接受染色。在“肿瘤样本评分标准”详细描述了结果解释和评分标准。
标准限度：
基于“肿瘤样本评分标准”详细描述的肿瘤细胞细胞质和细胞核中的AKR1C3表达，批间和批内一致性的标准限度保持在≥95％。
3.2试验结果
使用上述分析灵敏度研究的5个具有不同AKR1C3表达的样本，由2名操作员在2台仪器上非连续3天进行3次染色操作，以评估批间(日间/操作间、操作员间、仪器间)精密度和批内精密度。结果汇总在表10。
表10精密度研究总结结果
Figure PCTCN2021114774-appb-000035
AKR1C3 IHC测定由2名操作员在2台仪器上非连续3天对具有不同AKR1C3表达的5个样本进行3次染色操作，其精密度显示出可重现的结果。基于上文细描述的肿瘤细胞细胞质和细胞核中AKR1C3表达，操作间和操作内显示100％一致性，其符合≥95％的标准限度。
实施例4分析方法的一致性
4.1试验方法
两位病理学家(GH和KZ)独立评价了用于分析灵敏度研究的46个组织样本。
“肿瘤样本评分标准”详细描述了结果解释和评分标准。
标准限度：
基于“肿瘤样本评分标准”详细描述的肿瘤细胞细胞质和细胞核中的AKR1C3表达，病理学家间一致性的标准限度为≥90％。
4.2试验结果
两位病理学家(GH和KZ)独立评价了用于分析灵敏度研究的46个组织样本。结果汇总在表11中。
表11病理学家间一致性结果
Figure PCTCN2021114774-appb-000036
Figure PCTCN2021114774-appb-000037
两位病理学家(GH和KZ)之间对AKR1C3 IHC检测的病理学家间一致性显示，基于在肿瘤细胞细胞质和细胞核中的AKR1C3表达的一致性为100％(46/46)，且符合≥90％的可标准限度。
为了说明该方法和这些标准的性能特征，以下显示了检测样本子集的IHC结果的代表性图像(原始放大倍数为20倍)，如图14至19所示。
实施例5 AKR1C3检测的诊断试剂盒
在本实施例中，AKR1C3检测的诊断试剂盒(Kit)包括：
pH 6.0的抗原修复液；
1.2ug/ml浓度的AKR1C3单克隆抗体溶液，含有NaN 3、H +离子、Cl -离子以及氨丁三醇；
1.2ug/ml浓度的二抗溶液，含有NaN 3、H +离子、Cl -离子以及氨丁三醇；
封闭液：小鼠血清；
阴性质控试剂溶液；以及
记载相关操作方法的说明书。
1.2ug/ml浓度的小鼠AKR1C3单克隆抗体溶液和1.2ug/ml浓度的二抗溶液均由抗体稀释液稀释得到，抗体稀释液包括：
含有0.1％质量比的聚乙二醇或吐温，和0.015mol/L叠氮化钠的0.05mol/L的Tris-HCl缓冲液(氨丁三醇-盐酸缓冲液)；
二抗为山羊抗小鼠抗体、兔抗小鼠抗体、马抗小鼠抗体或驴抗小鼠抗体。
阴性质控试剂溶液为市售的产品：DAKO公司的FLEX阴性质控，小鼠，(Link)。
AKR1C3检测的诊断试剂盒，其被作为AKR1C3活化的抗癌前药的药物一同使用，通常对患者进行筛选。使用该试剂盒，便于医务人员在决定对患者施用药物前使用统一的检测试剂盒标准操作(SOP)在不同的实验室中进行检测，这样经过同样的试剂、同样的操作得到的AKR1C3检测结果才能与AKR1C3活化的抗癌前药的药物的药品说明书中针对特定癌症的推荐检测结果相匹配。
在说明书中记载了试剂盒的具体操作方法，即上述说明书中的具体操作条件。可选的或作为一种优选方案，在该说明书中还针对不同的癌(肿瘤)类型给出了使用AKR1C3活化的抗癌前药的IHC染色检测方法的打分值。比如说，针对胃癌，某个患者的胃癌组织切片使用上述试剂盒检测打分后得出的分数(例如H评分)为209，而根据统计得出胃癌患者使用AKR1C3活化的抗癌前药的IHC染色检测方法的打分值不能低于165，为此医生对于该患者是可以开出使用AKR1C3活化的抗癌前药的处方的。再比如说，针对食道癌，某个患者的食道癌组织切片使用上述试剂盒检测打分后得出的分数为105，而根据统计得出食道癌患者使用AKR1C3活化的抗癌前药的IHC染色检测方法的打分值不能低于115，为此医生对于该患者是不可以开出使用AKR1C3活化的抗癌前药的处方的。
实施例6 AKR1C3检测方法和AKR1C3检测的诊断试剂盒在治疗癌症、肿瘤或细胞增生性疾病中的用途
某胃癌患者离体的福尔马林固定石蜡包埋人体组织切片使用实施例1建立的AKR1C3检测方法或实施例5的AKR1C3检测的诊断试剂盒检测后得出的打分值(例如H评分)为209，大于预定打分值165；
对该胃癌患者施加AKR1C3活化的抗癌药物。
经过已有的试验验证，选自以下结构的AKR1C3活化的抗癌药物可能具有最好的治疗效果：
Figure PCTCN2021114774-appb-000038
Figure PCTCN2021114774-appb-000039
或者
Figure PCTCN2021114774-appb-000040
或其药学上可接受的盐或其异构体。

Claims (28)

  1. 一种AKR1C3检测方法，其中，使用免疫组织化学染色法检测离体的福尔马林固定石蜡包埋人体组织切片中AKR1C3的表达水平，包括以下步骤：
    a)抗原修复
    在抗原修复液的存在下，将福尔马林固定石蜡包埋人体组织切片于90～115℃加热17～30分钟进行抗原修复；
    b)一抗孵育
    将抗原修复后的福尔马林固定石蜡包埋人体组织切片与浓度为0.5～5.0μg/ml的AKR1C3单克隆抗体溶液混合后孵育25～700分钟；
    c)二抗孵育
    将一抗孵育后的福尔马林固定石蜡包埋人体组织切片与浓度为0.5～5.0μg/ml的二抗溶液混合后孵育25～700分钟。
  2. 根据权利要求1所述的AKR1C3检测方法，其中，在步骤a)抗原修复中，所述抗原修复液的pH值为2.0～9.0；
    更优选地，所述抗原修复液的pH值为6.0～9.0；
    进一步优选地，所述抗原修复液的pH值为6.0。
  3. 根据权利要求1或2所述的AKR1C3检测方法，其中，在步骤a)抗原修复中，所述抗原修复液包括柠檬酸钠抗原修复液或EDTA抗原修复液。
  4. 根据权利要求1～3任一项所述的AKR1C3检测方法，其中，在步骤a)抗原修复中，所述福尔马林固定石蜡包埋人体组织切片于92～102℃加热18～25分钟；
    更优选地，所述福尔马林固定石蜡包埋人体组织切片于97℃加热20分钟。
  5. 根据权利要求1～4任一项所述的AKR1C3检测方法，其中，在步骤b)一抗孵育中，所述AKR1C3单克隆抗体溶液的浓度为1.0～3.0μg/ml；
    更优选地，所述AKR1C3单克隆抗体溶液的浓度为1.2μg/ml；
    和/或，在步骤c)二抗孵育中，所述二抗溶液的浓度为1.0～3.0μg/ml；
    更优选地，所述二抗溶液的浓度为1.2μg/ml。
  6. 根据权利要求1～5任一项所述的AKR1C3检测方法，其中，所述AKR1C3单克隆抗体溶液和所述二抗溶液均含有NaN 3、H +离子、Cl -离子和氨丁三醇。
  7. 根据权利要求6所述的AKR1C3检测方法，其中，所述AKR1C3单克隆抗体溶液和所述二抗溶液由抗体稀释缓冲液稀释得到，所述抗体稀释缓冲液包含以下成分：
    0.02～0.08mol/L的Tris-HCl缓冲液，
    含有0.05～0.15％质量浓度的聚乙二醇或吐温，和
    0.010～0.020mol/L叠氮化钠；
    更优选地，所述抗体稀释缓冲液包含以下成分：
    0.05mol/L的Tris-HCl缓冲液，
    含有0.1％质量浓度的聚乙二醇或吐温，和
    0.015mol/L叠氮化钠。
  8. 根据权利要求1～7任一项所述的AKR1C3检测方法，其中，在步骤b)一抗孵育 中，所述抗原修复后的福尔马林固定石蜡包埋人体组织切片与AKR1C3单克隆抗体溶液的孵育时间为30～45分钟；
    更优选地，所述抗原修复后的福尔马林固定石蜡包埋人体组织切片与AKR1C3单克隆抗体溶液的孵育时间为45分钟。
  9. 根据权利要求1～8任一项所述的AKR1C3检测方法，其中，在步骤c)二抗孵育中，所述一抗孵育后的福尔马林固定石蜡包埋人体组织切片与二抗溶液的孵育时间为30～45分钟；
    更优选地，所述一抗孵育后的福尔马林固定石蜡包埋人体组织切片与二抗溶液的孵育时间为30分钟。
  10. 根据权利要求1～9任一项所述的AKR1C3检测方法，其中，在步骤b)一抗孵育中，所述AKR1C3单克隆抗体为小鼠单克隆抗体；
    和/或，在步骤c)二抗孵育中，所述二抗为山羊抗小鼠抗体、兔抗小鼠抗体、马抗小鼠抗体或驴抗小鼠抗体。
  11. 根据权利要求1～10任一项所述的AKR1C3检测方法，其中，在步骤c)二抗孵育之后，还包括：
    d)染色封固
    使用苏木精对福尔马林固定石蜡包埋人体组织切片进行染色，染色后脱水封片。
  12. 根据权利要求1～11任一项所述的AKR1C3检测方法，其中，在步骤a)抗原修复之前，还包括：
    a1)脱蜡复水
    将福尔马林固定石蜡包埋人体组织切片使用有机溶剂脱蜡，脱蜡后的切片经过不同水含量的醇依次洗涤，最后使用水洗涤；
    优选地，所述有机溶剂为丙酮、甲苯或二甲苯；更优选地，所述有机溶剂为二甲苯；
    和/或，优选地，所述醇为乙醇或甲醇；更优选地，所述醇为乙醇；
    和/或，优选地，所述脱蜡后的切片先经过无水乙醇洗涤，再经过体积分数为90～97％的乙醇洗涤。
  13. 根据权利要求1～12任一项所述的AKR1C3检测方法，其中，在步骤a)抗原修复和步骤b)一抗孵育之间，还包括：
    b1)封闭非特异性抗原
    将抗原修复后的福尔马林固定石蜡包埋人体组织切片与封闭液共同孵育以封闭非特异性抗原；
    优选地，所述封闭液为AKR1C3单克隆抗体来源动物的血清；
    更优选地，所述封闭液为小鼠血清。
  14. 根据权利要求1～13任一项所述的AKR1C3检测方法，其中，所述福尔马林固定石蜡包埋人体组织切片为乳腺癌组织切片、结直肠癌组织切片、食管癌组织切片、胃癌组织切片、肝细胞癌组织切片、非小细胞肺癌组织切片、前列腺癌组织切片、肾细胞癌切片、外周性T细胞淋巴瘤切片或结节性NK/T细胞淋巴瘤切片。
  15. 一种检测AKR1C3的诊断试剂盒，其中，包括：
    抗原修复液；
    浓度为0.5～5.0μg/ml的AKR1C3单克隆抗体溶液；
    浓度为0.5～5.0μg/ml的二抗溶液。
  16. 根据权利要求15所述的检测AKR1C3的诊断试剂盒，其中，所述抗原修复液的pH值为2.0～9.0；
    更优选地，所述抗原修复液的pH值为6.0～9.0；
    进一步优选地，所述抗原修复液的pH值为6.0。
  17. 根据权利要求15或16所述的检测AKR1C3的诊断试剂盒，其中，所述抗原修复液包括柠檬酸钠抗原修复液或EDTA抗原修复液。
  18. 根据权利要求15-17之一所述的检测AKR1C3的诊断试剂盒，其中，所述AKR1C3单克隆抗体溶液的浓度为1.0～3.0μg/ml；
    更优选地，述AKR1C3单克隆抗体溶液的浓度为1.2μg/ml；
    和/或，所述二抗溶液的浓度为1.0～3.0μg/ml；
    更优选地，所述二抗溶液的浓度为1.2μg/ml。
  19. 根据权利要求15-18之一所述的检测AKR1C3的诊断试剂盒，其中，所述AKR1C3单克隆抗体溶液和所述二抗溶液均含有NaN 3、H +离子、Cl -离子和氨丁三醇。
  20. 根据权利要求19所述的检测AKR1C3的诊断试剂盒，其中，所述AKR1C3单克隆抗体溶液和所述二抗溶液由抗体稀释缓冲液稀释得到，所述抗体稀释缓冲液包含以下成分：
    0.02～0.08mol/L的Tris-HCl缓冲液，
    含有0.05～0.15％质量浓度的聚乙二醇或吐温，和
    0.010～0.020mol/L叠氮化钠；
    更优选地，所述抗体稀释缓冲液包含以下成分：
    0.05mol/L的Tris-HCl缓冲液，
    含有0.1％质量浓度的聚乙二醇或吐温，和
    0.015mol/L叠氮化钠。
  21. 根据权利要求15-20之一所述的检测AKR1C3的诊断试剂盒，其中，所述AKR1C3单克隆抗体为小鼠单克隆抗体；
    和/或，所述二抗为山羊抗小鼠抗体、兔抗小鼠抗体、马抗小鼠抗体或驴抗小鼠抗体。
  22. 根据权利要求15-21之一所述的检测AKR1C3的诊断试剂盒，其中，还包括：
    封闭液，优选地，所述封闭液为AKR1C3单克隆抗体来源动物的血清；
    更优选地，所述封闭液为小鼠血清。
  23. 根据权利要求15-22之一所述的检测AKR1C3的诊断试剂盒，其中，还包括：
    阴性质控试剂溶液；以及
    说明书。
  24. 权利要求15-23之一所述的检测AKR1C3的诊断试剂盒在制备治疗癌症、肿瘤或细胞增生性疾病的药物中的用途。
  25. 根据权利要求24所述的用途，其中，包括以下步骤：
    使用所述检测AKR1C3的诊断试剂盒获得患者离体的福尔马林固定石蜡包埋人体组织切片中AKR1C3的表达水平；
    对AKR1C3的表达水平大于或等于预定表达水平的患者施加AKR1C3活化的抗癌药物。
  26. 根据权利要求25所述的用途，其中，所述AKR1C3活化的抗癌药物满足以下定义中的至少一项：
    A.在存在AKR1C3抑制剂的环境下，检测得到的某化合物的癌细胞增殖抑制作用小于不存在AKR1C3抑制剂的环境下检测得到的该化合物的癌细胞增殖抑制作用；
    B.某化合物对AKR1C3酶不同表达水平的癌细胞的增殖抑制作用具有显著差异，且对高表达AKR1C3酶的癌细胞增殖抑制作用远大于低表达AKR1C3酶的癌细胞增殖抑制作用；
    C.当某些含有碳氧双键的醛酮化合物对AKR1C3酶不同表达水平的癌细胞增殖抑制作用具有显著差异时，且对高表达AKR1C3酶的癌细胞增殖抑制作用远大于低表达AKR1C3酶的癌细胞增殖抑制作用，且对应的含有羟基的醇类化合物对于AKR1C3酶不同表达水平的癌细胞增殖抑制作用差距不大或近似，则该含有碳氧双键的醛酮化合物为AKR1C3活化的抗癌药物，对应的含有羟基的醇类化合物为原体药物。
  27. 根据权利要求25或26所述的用途，其中，所述AKR1C3活化的抗癌药物选自以下结构的化合物：
    Figure PCTCN2021114774-appb-100001
    Figure PCTCN2021114774-appb-100002
    Figure PCTCN2021114774-appb-100003
    Figure PCTCN2021114774-appb-100004
    或者
    Figure PCTCN2021114774-appb-100005
    Figure PCTCN2021114774-appb-100006
    或其药学上可接受的盐或其异构体；或者
    Figure PCTCN2021114774-appb-100007
    或其药学上可接受的盐或其异构体；
    优选地，所述AKR1C3活化的抗癌药物选自以下结构的化合物：
    Figure PCTCN2021114774-appb-100008
    Figure PCTCN2021114774-appb-100009
    或者
    Figure PCTCN2021114774-appb-100010
    或者
    Figure PCTCN2021114774-appb-100011
    或其药学上可接受的盐或其异构体。
  28. 根据权利要求24～27任一项所述的用途，其中，所述癌症、肿瘤或细胞增生性疾病包括：
    肺癌、非小细胞肺癌、肝癌、胰腺癌、乳腺癌、胃癌、骨癌、食管癌、乳房癌、前列腺癌、睾丸癌、结肠癌、卵巢癌、膀胧癌、宫颈癌、肝细胞癌、黑色素瘤、鳞状细胞癌、基底细胞癌、腺癌、汗腺癌、皮脂腺癌、乳头状癌、乳头状腺癌、肾细胞癌、囊性腺癌、囊性癌、髓状癌、支气管癌、骨细胞癌、上皮癌、胆管癌、绒毛膜癌、胚癌、精原细胞癌、维尔姆斯癌、胶质细胞癌、星形细胞瘤、成神经管细胞瘤、颅咽管瘤、室管膜瘤、松果体瘤、成血细胞瘤、声带神经瘤、脑膜瘤、成神经细胞瘤、成视神经细胞瘤、成视网膜细胞瘤、神经纤维瘤、纤维肉瘤、成纤维细胞瘤、纤维瘤、纤维腺瘤、纤维软骨瘤、纤维囊瘤、纤维粘液瘤、纤维骨瘤、纤维粘液肉瘤、纤维乳头状瘤、粘液肉瘤、粘液囊瘤、粘液软骨瘤、粘液软骨肉瘤、粘液软骨纤维肉瘤、粘液腺瘤、成粘液细胞瘤、脂肉瘤、脂肪瘤、脂肪腺瘤、成脂细胞瘤、脂肪软骨瘤、脂肪纤维瘤、脂肪血管瘤、粘液脂瘤、软骨肉瘤、软骨瘤、软骨肌瘤、脊索瘤、绒毛膜腺瘤、绒毛上皮瘤、成绒毛膜细胞瘤、骨肉瘤、成骨细胞瘤、骨软骨纤维瘤、骨软骨肉瘤、骨软骨瘤、骨囊瘤、骨牙质瘤、骨纤维瘤、骨纤维肉瘤、血管肉瘤、血管瘤、血管脂肪瘤、血管软骨瘤、成血管细胞瘤、血管角质瘤、血管神经胶质瘤、血管内皮瘤、血管纤维瘤、血管肌瘤、血管脂肪瘤、血管淋巴管瘤、血管脂肪平滑肌瘤、血管肌脂瘤、血管肌神经瘤、血管粘液瘤、血管网状内皮瘤、淋巴管肉瘤、淋巴肉芽瘤、淋巴管瘤、淋巴瘤、淋巴粘液瘤、淋巴肉瘤、淋巴管纤维瘤、淋巴细胞瘤、淋巴上皮瘤、成淋巴细胞瘤、外周性T细胞淋巴瘤、结节性NK/T细胞淋巴瘤、内皮瘤、成内皮细胞瘤、滑膜瘤、滑膜肉瘤、间皮瘤、结缔组织瘤、尤因瘤、平滑肌瘤、平滑肌肉瘤、成平滑肌瘤、平滑肌纤维瘤、横纹肌瘤、横纹肌肉瘤、横纹肌粘液瘤、急性淋巴白血病、急性骨髓性白血病、慢性病细胞、红细胞增多症、淋巴瘤、子宫内膜癌、胶质瘤、结直肠癌、甲状腺癌、尿路上皮癌或多发性骨髓瘤；优选地，所述癌症、肿瘤或细胞增生性疾病包括：卵巢癌、宫颈癌、胰腺癌、乳腺癌、结直肠癌、食管癌、胃癌、肝细胞癌、非小细胞肺癌、前列腺癌、肾细胞癌、外周性T细胞淋巴瘤或结节性NK/T细胞淋巴瘤。
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116086919A (zh) * 2023-02-20 2023-05-09 深圳裕策生物科技有限公司 一种肺癌和/或胰腺癌样本的染色方法及试剂盒
WO2024023641A1 (en) * 2022-07-26 2024-02-01 Novartis Ag Methods of tricyclic akr1c3 dependent kars inhibitor dosing field of the invention

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112904026A (zh) * 2019-12-03 2021-06-04 深圳艾欣达伟医药科技有限公司 通过前列腺素含量来关联akr1c3酶表达水平及筛选给药用途

Citations (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005098450A2 (en) * 2004-04-02 2005-10-20 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for use in evaluating and treating neoplastic disease conditions
WO2011121028A1 (en) * 2010-03-30 2011-10-06 Siemens Healthcare Diagnostics Inc. Algorithm for prediction of benefit from addition of taxane to standard chemotherapy in patients with breast cancer
WO2016145092A1 (en) 2015-03-10 2016-09-15 Threshold Pharmaceuticals, Inc. Dna alkylating agents
WO2016161342A2 (en) 2015-04-02 2016-10-06 Threshold Pharmaceuticals, Inc. Nitrobenzyl derivatives of anti-cancer agents
WO2017087428A1 (en) 2015-11-16 2017-05-26 Threshold Pharmaceuticals, Inc. (r)- and (s)-1-(3-(3-n,n-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-n,n'-bis (ethylene)phosphoramidate, compositions and methods for their use and preparation
US20170268066A1 (en) * 2016-03-15 2017-09-21 Chalmers Ventures Ab Cancer biomarkers
US20170315127A1 (en) * 2013-10-28 2017-11-02 The Regents Of The University Of California Treatment of metastatic prostate cancer
CN109387630A (zh) * 2017-08-14 2019-02-26 杭州源昶医药科技有限公司 用于预测吉西他滨药物敏感性的生物标记物及其用途
WO2019190331A1 (en) 2018-03-29 2019-10-03 Achilles Medical Limited Prodrug compounds activated by akr1c3 and their use for treating hyperproliferative disorders
WO2020082037A1 (en) * 2018-10-19 2020-04-23 Auransa Inc. Methods for treating a subtype of small cell lung cancer
CN111435138A (zh) * 2019-01-15 2020-07-21 中国科学院分子细胞科学卓越创新中心 胰腺癌蛋白生物标记物及其应用
WO2021005586A1 (en) 2019-08-01 2021-01-14 Novartis Ag Tricyclic akr1c3 dependent kars inhibitors
WO2021068952A1 (zh) 2019-10-12 2021-04-15 南京明德新药研发有限公司 靶向醛酮还原酶1c3的苯并二氢吡喃类化合物

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005098450A2 (en) * 2004-04-02 2005-10-20 The Board Of Trustees Of The Leland Stanford Junior University Methods and compositions for use in evaluating and treating neoplastic disease conditions
WO2011121028A1 (en) * 2010-03-30 2011-10-06 Siemens Healthcare Diagnostics Inc. Algorithm for prediction of benefit from addition of taxane to standard chemotherapy in patients with breast cancer
US20170315127A1 (en) * 2013-10-28 2017-11-02 The Regents Of The University Of California Treatment of metastatic prostate cancer
CN107530556A (zh) 2015-03-10 2018-01-02 深圳艾衡昊医药科技有限公司 Dna烷化剂
WO2016145092A1 (en) 2015-03-10 2016-09-15 Threshold Pharmaceuticals, Inc. Dna alkylating agents
WO2016161342A2 (en) 2015-04-02 2016-10-06 Threshold Pharmaceuticals, Inc. Nitrobenzyl derivatives of anti-cancer agents
CN108136214A (zh) 2015-04-02 2018-06-08 深圳艾衡昊医药科技有限公司 硝基苄基衍生物抗癌试剂
WO2017087428A1 (en) 2015-11-16 2017-05-26 Threshold Pharmaceuticals, Inc. (r)- and (s)-1-(3-(3-n,n-dimethylaminocarbonyl)phenoxyl-4-nitrophenyl)-1-ethyl-n,n'-bis (ethylene)phosphoramidate, compositions and methods for their use and preparation
CN108290911A (zh) 2015-11-16 2018-07-17 深圳艾衡昊医药科技有限公司 (r)-及(s)-1-(3-(3-n,n-二甲基胺基羰基)苯氧基-4-硝苯基)-1-乙基-n,n’-双(伸乙基)胺基磷酸酯、组合物及其使用及制备方法
US20170268066A1 (en) * 2016-03-15 2017-09-21 Chalmers Ventures Ab Cancer biomarkers
CN109387630A (zh) * 2017-08-14 2019-02-26 杭州源昶医药科技有限公司 用于预测吉西他滨药物敏感性的生物标记物及其用途
WO2019190331A1 (en) 2018-03-29 2019-10-03 Achilles Medical Limited Prodrug compounds activated by akr1c3 and their use for treating hyperproliferative disorders
CN111918864A (zh) 2018-03-29 2020-11-10 阿基利斯医疗有限公司 通过akr1c3活化的前药化合物及其治疗过度增殖性失调的用途
WO2020082037A1 (en) * 2018-10-19 2020-04-23 Auransa Inc. Methods for treating a subtype of small cell lung cancer
CN111435138A (zh) * 2019-01-15 2020-07-21 中国科学院分子细胞科学卓越创新中心 胰腺癌蛋白生物标记物及其应用
WO2021005586A1 (en) 2019-08-01 2021-01-14 Novartis Ag Tricyclic akr1c3 dependent kars inhibitors
WO2021068952A1 (zh) 2019-10-12 2021-04-15 南京明德新药研发有限公司 靶向醛酮还原酶1c3的苯并二氢吡喃类化合物

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
AZZARELLO JOSEPH, FUNG KAR-MING, LIN HSUEH-KUNG: "Tissue Distribution of Human AKR1C3 and Rat Homolog in the Adult Genitourinary System", JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, vol. 56, no. 9, 1 September 2008 (2008-09-01), US , pages 853 - 861, XP055906686, ISSN: 0022-1554, DOI: 10.1369/jhc.2008.951384 *
CAI GUANGLINGYU GUANGYINZHAO YANG.: "Influence of pH of antigen retrieval solution on immunohistochemical staining of lymphoid tissues [J", ACTA MEDICINAE SINICA, vol. 18, no. 4, 2005, pages 501 - 502
CAS , no. 2097713-69-2
DU JUANSHI XUEYINGZHENG JIE ET AL.: "Influence of pH and retrieval time of antigen retrieval solution on the immunohistochemical staining effects [J", JOURNAL OF PEKING UNIVERSITY (HEALTH SCIENCES, vol. 037, no. 002, 2005, pages 195 - 197
FANG JIEDIWANG XIAOXINGZHANG MENGLING ET AL.: "Influence of water quality on immunohistochemical staining results [J", JOURNAL OF CLINICAL AND EXPERIMENTAL PATHOLOGY, vol. 35, no. 04, 2019, pages 111 - 113
FLANAGAN, MEDICINAL CHEMISTRY, 2014, pages 962 - 977
JUANRAN: "AKR1C3 Expression in Gastric Cancer Tissue And Its Relationship with Prognosis", CHINESE MASTER'S THESES FULL-TEXT DATABASE, 1 May 2016 (2016-05-01), pages 1 - 44, XP055907536 *
K WAKO , T KAWASAKI , K YAMANA , K SUZUKI , S JIANG , H UMEZU , T NISHIYAMA , K TAKAHASHI , T HAMAKUBO , T KODAMA , M NAITO: "Expression of androgen receptor through androgen-converting enzymes is associated with biological aggressiveness in prostate cancer.", JOURNAL OF CLINICAL PATHOLOGY, vol. 61, no. 4, 24 August 2007 (2007-08-24), GB , pages 448 - 454, XP009535008, ISSN: 0021-9746, DOI: 10.1136/jcp.2007.050906 *
KUHN ELISABETTA, AYHAN AYSE, SHIH IE-MING, SEIDMAN JEFFREY D., KURMAN ROBERT J.: "Ovarian Brenner tumour: A morphologic and immunohistochemical analysis suggesting an origin from fallopian tube epithelium", EUROPEAN JOURNAL OF CANCER, vol. 49, no. 18, 1 December 2013 (2013-12-01), Amsterdam NL , pages 3839 - 3849, XP055907145, ISSN: 0959-8049, DOI: 10.1016/j.ejca.2013.08.011 *
LIU HAIYANGWANG XIAOJUNZHANG HAIYU ET AL.: "Influence of microwave heating retrieval and hydrochloric acid hydrolysis retrieval methods on immunohistochemical staining results of rat brain tissue [J", JOURNAL OF NINGXIA MEDICAL UNIVERSITY, no. 11, 2011, pages 1115 - 1116
LIU XIANYANYANG JIANCHEN YING ET AL.: "Influence of different antigen retrieval solutions and retrieval methods on immunohistochemical staining results [J", JOURNAL OF GUANGDONG MEDICAL COLLEGE, no. 05, 2013, pages 43 - 44
LUO XINLANLIN XINGTAOLUO DONGLAN ET AL.: "Influence of antigen retrieval solution with different components at pH 9.0 on immunohistochemical staining results [J].", CHINESE JOURNAL OF PATHOLOGY, vol. 41, no. 003, 2012, pages 192 - 194
MA YIQUN: "The Mechanism of the Notoginsenoside R1 on ROS, PI3K/AKT Signal Transduction Pathway and AKR1-C3 of Keloid Fibroblasts in Vitro and Tissue", CHINA MASTER’S THESES FULL-TEXT DATABASE, 1 May 2018 (2018-05-01), pages 1 - 68, XP055907543 *
MENG KUIZHOU XIAOJUN: "The role of antigen retrieval techniques in immunohistochemistry [J", CHINESE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, vol. 10, no. 001, 2001, pages 109 - 111
NOGRADY: "Medicinal Chemistry A Biochemical Approach", 1985, OXFORD UNIVERSITY PRESS, pages: 388 - 392
TOMO TAHARA: "Study on Berberine Inhibiting the Development of Castration-Resistant Prostate Cancer by Targeting AKR1C3 and Its Mechanisms", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, vol. 2014, no. 9, 1 April 2014 (2014-04-01), pages 1 - 102, XP055907529 *
WANG, HAILI: "The Expressions and Clinical Significance of Androgen Receptor and Androgen Metabolism-related Enzymes in Endometrial Carcinoma", CHINA MASTER’S THESES FULL-TEXT DATABASE, 1 May 2018 (2018-05-01), pages 1 - 51, XP055907547 *
YANG, LEI: "Global and Quantitative Evaluation Towards Expression Status of Aldo-keto Reductase Genes in Liver Cancer", CHINA DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, 1 October 2012 (2012-10-01), pages 1 - 137, XP055907556 *
ZHANG WEILIANG YINGJIE: "Influence of different antigen retrieval methods and staining conditions on the results of P53 protein immunohistochemical methods [J", CHINESE JOURNAL OF HISTOCHEMISTRY AND CYTOCHEMISTRY, no. 2, 2002

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024023641A1 (en) * 2022-07-26 2024-02-01 Novartis Ag Methods of tricyclic akr1c3 dependent kars inhibitor dosing field of the invention
CN116086919A (zh) * 2023-02-20 2023-05-09 深圳裕策生物科技有限公司 一种肺癌和/或胰腺癌样本的染色方法及试剂盒
CN116086919B (zh) * 2023-02-20 2024-01-26 深圳裕策生物科技有限公司 一种肺癌和/或胰腺癌样本的染色方法及试剂盒

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